WO2006062200A1 - Nucleic acid library and protein library - Google Patents

Abstract

It is intended to provide a nucleic acid library by which proteins of multiple types can be provided at once in coordination with nucleic acids encoding the same, the proteins of multiple types can be analyzed in parallel immediately after providing these proteins, and a denatured or inactivated protein can be easily regenerated, and which can be efficiently prepared without resorting to the coordination of the positional relation between a nucleic acid and a protein capturer in the course of the preparation thereof. To achieve this object, a nucleic acid library (1) which has a first solid support (2), multiple nucleic acids (3) having been immobilized in a the state of being separated from each other in a definite area of the surface of the first solid support (2), a second solid support (4) spirally winding around the first solid support (2), and multiple protein capturers (5) having been immobilized at a high density in a definite area of the surface of the second solid support (4) is provided.

Description

 Nucleic acid library and protein library

 Technical field

 [0001] The present invention relates to a nucleic acid library (aggregate of nucleic acids) containing a plurality of nucleic acids immobilized on the surface of a solid support, and a plurality of proteins displayed (presented) on the surface of the solid support. Containing protein libraries (aggregates of proteins).

 Background art

 In proteomics, it is necessary to perform a wide variety of analyzes on the structure, function, etc. of multiple types of proteins in parallel (ie, high-throughput analysis). The usefulness of protein arrays (protein chips), in which multiple types of proteins are immobilized on the surface of a solid support, is attracting attention when analyzing multiple types of proteins in parallel. In the protein array, a plurality of types of proteins are fixed in a predetermined region on the surface of the solid support, and the types of proteins can be identified by the fixing positions of the proteins. The protein array is useful, for example, for screening proteins that can bind to a target substance (for example, protein, DNA, etc.) from a plurality of types of proteins.

 [0003] However, in the conventional protein array, when the protein is immobilized on the surface of the solid support, it is necessary to perform inefficient spotting of the protein on the surface of the solid support.

 [0004] In the conventional protein array, the protein immobilized on the surface of the solid support is denatured and deactivated by drying, and the protein immobilized on the surface of the solid support is immediately denatured and deactivated. When this occurs, it is difficult to regenerate the protein, so it has been difficult to store and use it for a long time.

[0005] Furthermore, in the assembly system using the conventional protein array, the analysis can be started immediately after the protein is supplied because the analysis can be performed only after the supplied protein is fixed on the surface of the solid support. In addition, the protein supply power required a lot of time and effort to start the analysis. That is, protein supply and protein It was difficult to efficiently analyze proteins because it was a separate and independent technology.

 [0006] Under such circumstances, a nucleic acid library having two or more types of library sets that exist in a state of being separated from each other, wherein each library unit has an in vitro transcription'translation system or in vitro translation. The system comprises a nucleic acid capable of expressing one or more kinds of proteins and a protein trap provided in the vicinity of the nucleic acid. Each library unit is provided on the surface of a solid support, and A nucleic acid library in which nucleic acids contained in a rally unit are fixed on the surface of a solid support provided with each library unit has been developed (see Patent Document 1).

 [0007] In the nucleic acid library, different types of library units exist in a state of being separated from each other, and protein capture bodies are provided in close proximity to the nucleic acid in each library unit. The distance between the nucleic acid contained in the library unit and the protein capture body is overwhelmingly larger than the distance between the nucleic acid contained in the same library unit and the protein capture body. Therefore, proteins that also express nucleic acid strength in each library unit are preferentially captured by protein catchers contained in the same library unit over protein catchers contained in different types of library units. Thus, cross-contamination of proteins expressed in different types of library units can be effectively prevented. Therefore, when two or more types of library units possessed by the above-mentioned nucleic acid library are subjected to an in vitro transcription 'translation system or an in vitro translation system, the nucleic acid-expressed proteins contained in each library unit are identical to the same library. It is displayed (presented) on the solid support while being captured by the protein trap contained in the unit. That is, according to the nucleic acid library, a plurality of types of proteins can be supplied at a time in a state in which they are associated with nucleic acids encoding them and displayed on a solid support. Protein analysis can be performed efficiently in parallel.

[0008] The nucleic acid library is immediately converted into a protein library by subjecting each library unit to an in vitro transcription / translation system or an in vitro translation system. In other words, in the nucleic acid library, protein supply and protein assembly are Since they are integrated, the plurality of types of proteins can be analyzed immediately after the supply of a plurality of types of proteins.

 [0009] Furthermore, in the above-described nucleic acid library, even if the protein displayed on the solid support is denatured or inactivated, the protein can be easily expressed by re-expressing the nucleic acid corresponding to the protein. Can be played.

 Patent Document 1: International Publication WO03Z083111 Pamphlet

 Disclosure of the invention

 Problems to be solved by the invention

 [0010] In Patent Document 1, for example, in order to provide a nucleic acid and a protein capture body close to each library unit, one end of the nucleic acid is fixed to a solid support, and a protein capture body is provided at the other end of the nucleic acid. The positional relationship between the nucleic acid and the protein capturing body is associated. Thus, when the library unit is formed by associating the positional relationship between the nucleic acid and the protein trap for each library unit, the efficiency of producing the nucleic acid library is lowered.

 [0011] Thus, the present invention can supply a plurality of types of proteins at once in association with the nucleic acids that encode them, and immediately analyze the plurality of types of proteins after supplying the plurality of types of proteins. It is a nucleic acid library that can be performed in parallel and can easily regenerate proteins that have been denatured or inactivated, and it is necessary to associate the positional relationship between the nucleic acid and the protein trap in the production. It is an object of the present invention to provide a nucleic acid library that can be efficiently produced and a method for producing the same.

 [0012] Another object of the present invention is to provide a protein library prepared using the nucleic acid library of the present invention, and a method for preparing a protein library using the nucleic acid library of the present invention. .

 Means for solving the problem

[0013] In order to solve the above-mentioned problems, the present invention provides a first solid support and an in vitro transcription / translation system or in vitro translation system fixed to a predetermined surface region of the first solid support. A plurality of nucleic acid groups capable of expressing different types of proteins in the second solid support, and the plurality of nucleic acid groups fixed to the predetermined surface region of the second solid support at a high density. With multiple protein traps that can capture any protein Each of the plurality of nucleic acid groups is fixed to one partial region of the plurality of partial regions that are separated from each other on the predetermined surface region, and the plurality of protein capture bodies The second solid support is arranged with respect to the first solid support so that the predetermined surface region to which the plurality of nucleic acid groups are fixed is close to the predetermined surface region to which the plurality of nucleic acid groups are fixed. A nucleic acid library is provided.

 [0014] In the nucleic acid library of the present invention, there is a case where a predetermined surface region of the second solid support (hereinafter referred to as "protein capturer fixed surface region"!) Is fixed. ) Is close to a predetermined surface region (hereinafter sometimes referred to as “nucleic acid immobilization surface region”) of the first solid support in which a plurality of nucleic acid groups are immobilized in a state of being separated from each other. The different protein capture bodies for each nucleic acid group are close to each other, and the distance between one nucleic acid group and a protein capture body close to one nucleic acid group It is overwhelmingly smaller than the distance to the body. Since each protein capturer can capture any protein expressed from each nucleic acid group, each nucleic acid group may be in close proximity to a different protein capturer.

 [0015] Therefore, when the nucleic acid library of the present invention is subjected to an in vitro transcription / translation system or an in vitro translation system, the protein expressed from each nucleic acid group has priority over the protein catcher adjacent to each nucleic acid group. Therefore, cross-contamination of proteins expressed from each nucleic acid group is prevented. That is, according to the nucleic acid library of the present invention, a plurality of types of proteins can be displayed on the second solid support in a state in which they are associated with the nucleic acids that encode them, thereby analyzing a plurality of types of proteins. Can be performed efficiently in parallel.

 [0016] Further, the nucleic acid library of the present invention is immediately converted into a protein library by subjecting it to an in vitro transcription / translation system or an in vitro translation system. That is, in the nucleic acid library of the present invention, the protein supply and the protein assembly are in the form of a body. Therefore, according to the nucleic acid library of the present invention, it is possible to analyze the plurality of types of proteins immediately after supplying the plurality of types of proteins.

[0017] Furthermore, according to the nucleic acid library of the present invention, even if the protein displayed on the second solid support is denatured or inactivated, the nucleic acid group corresponding to the protein is collected. By re-expressing the protein, the protein can be easily regenerated.

 [0018] Further, in preparing the nucleic acid library of the present invention, a nucleic acid fixing surface region is formed by a step of fixing a plurality of nucleic acid groups in a state of being separated from each other to a predetermined surface region of the first solid support. The protein capturer immobilization surface region is formed by fixing a plurality of protein capturers at a high density on the predetermined surface region of the solid support 2 so that the protein capturer immobilization surface region is close to the nucleic acid immobilization surface region. The second solid support may be disposed with respect to the first solid support. That is, the protein traps need not be arranged in association with the positions of the nucleic acid groups, and may be arranged randomly regardless of the positions of the nucleic acid groups. Therefore, the nucleic acid library of the present invention can be produced efficiently.

 [0019] In the nucleic acid library of the present invention, the protein capture body is overwhelmed by the distance between adjacent protein capture bodies than the distance between adjacent partial areas among a plurality of partial areas that are separated from each other. The second solid support is fixed to a predetermined surface area of the second solid support with such a density that the distance between the nucleic acid fixation surface area and the protein capturer fixation surface area is separated from each other. It arrange | positions with respect to a 1st solid support body so that it may become overwhelmingly smaller than the distance between adjacent partial areas among several existing partial areas. This effectively prevents cross-contamination of proteins in which each nucleic acid group force is also expressed.

 [0020] The distance between adjacent partial areas among a plurality of partial areas that are separated from each other is not particularly limited, but is usually not less than 0. Olmm, preferably not less than 0.1 mm, more preferably lmm. That's it. The upper limit value of the distance between the adjacent partial regions is not particularly limited, and can be appropriately adjusted according to the size of the predetermined surface region of the first solid support, but is usually 10 mm. The “distance between partial areas” means the distance between the two closest points in the periphery of the two partial areas.

[0021] The protein capture body has a density such that the distance between adjacent protein capture bodies is 1/100 or less of the distance between adjacent partial areas among a plurality of partial areas that are separated from each other. The second solid support is preferably fixed to a predetermined surface region. The distance between adjacent protein traps is the distance between multiple partial regions that are separated from each other. 1/100, 1 / 10,000, 1 / 10,000 or less, 1 / 100,000, 1 / 1,000,000 or less, 1 / 10,000,000 or less. The better.

 [0022] The second solid support is such that the distance between the protein capturer-immobilized surface region and the nucleic acid-immobilized surface region is 5 minutes of the distance between adjacent partial regions among a plurality of partial regions that are separated from each other. It is preferable that it is arranged with respect to the first solid support so that it is 1 or less. The distance between the surface area for immobilizing the protein capturer and the surface area for immobilizing the nucleic acid is 1/5 or less, 50 minutes or less, and 500 minutes or less of the distance between adjacent partial areas that are separated from each other. The smaller it is, the better it is. The lower limit of the distance between the protein capturer fixed surface region and the nucleic acid fixed surface region is zero.

 [0023] In the nucleic acid library of the present invention, the shape of the first and second solid supports, the arrangement of the second solid support relative to the first solid support, and the like are as follows. The region is not particularly limited as long as the region can be brought close to the nucleic acid immobilization surface region, but in a preferred embodiment of the nucleic acid library of the present invention, the second solid support is made of a flexible long member. The second solid support is spirally mounted on the first solid support (this embodiment is hereinafter referred to as “first embodiment”).

 [0024] According to the first aspect, by adjusting the number of turns of the second solid support, the sequence density of the protein capture body relative to the nucleic acid-fixed surface region can be adjusted, and expression from each nucleic acid group can be achieved. Cross contamination of the produced protein can be effectively prevented. In the first embodiment, the density of the protein traps on the nucleic acid-fixed surface region is larger than the density of the protein traps on the surface of the second solid support.

 [0025] Further, according to the first aspect, the second solid support interposed between adjacent nucleic acid groups serves as a barrier, and effectively prevents cross-contamination of proteins expressed by each nucleic acid force. can do.

[0026] In the first aspect, the shape of the flexible long member is not particularly limited as long as it is long, and examples thereof include a thread shape, a string shape, and a tape shape. [0027] In the first aspect, the shape of the first solid support is not particularly limited as long as the flexible long member can be equipped. For example, a flat plate shape, a cylindrical shape, a cylindrical shape, a prismatic shape , Rectangular tube shape, rod shape, thread shape, string shape, tape shape and the like.

 [0028] In the first embodiment, the number of windings of the second solid support is not particularly limited as long as the protein-immobilized surface region is close to each nucleic acid group. The number of turns of the second solid support per unit length in the winding axis direction is preferably when the shortest length is the unit length in the winding axis direction. Is at least once, more preferably at least twice. The upper limit value of the number of turns of the second solid support per unit length in the winding axis direction is not particularly limited and can be appropriately adjusted according to the width of the second solid support. The length of each partial region in the winding axis direction is not particularly limited, but is usually 0.05 mm or more, preferably 2 mm or more. The upper limit of the length in the winding axis direction of each partial region is not particularly limited, and is a force that can be appropriately adjusted according to the size of the first solid support and the like, which is usually 10 mm. The width of the second solid support is less than or equal to the unit length in the winding axis direction, and the second solid support is mounted on the first solid support so that different regions of the protein immobilization surface region do not overlap each other. It is preferable to do. The “winding shaft” means a line passing through the center of the spiral formed by the second solid support mounted on the first solid support.

 [0029] In the nucleic acid library of the present invention, the protein displayed on the second solid support is analyzed while the second solid support is arranged with respect to the first solid support. Depending on the shape of the second solid support, the arrangement of the second solid support relative to the first solid support, etc., the second solid support may become an obstacle to protein analysis. is there. For example, when the entire nucleic acid immobilization surface region is covered by the second solid support, the second solid support remains in a state in which the second solid support is arranged with respect to the first solid support. Access to the protein displayed on the support is difficult. In such a case, it is difficult to automate a series of operations up to protein analysis using the protein library alone as a protein library production ability.

[0030] In the nucleic acid library of the present invention, when the entire nucleic acid immobilization surface region is covered with the second solid support, for example, the second solid support has a hollow part. Material (for example, a flexible sheet-like member curved or bent into a cylindrical shape, a rectangular tube shape, a `` c '' shape, a `` U '' shape, etc.), and the first solid support is the second The case where it is accommodated in the hollow part of a solid support is mentioned.

 [0031] On the other hand, in the first aspect, the second solid support is displayed on the second solid support even when the second solid support remains arranged with respect to the first solid support. Easy access to proteins. Therefore, according to the first aspect, it is possible to easily automate a series of operations up to protein analysis using a protein library.

 [0032] In the nucleic acid library of the present invention, the method for identifying the type of nucleic acid immobilized on the surface of the first solid support is not particularly limited, but in a preferred embodiment of the nucleic acid library of the present invention, The position of each nucleic acid group on the surface of the first solid support is associated with the type of nucleic acid contained in each nucleic acid group (this embodiment is hereinafter referred to as “second embodiment”).

 [0033] According to the second aspect, the type of nucleic acid contained in each nucleic acid group and the type of protein expressed from each nucleic acid group can be easily identified based on the position of each nucleic acid group. In addition, since the protein expressed from each nucleic acid group is captured by a protein capture body close to each nucleic acid group, the type of protein captured by the protein capture body is at the position of the nucleic acid group close to the protein capture body. Based on this, it can be easily identified.

 [0034] In the nucleic acid library of the present invention, the type of the protein capture body is not particularly limited as long as any protein expressed from each nucleic acid group can be captured, but in a preferred embodiment of the present invention, The protein expressed from each nucleic acid group is a fusion protein of a target protein of a different type between the nucleic acid groups and a tag protein of the same type between the nucleic acid groups, and binds to each protein capture force tag protein (This mode is hereinafter referred to as “third mode”).

 [0035] According to the third aspect, the binding between the protein expressed from each nucleic acid group and the protein trap is ensured. The “target protein” means a protein to be analyzed that is displayed on the second solid support.

[0036] In the third embodiment, the fusion proteins expressed from each nucleic acid group have the same orientation. U, preferred to bind with protein traps. By making the direction of the fusion protein expressed from each nucleic acid group the same, the reactivity of the fusion protein displayed on the second solid support can be made uniform and optimized.

 [0037] The present invention is a method for producing the nucleic acid library of the present invention, wherein each of the plurality of nucleic acid groups in each of the plurality of site regions of the predetermined surface region of the first solid support is provided. A step of fixing one of the nucleic acid groups, a step of fixing the plurality of protein capturing bodies at a high density to a predetermined surface region of the second solid support, and the plurality of protein capturing bodies being fixed. A method comprising the step of arranging the second solid support relative to the first solid support so that the predetermined surface area is close to the predetermined surface area to which the plurality of nucleic acid groups are fixed. provide.

 [0038] According to the method of the present invention, the nucleic acid-immobilized surface region and the protein capturer-immobilized surface region are separately formed on the first solid support and the second solid support, respectively. The nucleic acid library of the present invention can be prepared by arranging the second solid support relative to the first solid support so as to be close to the nucleic acid immobilization surface region. That is, in producing the nucleic acid library of the present invention, the protein capture bodies need not be arranged in association with the positions of the nucleic acids, but may be arranged at random regardless of the positions of the nucleic acids. Therefore, according to the nucleic acid library of the present invention, the nucleic acid library of the present invention can be efficiently produced.

 [0039] The present invention provides a protein obtained by subjecting the nucleic acid library of the present invention to an in vitro transcription / translation system or an in vitro translation system to express a plurality of nucleic acid groups contained in the nucleic acid library at once. Provide a library.

 [0040] Even if the nucleic acid library of the present invention is subjected to an in vitro transcription / translation system or an in vitro translation system all at once, cross-contamination of proteins in which each nucleic acid group force is expressed is prevented. Are displayed at once on a second solid support in association with the nucleic acid encoding them. Therefore, according to the protein library of the present invention, it is possible to efficiently analyze a plurality of types of proteins in parallel.

[0041] In addition, since the protein library of the present invention is immediately prepared by expressing the nucleic acid group contained in the nucleic acid library of the present invention, a plurality of types of proteins can be supplied. Thereafter, the plurality of types of proteins can be analyzed immediately.

 [0042] Furthermore, in the protein library of the present invention, even if the protein displayed on the solid support is denatured or inactivated, the protein group is expressed by re-expressing the nucleic acid group corresponding to the protein. Can be reproduced easily.

 [0043] Furthermore, the protein library of the present invention is stored in the state of a nucleic acid library, and can be repeatedly used over a long period of time by regenerating the protein library when necessary.

 [0044] The present invention provides a protein comprising the step of subjecting the nucleic acid library of the present invention to an in vitro transcription / translation system or in vitro translation system to express a plurality of nucleic acid groups contained in the nucleic acid library at one time. A method for producing a library is provided.

 [0045] According to the method for producing a protein library of the present invention, the protein library of the present invention can be produced in a state in which cross-contamination of the protein expressed from each nucleic acid group is prevented.

 [0046] The present invention relates to a solid support and different types of proteins immobilized in a predetermined surface region of the solid support in a state of being separated from each other in an in vitro transcription'translation system or an in vitro translation system. A method for producing a nucleic acid library comprising a plurality of nucleic acid groups to be expressed and a protein trap fixed to the predetermined surface region in the vicinity of each nucleic acid group, wherein the nucleic acid library is formed on the entire predetermined surface region. A step of fixing a plurality of protein capture bodies capable of capturing any protein expressed from a plurality of nucleic acid groups at a high density, and a plurality of partial regions existing apart from each other on the predetermined surface region, respectively. A method comprising the step of immobilizing one nucleic acid group among the plurality of nucleic acid groups is provided.

 [0047] According to the method of the present invention, when the nucleic acid and the protein capturing body are immobilized on the surface of the same solid support, it is not necessary to arrange the protein capturing body in association with the position of the nucleic acid. If the sequences are randomly arranged, the nucleic acid library can be efficiently produced.

[0048] In the method of the present invention, the order of immobilization of the nucleic acid and the protein catcher to the predetermined surface region of the solid support is not particularly limited, and the nucleic acid may be immobilized after immobilizing the protein capturer. And you can fix the protein trap after fixing the nucleic acid. [0049] In the method of the present invention, a plurality of protein capture bodies are separated by 100 minutes of the distance between adjacent partial areas of the plurality of partial areas. It is preferable to fix at a density such that it is 1 or less. Thereby, cross-contamination of proteins expressed by each nucleic acid group force can be effectively prevented. The distance between adjacent protein capture bodies is less than 1/100, less than 1/1000, less than 1 / 10,000, and 100,000 minutes Less than 1 / 1,000,000 / 1,000,000 / 1,000,000 or less

The smaller it is, the better.

 [0050] In the method of the present invention, the solid support is preferably porous. Since the fluidity of the liquid (for example, in vitro transcription, translation system or in vitro translation system) is low in the pores of the solid support, the group of nucleic acids immobilized on the internal surface of the pores of the solid support is expressed. Tungsten is not easily released from the pores. Therefore, the protein expressed from the nucleic acid group can be surely captured by the protein trap, and cross-contamination of the protein in which each nucleic acid group force is expressed can be effectively prevented.

 The invention's effect

 [0051] According to the present invention, a plurality of types of proteins can be supplied at once in association with the nucleic acids that encode them, and the plurality of types of proteins are analyzed immediately after the supply of the plurality of types of proteins. Is a nucleic acid library that can easily regenerate proteins that have undergone denaturation or inactivation, and associates the positional relationship between the nucleic acid and the protein trap during the production. Nucleic acid libraries that can be efficiently produced without need are provided. In addition, according to the present invention, there is provided a protein library prepared using the nucleic acid library of the present invention. Furthermore, according to the present invention, a method for producing a protein library using the nucleic acid library of the present invention is provided.

 Brief Description of Drawings

FIG. 1 is a perspective view of a nucleic acid library according to one embodiment of the present invention.

 FIG. 2 is a schematic diagram of the structures of various cage DNAs.

[Figure 3] (a) shows the protein immobilized on the composite yarn using anti-His antibody and anti-GFP antibody. It is a figure which shows the detected result, (b) is a schematic diagram of a composite yarn.

 [Fig. 4] (a) shows the protein immobilized on the composite yarn with anti-His antibody, anti-GFP antibody and anti-GST.

 6

 It is a figure which shows the result detected using the antibody, (b) is a schematic diagram of a composite yarn.

 FIG. 5 is a diagram showing the results of detecting proteins immobilized on cotton yarn using Cy5-labeled anti-rabbit IgG (H + L) antibody and AF488-labeled anti-murine IgG (H + L) antibody.

 [FIG. 6] (a) is a schematic diagram of a cotton yarn in which nickel chelate and cage DNA are immobilized, and (b) shows the result of detecting the protein immobilized on the cotton yarn using a fluorescently labeled antibody. It is a figure.

 FIG. 7 is a schematic diagram of the structure of various cage DNAs.

 [Fig. 8] (a) is a diagram showing the detection results of GST protein for binding to dartathione, (b) is a diagram showing the results of detection of alkaline phosphatase activity, and (c) is a detection of the anti-β galactosidase ScFv antibody activity. It is a figure which shows a result.

 FIG. 9 is a diagram showing the results of detecting the protein immobilized on a cellulose membrane using an antibody.

 Explanation of symbols

[0053] 1 · · · Nucleic acid library

 2—First solid support

 3 ... Nucleic acid group 3

 4 · · · Second solid support

 5 ··· Protein Capturer BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, embodiments of the present invention will be described with reference to the drawings.

 As shown in FIG. 1, the nucleic acid library 1 according to this embodiment includes a first solid support 2 and a plurality of nuclei fixed to a predetermined surface region of the first solid support 2 in a state of being separated from each other. A plurality of acid groups 3; a second solid support 4 spirally mounted on the first solid support 2; and a plurality of high-density anchors fixed to a predetermined surface region of the second solid support 4 And protein trap 5.

[0055] As shown in FIG. 1, the solid support 2 has a cylindrical shape, but the solid support 2 has a shape of For example, the solid support 4 is not limited to a columnar shape as long as it can be fitted, for example, a flat plate shape, a cylindrical shape, a prismatic shape, a rectangular tube shape, a rod shape, a string shape, a thread shape, a tape shape, etc. It may be in shape.

 [0056] As shown in FIG. 1, the solid support 4 has a thread-like or string-like force. The solid support 4 is not limited to a thread-like or string-like shape as long as it is long, for example, It may be in the form of a tape. The thickness (diameter) of the solid support 4 is usually 0.05 to 2 mm, preferably 0.2 to lmm.

 [0057] The material constituting the solid support 2 is not particularly limited as long as it is insoluble in the liquid to which the nucleic acid library 1 is provided (for example, in vitro transcription / translation system or in vitro translation system solution). It is not something. Further, the material constituting the solid support 4 is not particularly limited as long as it is insoluble in the liquid to which the nucleic acid library 1 is provided and can give the solid support 4 flexibility. The material constituting the solid support 2 may be a material that can impart flexibility to the solid support 2 or a material that cannot be imparted!

 [0058] Examples of the material constituting the solid support 2 or 4 include glass; silicone; ceramics; polystyrene-based resin such as polystyrene; (meth) acrylic resin such as polymethylmetatalate; nylon 6, Polyamides such as nylon 66, polyesters such as polyethylene terephthalate, synthetic resins such as polycarbonate; polysaccharides such as agarose, dextran, and cellulose; proteins such as gelatin, collagen, and casein. The materials can be used alone or in combination of two or more materials.

 [0059] The solid support 2 or 4 may be either porous or nonporous, but is preferably porous. Examples of the porous solid support include those obtained by treating the surface of a solid support made of a synthetic resin such as nylon with an acid such as hydrochloric acid, and an aggregate of fibers (for example, a twisted fiber). It is done. When the solid support 2 or 4 is porous, the surface area of the solid support 2 or 4 increases, so that more nucleic acid groups 3 or protein capture bodies 5 can be immobilized on the surface.

[0060] As shown in FIG. 1, there are a plurality of partial regions that are separated from each other in the predetermined surface region of the solid support 2, and each of the plurality of nucleic acid groups 3 includes a plurality of portions. It is fixed to one of the areas. As a result, the plurality of nucleic acid groups 3 of the solid support 2 Fixed to a predetermined surface area in a state of being separated from each other.

 [0061] The surface of the solid support 2 is infiltrated with the liquid in addition to the outer surface (external surface) of the solid support 2 as long as it comes into contact with the liquid (for example, in vitro transcription'translation system or solution of in vitro translation system). The inner surface (inner surface) of the solid support 2 (for example, the inner surface of the pores of the solid support 2) is also included.

 [0062] The distance between adjacent partial regions among the plurality of partial regions that are separated from each other is usually 0.01 mm or more, preferably 0.1 mm or more, and more preferably 1 mm or more. Note that the upper limit of the distance between adjacent partial regions among a plurality of partial regions that are separated from each other is not particularly limited, depending on the area of the predetermined surface region to which a plurality of nucleic acid groups 3 are fixed. The force that can be adjusted appropriately is usually 10mm.

 [0063] Each nucleic acid group 3 expresses different types of proteins in an in vitro transcription / translation system or in vitro translation system. When a protein expressed from one nucleic acid group 3 (at least one protein when two or more proteins are expressed from one nucleic acid group 3) is not expressed from another nucleic acid group 3. It can be said that one nucleic acid group 3 and another nucleic acid group 3 express different types of proteins. If a protein expressed from one nucleic acid group 3 is not expressed from another nucleic acid group 3, there may be a problem of cross-contamination of proteins expressed from each nucleic acid group 3. This problem has been resolved.

 [0064] In addition to a plurality of nucleic acid groups 3 that express different types of proteins, a nucleic acid group that expresses only the same type of protein as one nucleic acid group 3 is fixed to the predetermined surface region of the solid support 2 It may be. A nucleic acid group that expresses only the same type of protein as one nucleic acid group 3 may be fixed to a predetermined surface region of the solid support 2 in the vicinity of the one nucleic acid group 3, or one nucleic acid group 3 It may be fixed to a predetermined surface region of the solid support 2 in a separated state.

[0065] Each nucleic acid group 3 expresses a plurality of nucleic acids that express only the same type of protein and / or different types of proteins so that one or more types of proteins are expressed from each nucleic acid group 3. A plurality of nucleic acids. A plurality of nucleic acids expressing only the same type of protein may have either the same structure or different structures. Different structure For example, a force transcription control region or translation control region having the same open reading frame may have a different structure.

 [0066] From the viewpoint of efficient protein analysis, it is preferable that only one type of protein is expressed from each nucleic acid group 3. When there is one type of protein expressed from each nucleic acid group 3, each nucleic acid group 3 usually includes a plurality of nucleic acids that express only the same type of protein, but a plurality of nucleic acids that express different types of proteins. Nucleic acids may be included. For example, when a protein expressed from a certain nucleic acid group 3 is composed of a plurality of different subunits, the nucleic acid group 3 includes a plurality of nucleic acids that express each subunit.

 [0067] The protein that each nucleic acid group 3 expresses in an in vitro transcription'translation system or in vitro translation system is a fusion protein of a target protein and a tag protein.

 [0068] The target protein is displayed on the solid support 4 and is the protein to be analyzed. The type of the target protein differs between the nucleic acid groups 3.

 [0069] The tag protein is, for example, a piotin-binding protein such as avidin or streptavidin, a maltose-binding protein, a polyhistidine peptide, a glutathione S-transferase, a calmodulin, an ATP-binding protein, or a receptor protein. The type is the same among nucleic acid groups 3.

 [0070] The nucleic acid group that expresses a protein in an in vitro transcription / translation system includes DNA and Z or RNA, and the nucleic acid group that expresses a protein in an in vitro translation system includes RNA.

[0071] The in vitro transcription / translation system includes an in vitro transcription system capable of performing transcription from DNA to mRNA in vitro, and an in vitro translation system capable of performing translation from mRNA to protein in vitro. In fact, the in vitro transcription / translation system contains all the elements necessary for transcription and translation (eg, RNA polymerase, ribosome, tRNA, etc.). Examples of in vitro transcription / translation systems include cell-free transcription / translation systems prepared from eukaryotic and prokaryotic cell extracts. Examples of cell-free transcription / translation systems include E. coli (for example, Examples include cell-free transcription / translation systems prepared from cell extracts such as E. coli S30), wheat germ, rabbit rabbit reticulocytes, mouse L cells, Ehrlich ascites tumor cells, HeLa cells, CHO cells, and budding yeast. [0072] The structure of the nucleic acid contained in each nucleic acid group 3 is the same in the in vitro transcription / translation system or in vitro translation system, and the target protein is different in the nucleic acid group 3 and the same in the nucleic acid group 3. It is not particularly limited as long as a fusion protein with the tag protein can be expressed. Examples of nucleic acids that express a target protein in an in vitro transcription / translation system include a transcriptional regulatory region, a translational regulatory region, an open reading frame (ORF) encoding the target protein, and an open reading frame. DNA comprising the stop codon provided on the 3 'side of Examples of nucleic acids that express a target protein in an in vitro translation system are the same except for DNA that expresses the target protein in an in vitro transcription / translation system and thymine (T) force uracil (U). And RNA having a structure. However, in the case of RNA, a transcriptional regulatory region is not necessary.

 [0073] The transcriptional regulatory region and the translational regulatory region are not particularly limited as long as transcription from DNA to mRNA and translation from mRNA to protein are possible. Examples of transcriptional regulatory regions include promoters, Terminator 1, Enno, Nsa, etc. Examples of translation regulatory regions include Kozak sequence, Shine * Dalgarno (SD) sequence, etc. Types of in vitro transcription systems, in vitro translation systems It can be selected appropriately according to the type. The transcriptional regulatory region and the translational regulatory region may exist as separate regions !, or may overlap and exist! /.

 [0074] An open reading frame (ORF) encoding a protein of interest includes an open reading frame encoding a target protein and an open reading frame encoding a tag protein linked to each other.

 [0075] The specific structure of the nucleic acid contained in each nucleic acid group 3 includes, for example, a promoter and an SD sequence on the 5 'side of the open reading frame, and a stop codon on the 3' side of the open reading frame. Examples include a structure having a terminator.

 [0076] The nucleic acid contained in each nucleic acid group 3 may be double-stranded or single-stranded, and may be immobilized on the surface of the solid support 2 in a shifted state. However, when the nucleic acid is DNA, double-stranded In this state, it is preferably fixed to the surface of the solid support 2. This is because transcription of DNA by polymerase is efficiently performed using double-stranded DNA as a substrate.

[0077] In each nucleic acid group 3, no matter what part of the nucleic acid is immobilized on the surface of the solid support 2 However, it is preferable that the 3 ′ end or 5 ′ end of the nucleic acid is fixed to the surface of the solid support 2. When the 3 ′ end or 5 ′ end of the nucleic acid is immobilized on the surface of the solid support 2, the protein can be efficiently expressed with the nuclear acidity.

[0078] The position of each nucleic acid group 3 is associated with the type of nucleic acid contained in each nucleic acid group 3, and the type of nucleic acid contained in each nucleic acid group 3 and the type of protein expressed from each nucleic acid group 3. Can be identified based on the position of each nucleic acid group 3.

As shown in FIG. 1, a plurality of protein capture bodies 5 are fixed at a high density in a predetermined surface region of the solid support 4.

[0080] The surface of the solid support 4 is infiltrated with the liquid in addition to the outer surface (external surface) of the solid support 4 as long as it comes into contact with the liquid (for example, in vitro transcription'translation system or in vitro translation system solution). The inner surface (inner surface) of the solid support 4 (for example, the inner surface of the pores of the solid support 4) is also included.

 [0081] The protein trap 5 can bind to a tag protein contained in the fusion protein expressed from each nucleic acid group 3. Examples of combinations of tag protein Z protein traps include metal ions such as avidin, streptavidin, etc., Piotin-binding protein Z-Piotin, maltose-binding protein Z-maltose, polyhistidine peptide Z nickel-cobalt, glutathione s-transferase Z Glutathione, calmodulin Z calmodulin binding peptide, ATP binding protein ZATP, receptor protein Z ligand and the like. Since the fusion protein expressed from each nucleic acid group 3 includes tag proteins of the same type between the nucleic acid groups 3, the protein trap 5 is expressed from each nucleic acid group 3 through binding to the tag protein. It is possible to capture a few fusion proteins.

[0082] As the protein trap 5, a positively charged body or a negatively charged body may be used. Protein negatively charged at higher pH isoelectric point, since positively charged at low _P H than the isoelectric point, by using a positive charge, or negatively charged body as protein capture body 5, the protein capturing The prey 5 can capture any fusion protein expressed from each nucleic acid group 3 via electrical coupling (electrostatic interaction).

[0083] As the positively charged body and the negatively charged body, for example, a positively charged group (for example, an amino group, guazyl) And a substance having a negatively charged group (for example, a carboxyl group, a sulfonyl group, or a phosphate group).

 [0084] As the protein trap 5, a substance having a hydrophobic group such as an alkyl group or a derivative group thereof, a phenyl group or a derivative group thereof may be used. By using a substance having a hydrophobic group as the protein capturing body 5, the protein capturing body 5 can also capture a misaligned fusion protein expressed from each nucleic acid group 3 through a hydrophobic interaction. Can do.

 [0085] When a positively charged body, a negatively charged body, or a substance having a hydrophobic group is used as the protein capture body 5, the protein expressed from each nucleic acid group 3 is a fusion of the target protein and the tag protein. Regardless of whether it is a protein or not, the protein capture body 5 is expressed from each nucleic acid group 3 and can also capture a misaligned protein. Therefore, the protein expressed from each nucleic acid group 3 is, for example, a tag It may be a target protein that is not fused with a protein, a target protein that is fused with a protein other than a tag protein, or the like.

 [0086] The protein capture body 5 has a density such that the distance between adjacent protein capture bodies is much smaller than the distance between adjacent partial areas among a plurality of partial areas that are separated from each other. The solid support 4 is fixed to a predetermined surface area. The distance between adjacent protein catchers is a density that is not more than 1/100 of the distance between adjacent partial areas among a plurality of partial areas that are separated from each other. It is preferably fixed to the surface region. The distance between adjacent protein capture bodies is less than 1/100, less than 1/1000, less than 1 / 10,000, and less than 100,000 minutes of the distance between adjacent partial areas among multiple partial areas that are separated from each other. 1 / less, 1 / 1,000,000 or less, 1 / 1,000,000 or less! /, The smaller, the better.

 [0087] As shown in FIG. 1, the solid support 4 is spirally mounted on the solid support 2!

[0088] The solid support 4 includes a predetermined surface region (nucleic acid fixation surface region) of the solid support 2 to which a plurality of nucleic acid groups 3 are fixed and a predetermined support of the solid support 4 to which a plurality of protein capture bodies 5 are fixed. The solid support so that the distance to the surface area (surface area on which the protein capturer is fixed) is overwhelmingly smaller than the distance between adjacent partial areas among a plurality of partial areas that are separated from each other. 2 are outfitted. The distance between the nucleic acid immobilization surface area and the protein capturer immobilization surface area is the distance between the adjacent partial areas of the plurality of partial areas that are separated from each other. It is preferable to be less than one fifth of the distance between the zones. The distance between the nucleic acid-immobilized surface region and the protein catcher-immobilized surface region is less than one fifth, less than one-fifth, and less than one-fifth of the distance between adjacent partial regions among a plurality of partial regions that are separated from each other The smaller it is, such as 1 / min or 1/5000 min or less, the better. Note that the lower limit of the distance between the protein capturer fixed surface region and the nucleic acid fixed surface region is zero.

 [0089] The number of windings of the solid support 4 per unit length in the winding axis direction is not particularly limited as long as the protein capturer fixing surface region is close to each nucleic acid group 3, but the winding of a plurality of partial regions is not limited. When the shortest length in the axial direction is taken as the unit length in the winding axis direction, the number of windings of the solid support 4 per unit length in the winding axis direction is preferably 1 mm or more, more preferably 2 times Zmm or more. The upper limit value of the number of turns of the solid support 4 per unit length in the winding axis direction is not particularly limited and can be appropriately adjusted according to the width of the solid support 4. The length of each partial region in the winding axis direction is not particularly limited, but is usually 0.05 mm or more, preferably 2 mm or more. The upper limit value of the length of each partial region in the winding axis direction is not particularly limited, and is a force that can be appropriately adjusted according to the size of the first solid support and the like, which is usually 10 mm. The width of the solid support 4 is equal to or less than the unit length in the winding axis direction, and the solid support 4 is mounted on the first solid support so that different regions of the protein-immobilized surface region do not overlap each other. The “winding axis” means a line passing through the center of a spiral formed by the solid support 4 mounted on the solid support 2.

 [0090] Among the lengths of the plurality of partial regions in the winding axis direction, when the shortest length is taken as the winding axis direction unit length, the number of windings of the solid support 4 per winding axis direction unit length is 1 If it is Zmm or more, a different region of the protein trap body fixing surface region is close to each nucleic acid group 3, so that cross-contamination of proteins expressed from each nucleic acid group 3 can be effectively prevented. .

[0091] In the nucleic acid library 1, the protein capturing bodies 5 are arranged at high density close to the predetermined surface region of the solid support 2 to which the nucleic acid groups 3 are fixed. Different protein capture bodies 5 are close to each other in each of the nuclear acid groups 3. Further, since each nucleic acid group 3 is fixed to the surface of the solid support 2 in a state of being separated from each other, the distance between one nucleic acid group 3 and the protein capture body 5 adjacent to one nucleic acid group 3 is different from the other. In close proximity to one nucleic acid group 3 and one nucleic acid group 3 It is overwhelmingly smaller than the distance to the protein capture body 5 to be. Therefore, if the nucleic acid library 1 is subjected to an in vitro transcription / translation system or an in vitro translation system and a plurality of nucleic acid groups 3 contained in the nucleic acid library are expressed at once, the proteins expressed from each nucleic acid group 3 are expressed. Is preferentially captured by the protein capturing body 5 adjacent to each nucleic acid group 3, so that cross-contamination of the protein expressed from each nucleic acid group 3 is prevented. Particularly in the nucleic acid library 1, the solid support 4 interposed between the nucleic acid groups 3 serves as a barrier, and cross-contamination of proteins expressed from the nucleic acid groups 3 is effectively prevented.

[0092] That is, by subjecting the nucleic acid library 1 to an in vitro transcription / translation system or an in vitro translation system to express a plurality of nucleic acid groups 3 contained in the nucleic acid library 1 at a time, A protein library in which a plurality of types of expressed proteins are displayed on the solid support 4 in a state in which they are associated with the nucleic acid encoding them can be prepared. At this time, as long as there are two or more types of nucleic acid groups 3 to be expressed at one time, all of the nucleic acid groups 3 included in the nucleic acid library 1 may be used, or some of the nucleic acid groups 3 included in the nucleic acid library 1 may be included. May be.

 [0093] Nucleic acid library 1 is immediately converted into a protein library when subjected to an in vitro transcription / translation system or in vitro translation system, so the protein supply and protein assembly system are integrated. The analysis of the plurality of types of proteins can be started immediately after the supply of the plurality of types of proteins.

 [0094] In the protein library prepared from the nucleic acid library 1, the protein displayed on the solid support 4 can be accessed even when the solid support 4 is mounted on the solid support 2. Since it is easy, it is possible to easily automate a series of operations up to protein analysis using a protein library.

 [0095] In the protein library prepared from the nucleic acid library 1, even if the protein contained in the protein library is denatured or inactivated during the protein analysis, the nucleic acid encoding the protein is expressed again. Thus, the protein can be regenerated.

[0096] After the protein analysis is completed, the nucleic acid library 1 is maintained until the next use. It can be used repeatedly over a long period of time by regenerating the protein library when necessary.

 [0097] The nucleic acid library 1 is obtained by fixing one nucleic acid group of a plurality of nucleic acid groups 3 to a plurality of site regions that are separated from each other in a predetermined surface region of the solid support 2, and A plurality of protein capture bodies 5 are fixed at a high density to a predetermined surface area of the support 4, and a predetermined surface area where a plurality of protein capture bodies 5 are fixed is a predetermined surface area where a plurality of nucleic acid groups 3 are fixed. The solid support 4 can be manufactured by mounting the solid support 4 on the solid support 2 so as to be close to the substrate.

 [0098] The method for immobilizing a nucleic acid or protein trap on the surface of a solid support is not particularly limited. For example, specific interactions between specific substances, covalent bonds (for example, amide bonds, disulfide bonds, thioethers). (Bond) formation or the like can be used.

 [0099] Specific interactions between specific substances include, for example, avidin or a derivative thereof (eg, streptavidin, extraavidin, NeutrAvidin) Z piotin or a derivative thereof (eg, iminobiotin) Maltose binding protein Z maltose; metal ions such as polyhistidine peptide nickel and cobalt; glutathione S transferase Z glutathione; calmodulin Z calmodulin binding peptide; ATP binding protein ZATP; nucleic acid Z complementary nucleic acid; Specific interactions such as body protein Z ligand; enzyme Z substrate; antibody Z antigen; IgGZ protein A or G can be used. Substances that produce specific interactions can be bound to nucleic acids, protein capture bodies or solid supports according to conventional methods.

 [0100] The covalent bond includes a functional group (including a functional group artificially introduced into the nucleic acid or protein capture body) of the nucleic acid or protein capture body 5 included in the nucleic acid group 3, and the solid support 2 or Can be formed by reacting with a functional group possessed by 4 (including a functional group artificially introduced into the solid support 2 or 4). Examples of the functional group that forms a covalent bond include a carboxyl group, an amino group, a hydroxyl group, a sulfhydryl group, and an aldehyde group.

[0101] A crosslinking agent can be used for the formation of the covalent bond. Examples of cross-linking agents include N —Succinimidyl (4—iodoacetyl) aminobenzoate (SIAB), dimaleimide, dithio-bis-nitrobenzoic acid (DTNB), N- N-succinimidyl-S-acetyl-thioacetate (SATA), N-succinimidyl-3- (2-pyridyldithio) propionate ) (SPDP), succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), 6-hydrazinonicotimide And polyfunctional reagents such as (6-hydrazinonicotimide) (HYNIC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MB), and the like.

 [0102] When a specific interaction between specific substances (substances A and B) is used when immobilizing nucleic acids on the surface of the solid support 2, for example, a substance is placed in a predetermined region of the surface of the solid support. A solution containing nucleic acid to which A is fixed and substance B is bound is spotted on a predetermined region. In this case, if the material of the solid support 2 is a material that can penetrate the nucleic acid solution, contamination may occur between the nucleic acid solutions spotted on the adjacent region. Therefore, when the material of the solid support 2 is a material that can be penetrated by the nucleic acid solution, the nucleic acid solution force S prevents the nucleic acid from passing between the adjacent site regions. It is preferable to spot in advance a solution containing a substance that can be used. By pre-spotting such a solution, a barrier is formed inside the solid support 2 and it is possible to prevent contamination from occurring between the nucleic acid solutions spotted in the adjacent site regions. . Examples of substances that can prevent the passage of nucleic acids include protein substances such as casein, skim milk, gelatin, and BSA, sugars such as glucose, sucrose, raffinose, dextrin, and starch, dextran sulfate, PVP, glycerin, Examples include viscous substances such as PEG and substances A released from the solid support 2.

[0103] Among the site regions where the nucleic acid solution is spotted, between adjacent site regions, proteinaceous substances such as casein, skim milk, gelatin, BSA, and fusion proteins expressed from each nucleic acid group 3 are included. By pre-spotting a solution containing a protein trap that can bind to the tag protein to be expressed, the tamper expressed from each nucleic acid group 3 It is possible to prevent the soil from adsorbing to the solid support 2 nonspecifically.

[0104] When the solid support 4 is mounted on the solid support 2, the predetermined surface area of the solid support 2 to which the protein capturing body 5 is fixed is changed to the predetermined surface area of the solid support 4 to which the nucleic acid group 3 is fixed. What is necessary is just to make it adjoin to a predetermined surface area | region. In other words, it is not necessary to arrange the protein capturing body 5 in correspondence with the position of the nucleic acid group 3; Therefore, the nucleic acid library 1 can be produced efficiently.

 Example

[Example 1]

 In Example 1, a composite yarn composed of a cage DNA-immobilized nylon yarn and a nickel chelate-immobilized cotton yarn wound around the nylon yarn was subjected to an in vitro transcription / translation system and expressed from the cage DNA. In this example, a His-tagged protein was immobilized on a nickel chelate immobilized cotton thread.

6

 The

 [0106] 1. Fabrication of nickel chelate-immobilized cotton yarn

 (1) Cotton yarn activity

 Six 60th cotton yarns (diameter: about 0.2mm) cut to 30cm were prepared, sterilized in distilled water by autoclave (120 ° C, 15 minutes), and then washed 3 times with 3ml of 30% acetone. The following reactions were performed at 4 ° C unless otherwise stated. After washing 3 times with 3 ml of 70% acetone, 3 times with 3 ml of 100% acetone, 3 times with 3 ml of anhydrous acetone, and 3 times with 3 ml of anhydrous acetonitrile, 0.33M p--Trophe- 2 ml of chloroformate Z anhydrous acetonitrile was added and then 2 ml of 1.0M dimethylaminopyridine Z anhydrous acetonitrile was added and allowed to stand for 1 hour. Then wash 3 times with 3 ml of 100% acetone, 3 times with 3 ml of 5% acetic acid Z-dioxane, 3 times with 3 ml of methanol, and 3 times with 3 ml of anhydrous 2-propanol. Medium and stored at 4 ° C until use.

[0107] (2) Nickel chelate

Prepare three activated yarns, wash them with 3 ml of distilled water 3 times (4 ° C), and then add 10 mg / ml AB-NT A / 0.1M sodium phosphate buffer (pH 7.4) to 1 ml. The reaction was allowed to proceed for 48 hours at room temperature. Thereafter, the plate was washed 3 times with 3 ml of 0.1 M phosphate buffer (pH 7.5), 50 ml of 0.1 M monoethanolamine (pH 7.5) was added, and the mixture was reacted at room temperature for 24 hours. Then add 3 ml of 0.1M phosphate buffer (pH 7.5) After washing 3 times, 50 ml of 0.1M monoethanolamine (pH 7.5) was added and reacted at room temperature for 24 hours. Thereafter, it was washed 3 times with 3 ml of 0.1M phosphate buffer (pH 7.5), immersed in a 1% nickel acetate aqueous solution for 2 hours at room temperature, and washed 3 times with 3 ml of distilled water.

 [0108] As described above, a cotton yarn having a nickel chelate fixed on the surface thereof at high density was produced.

 Of the large amount of nickel chelate fixed to cotton yarn, the distance between adjacent nickel chelates is expected to be about 2 to 20 nm (estimated value). As will be described later, among the plurality of saddle DNA groups fixed to the nylon thread, the distance between adjacent saddle DNA groups is about 5 mm, so the distance between adjacent nickel chelates is adjacent. The distance between adjacent DNA groups is 1 / 2.5 million (about 0.000004 times), and the distance between adjacent nickel chelates is far smaller than the distance between adjacent DNA groups. .

 [0109] 2. Fabrication of saddle-shaped DNA-immobilized nylon thread

 (1) Preparation of vertical DNA

 His tag (tag protein consisting of 6 histidines) at the 5 'end of the streptavidin gene

 6

 The coding sequence is ligated, and the T7 promoter sequence and ribosome binding sequence (RBS) are attached to the 5 'end, and the T7 sequence is attached to the 3' end.铸 DNA encoding His-avidin fusion protein (hereinafter referred to as “His-a”)

 6 6 “Vidin-type DNA”. (See SEQ ID NO: 3).

[0110] GFP (green fluorescent protein) -His-encoded DNA (hereinafter “GFP-His-type D”)

 6 6

 NA ". ) Was prepared using the Rapid Translation System (Roche) control plasmid pIVEX control vector GFP (see SEQ ID NO: 4). This plasmid has a structure expressing a protein in which a His tag is fused to the C-terminal side of GFP.

 6

 [0111] STDNA encoding GST (glutathione s-transferase) -His (hereinafter referred to as “GST-His D D”

 6 6

 NA ". ) Was prepared by fusing the His tag coding sequence to the 3 'end of the GST gene using a Roche Linear template generation set, His-tag (see SEQ ID NO: 5).

 6

 See).

 [0112] Figure 2 shows a schematic diagram of the structure of each cage DNA.

Using each cage DNA as a cage, amplification of each cage DNA by PCR and addition of a digoxigenin (DIG) sequence to the 5 ′ end side were performed. The composition and reaction conditions of the PCR reaction solution are as follows: The The composition of the PCR reaction solution is 10 ng / μ 1 DNA 4.0 μ \, 20 μ Μ forward primer (DIG-17f, see SEQ ID NO: 1) 8.0 μ 20 μ Μ reverse primer (see l lr, SEQ ID NO: 2) 8.0 1, 10 X Ex Taq buffer 20 μ 1, 2.5 mM dNTPs 20 μ 1, LA Taq (5U / μ 1) 2.0 1, dH O 138 1 (

 2 The total amount is 200 μ 1). The reaction conditions were (0 94 ° C for 1 minute, (ii) 94 ° C for 1 minute, 94 ° C for 30 seconds, 55 ° C for 30 seconds and 72 ° C for 1 minute for 30 cycles. Yes, PCR products were stored at 4 ° C until use.

[0113] (2) Purification of vertical DNA

 Using a MicroSpin S-400 HR column, gel filtration purification was performed 100 μl at a time. Next, 60 μl of 7.5% ammonium acetate was added, 500 μl of EtoH was added, left at −80 ° C. for 20 minutes, centrifuged at 4 ° C. and 15000 rpm for 15 minutes, and the supernatant was discarded. Next, 800 1 of 70% EtoH was added, centrifuged at 4 ° C, 15 OOOrpm for 5 minutes, the supernatant was discarded and dried under reduced pressure. When used, it was dissolved in 1 TE.

[0114] (3) Immobilization of saddle DNA on nylon thread

 As the nylon thread, the surface was treated with 4-6N hydrochloric acid (20-25 ° C, 0.5-15 seconds) and the surface was made porous. 200 g / ml anti-DIG antibody (Roche) 0.41 was spotted on nylon thread at intervals of 5 mm and dried at room temperature for 40 minutes. On the spotted antibody, 0.41 spot of purified DIG-labeled vertical DNA was spotted and dried at room temperature for 90 minutes.

 As described above, a nylon thread having a plurality of cage DNA groups (one spot corresponding to one cage DNA group) fixed on the surface was produced.

[0115] 3. Preparation of composite yarn

 A cotton yarn with a nickel chelate fixed at a high density on the surface was wrapped around a nylon yarn with a hook-shaped DNA fixed on the surface to produce a composite yarn. At this time, since the diameter of the cotton yarn is 0.2 mm, the distance between the surface of the cotton yarn to which the nickel chelate is immobilized and the surface of the nylon yarn to which the cage DNA is immobilized is in the range of about 0 to 0.2 mm. Cotton yarn was wound three times per 5mm nylon yarn.

 The composite yarn was washed with 1% SDS / lxSSC, washed with 10 mM Tris-HCl (pH 7.5), and then used.

[0116] 4. In vitro transcription 'protein expression by translation system Two composite yarns were prepared in the same manner, and placed in the Rapid Translation System (RTS) reaction solution in the glass cabinet, and the protein was expressed by an in vitro transcription / translation system. The reaction solution was prepared according to the protocol attached to the Roche kit and reacted at 30 ° C for 2 hours. After washing the composite yarn after RTS reaction, peroxidase (POD) -labeled anti-His antibody and anti-GFP antibody are used.

 6

 Detection was performed.

 [0117] (1) Detection of protein immobilized on composite yarn (chemiluminescence method)

 The composite yarn after the RTS reaction was washed with 20 mM imidazole / PBS buffer, immersed in 0.5% blocking rea gent (Roche) for 30 minutes, and washed with PBS buffer. Then, immerse in POD (peroxidase) -labeled anti-His antibody, anti-GFP antibody, and anti-GST antibody diluted in PBS for 30 minutes, and relax with PBS.

 6

 After washing with the impulse solution, 10 µ 1 of ECL Detection Reagents (Amersham) was added, and luminescence was detected by CCD imaging device ARGUS (Hamamatsu Photonicus).

 [0118] (2) Detection of protein immobilized on composite yarn (fluorescence method)

 As in (1) above, the composite yarn subjected to the RTS reaction was unwound and only the cotton yarn was taken out, and the protein immobilized on the cotton yarn was detected using a fluorescently labeled antibody. Cotton yarn is washed with 20 mM imidazole / PBS, soaked in 1% block ace / PBS for 30 minutes, washed with PBS, and diluted 100-fold with 1% bio ace ace / PBS. Anti-GFP antibody and anti-GST antibody Soaked for 30 minutes. After washing with PBS, the cells were immersed in Cy5-labeled anti-rabbit Ig G (H + L) antibody and AF488-labeled anti-murine IgG (H + L) antibody diluted 100-fold with 2.5% block ace / PBS for 30 minutes. After washing with PBS, the fluorescence of the cotton yarn was measured with a fluorescence scanner (STORM, FL595: ABI).

 [0119] (3) Results

 Schematic diagram of a composite thread with immobilized His-avidin-type DNA or GFP-His-type DNA

6 6

 3 As shown in (b). In this composite yarn, as shown in Fig. 3 (b), GFP-His 铸 DNA (spot

 6

 1, 3, 5, 7, 9) and His-avidin-type DNA (spots 2, 4, 6, 8), 5 and 4 respectively.

 6

 It is spotted alternately at the place. The protein immobilized on this composite yarn

 As a result of detection using 6 antibodies and anti-GFP antibody, detection with anti-His antibody was performed as shown in Fig. 3 (a).

 6

9 spots (spots 1-9) were confirmed, and 5 spots (spots 1, 3, 5, 7, 9) were confirmed every other spot when detected with anti-GFP antibody. It was. Since each signal is detected independently, protein contamination between spots is None or very low was expected.

[0120] Figure 4 (b) shows a schematic diagram of a composite thread with immobilized GFP-His 铸 type DNA or GST-His 铸 type DNA.

 6 6

 ). In this composite yarn, as shown in Fig. 4 (b), GFP-His 铸 DNA (spot 1,

 6

 3,5,7,9) and GST-His-type DNA (spots 2,4,6,8) alternately in 5 and 4 locations, respectively.

 6

 Have been spotted. Anti-His antibody, anti-GFP antibody

 6

 As a result of detection using anti-GST antibody, as shown in Fig. 4 (a), detection was performed using anti-His antibody.

 6

 9 spots (spots 1-9) were confirmed, and 5 spots (spots 1, 3, 5, 7, 9) were confirmed every other spot when detected with anti-GFP antibody. When detected with anti-GST antibody, four spots (spots 2, 4, 6, 8) were confirmed every other place. Moreover, since each signal was detected independently, it was predicted that there was no or very low protein contamination between spots.

[0121] As a result of detecting the protein immobilized on the cotton thread using the Cy5-labeled anti-rabbit IgG (H + L) antibody and the AF488-labeled anti-murine IgG (H + L) antibody, FIG. 5 (a) and As shown in (b), it was confirmed that both GFP-His and GST-His proteins could be detected simultaneously. Ie

6 6

 It was confirmed that the detection system of the protein fixed to the composite yarn can be multiplexed.

[Example 22]

 Example 2 uses an in vitro transcription / translation system with a single cotton thread in which nickel chelate and cage DNA are immobilized, and the His-tagged protein expressed from the cage DNA is immobilized on the cotton thread.

 6

 This is an example.

[0123] 1. Preparation of nickel chelate and cocoon-type DNA-fixed cotton yarn

 The cotton yarn activity was carried out in the same manner as in Example 1, and the chelate yarn and antibody coupling were carried out as follows.

[0124] (1) Chelating chelate and antibody coupling

Prepare three activated yarns, wash them with 3 ml of distilled water 3 times (4 ° C), and then add 10 ml of AB-NTA / 0.1M phosphate buffer (pH 7.5) The reaction was allowed to proceed for 24 hours at room temperature. 0.1M phosphate buffer (pH 7.5) Wash 3 times with 3 ml, and then add 100 ml of 0.1M phosphate buffer (pH 7.5) containing 10 mg of anti-digoxigenin (DIG) antibody for 24 hours at room temperature. Reacted. 0.1M phosphate buffer (p After washing 3 times with 3 ml of H 7.5), 50 ml of 0.1M monoethanolamine (pH 7.5) was added and allowed to react at room temperature for 24 hours. After washing 3 times with 3 ml of 0.1 M phosphate buffer (pH 7.5), 50 ml of 0.1 M monoethanolamine (pH 7.5) was added and reacted at room temperature for 24 hours. The plate was washed 3 times with 3 ml of 0.1 M phosphate buffer (pH 7.5), then immersed in a 1% aqueous nickel acetate solution at room temperature for 2 hours, and washed 3 times with 3 ml of distilled water.

 As described above, a cotton thread having an anti-DIG antibody and nickel chelate immobilized on the surface was produced.

 [0125] (2) Fixation of cocoon-shaped DNA to cotton thread

 10% Block ace, 10% Ficoll was spotted on a cotton thread with anti-DIG antibody and nickel chelate immobilized on the surface at intervals of 6-10 mm and dried at room temperature for 30 minutes. After spotting 0.21 of DIG-labeled vertical DNA while spotting Blockace and Ficoll, it was dried at room temperature for 30 minutes, washed with PBS, and washed with 10 mM Tris-HCl (pH 7.5). When spotted with DIG-labeled saddle DNA, each saddle DNA solution may permeate into the cotton thread and cause contamination between the saddle DNA solutions. However, while spotting Block ace and Ficoll, By spotting DIG-labeled vertical DNA, contamination of each vertical DNA solution is prevented.

 [0126] As described above, a cotton yarn in which nickel chelate and cage DNA were immobilized was prepared (see FIG. 6 (a)). FIG. 6 (a) shows a schematic diagram of a cotton thread to which nickel chelate and cage DNA are immobilized.

 [0127] 2. In vitro transcription 'protein expression by translation system

 The cotton yarn in which the nickel chelate and the cocoon-type DNA were immobilized was used in the in vitro transcription / translation system using RTS in the same manner as in Example 1 to express the protein. The reaction solution was prepared according to the protocol supplied with the Roche kit and reacted at 37 ° C for 2 hours. After the reaction, detection with a fluorescent-labeled antibody was performed in the same manner as in Example 1.

 As a result, as shown in FIG. 6 (b), GFP-His and GST- that also expressed saddle-type DNA force even when nickel chelate and saddle-type DNA existed on the same thread. His protein

 6 6 The quality was fixed on the yarn as each spot. In addition, protein spots could be identified even when the distance between each type of DNA was reduced to 6 mm.

[Example 3] Example 3 uses an in vitro transcription / translation system with a single cotton thread with nickel chelate and anchored DNA immobilized on it, and the His-tagged protein expressed from the anchored DNA is immobilized on the cotton thread.

 6

 This is an example in which various activities of the protein immobilized on the cotton yarn were detected.

[0130] Activation of cotton yarn, chelating candy and antibody coupling were carried out in the same manner as in Example 2. Fig. 7 shows a schematic diagram of the structure of each cage DNA used in Example 3. His-GST-type DNA (sequence

 6

 No. 6), β gal-His type DNA (see SEQ ID No. 8), β gal (H)-His type DNA (sequence

 6 6

 No. 9), β gal (L) -His-type DNA (see SEQ ID No. 10) is glutathione s

 6

 The -transferase gene was prepared by cloning the H chain and L chain of anti-j8 galactosidase ScFv and anti-j8 galactosidase ScFv into pIVEX2.4c in pIVEX2.3c. Hi s -BAP-type DNA (see SEQ ID NO: 7) is a Linear template generation set, manufactured by Roche

6

 Fusion expression of His tag on the N-terminal side of alkaline phosphatase of Escherichia coli using His-tag

 6

 It was constructed as follows. Immobilization of silkworm DNA on cotton yarn and expression of the protein by an in vitro transcription / translation system were carried out in the same manner as in Example 2.

[0131] (l) Detection of daltathione binding ability of GST protein

 After RTS reaction, PBS-washed His-GST-type DNA fixation thread is blocked with 1% block ace

 6

 The sample was then immersed in 2 g / ml POD-labeled dartathione for 30 minutes. After washing with PBS buffer, 10 μl of EC L Detection Reagents was added and measurement was performed with ARGUS (Hamamatsu Photonicus).

[0132] (2) Detection of alkaline phosphatase activity

 After RTS reaction, add PBS-star reagent (Roche) to His-BAP-type DNA fixation thread washed with PBS

 6

 In addition, it was measured with ARGUS.

[0133] (3) Detection of antibody activity of anti-j8 galactosidase ScFv

 After RTS reaction, PBS washed β gal-His and β gal (H) -His + β gal (L) -His 铸 DNA

 6 6 6

 The fixed yarn was blocked with 1% block ace and immersed in 50 μg / ml j8 galactosidase for 30 minutes. After washing with PBS buffer, it was immersed in 100 times diluted anti | 8 galactosidase antibody for 30 minutes and washed with PBS buffer. Thereafter, the cells were immersed in a 100-fold diluted POD-labeled anti-rabbit IgG antibody for 30 minutes. After washing with PBS buffer, 10 μl of ECL Detection Reagents was added and measured with ARGUS.

[0134] (4) Results Daltathione binding of His-GST protein expressed from cotton thread and anchored on the same cotton thread

 6

 As a result of a binding experiment using POD-Daltathion, as shown in Fig. 8 (a), the POD-Daltathion-derived gene was generated only at the position where the His-GST vertical DNA was immobilized.

 6

 A light signal was detected.

 [0135] Alkaline phos of His-BAP protein expressed from cotton thread and immobilized on the same cotton thread

 6

 As for the phatase activity, CDP-Star reagent containing a luminescent substrate was added and the luminescence signal was measured. As a result, as shown in Fig. 8 (b), the luminescence signal was observed specifically for His-BAP-type DNA.

 6

 Was observed. The BAP protein has activity as a dimer, suggesting that the expressed protein can form a homodimer on the thread.

[0136] As a result of an attempt to detect the specific binding ability of the anti-β galactosidase ScFv expressed from cotton thread and immobilized on the same cotton thread, as shown in Fig. 8 (c), Anti-j8 galactosidase ScFv is not only bound specifically to j8 galactosidase, but also when mixed with spotted DNA constructed with H- and L-chains as individual His-tag fusion genes,

6

 In which a heterodimer was formed and was confirmed to have antibody activity.

[Example 4]

 Example 4 is an example in which protein expression and immobilization were examined in the case of using a planar carrier having cellulose membrane (cellophane) force made of cellulose similar to cotton yarn.

 [0138] Activation of the cellulose membrane, chelation, and antibody coupling were performed in the same manner as in Example 2.

The anchor-shaped fixation on the cellulose membrane and the expression of the protein by the in vitro transcription / translation system were performed in the same manner as in Example 2 except for the spots of 10% Block ace and 10% Ficoll. In addition, the RTS reaction was carried out in a state in which 25 1 RTS reaction solution per 1 cm ² was placed on the membrane, the surface was covered with a film to prevent drying, and the humidity was maintained.

 The detection of the protein immobilized on the cellulose membrane was performed in the same manner as in Example 2.

[0139] Immobilization of GFP-His 铸 type DNA and GST-His 铸 type DNA on cellulose membrane and RTS reaction

 6 6

As shown in Fig. 9 (a), each cell was detected using an antibody. The signal derived from GFP-His was detected on the loin membrane, but the signal of GST-His

6 6

Not detected. In addition, when the RTS reaction temperature (37 ° C → 30 ° C) was changed, as shown in Fig. 9 (b), the force at which the GFP signal was detected at either temperature in the cotton yarn was 37 ° in the cellulose membrane. Although a GFP signal was detected in C, it was not detected at 30 ° C. We have confirmed that GFP has higher RTS expression efficiency than GST by analysis such as Western blotting etc. As shown in Examples 1 and 2, these two types of expression experiments on yarn The 铸 -type DNA-derived protein is detected with almost the same signal intensity. This suggests that the cotton yarn with a more complex surface structure than the cellulose membrane is expected to fix the expressed protein efficiently and is less susceptible to the difference in protein expression efficiency in RTS. .

Claims

The scope of the claims  [1] a first solid support;  A plurality of nucleic acid groups capable of expressing different types of proteins in an in vitro transcription / translation system or in vitro translation system fixed to a predetermined surface region of the first solid support; and a second solid support Body,  A nucleic acid library comprising a plurality of protein capture bodies fixed at a high density on a predetermined surface region of the second solid support and capable of capturing any protein expressed from the plurality of nucleic acid groups. ,  Each of the plurality of nucleic acid groups is fixed to one partial region among the plurality of partial regions that are separated from each other in the predetermined surface region,  The second surface with respect to the first solid support is arranged such that the predetermined surface region to which the plurality of protein capturing bodies are fixed is close to the predetermined surface region to which the plurality of nucleic acid groups are fixed. A nucleic acid library in which a solid support is placed. [2] The density is such that the distance between adjacent protein capture bodies among the plurality of protein capture bodies is 1/100 or less of the distance between adjacent partial areas of the plurality of partial areas. The nucleic acid library according to claim 1, wherein a plurality of protein capture bodies are immobilized on the predetermined surface region.  [3] The distance between the predetermined surface region to which the plurality of protein capturing bodies are fixed and the predetermined surface region to which the plurality of nucleic acid groups are fixed is between adjacent partial regions of the plurality of partial regions. The nucleic acid library according to claim 1 or 2, wherein the second solid support is arranged with respect to the first solid support so as to be equal to or less than one fifth of the distance.  [4] The method according to any one of claims 1 to 3, wherein the second solid support is made of a flexible elongated member, and the second solid support is spirally mounted on the first solid support. A nucleic acid library according to any one of the above.  [5] Of the lengths in the winding axis direction of the plurality of partial regions, the shortest length is defined as the unit length in the winding axis direction. The nucleic acid library according to claim 4, wherein the number of turns is 1 or more. [6] The position of each nucleic acid group on the surface of the first solid support and the nucleus contained in each nucleic acid group The nucleic acid library according to any one of claims 1 to 5, which is associated with an acid type. [7] A protein that also expresses the strength of each nucleic acid group is a fusion protein of a target protein that is different in type between nucleic acid groups and a tag protein that is the same type in the nucleic acid group, and each protein catcher is the tag protein. The nucleic acid library according to any one of claims 1 to 6, which can be bound.  [8] A method for producing a nucleic acid library according to any one of claims 1 to 7,  Immobilizing one nucleic acid group of the plurality of nucleic acid groups to each of the plurality of site regions of the predetermined surface region of the first solid support,  Fixing the plurality of protein capture bodies to a predetermined surface region of the second solid support at a high density; and  The second surface with respect to the first solid support is arranged such that the predetermined surface region to which the plurality of protein capturing bodies are fixed is close to the predetermined surface region to which the plurality of nucleic acid groups are fixed. Comprising a step of disposing a solid support. [9] The nucleic acid library according to any one of claims 1 to 7 is subjected to an in vitro transcription / translation system or an in vitro translation system to express a plurality of nucleic acid groups contained in the nucleic acid library at a time. A tannologic library obtained from Tokoko. [10] The nucleic acid library according to any one of claims 1 to 7 is subjected to an in vitro transcription / translation system or an in vitro translation system to express a plurality of nucleic acid groups contained in the nucleic acid library at a time. A method for producing a protein library comprising the step of allowing [11] A solid support and a plurality of different types of proteins that can be expressed in an in vitro transcription'translation system or an in vitro translation system that are fixed to a predetermined surface region of the solid support in a state of being separated from each other. A method for producing a nucleic acid library comprising a nucleic acid group and a protein trap immobilized in the vicinity of each nucleic acid group in the predetermined surface region, wherein the entire predetermined surface region includes the plurality of nucleic acid groups. A step of fixing at a high density a plurality of protein capture bodies capable of capturing even a few proteins; and A method comprising the step of immobilizing one nucleic acid group of the plurality of nucleic acid groups to each of a plurality of partial regions existing apart from each other on the predetermined surface region. The distance between adjacent protein capture bodies of the plurality of protein capture bodies is less than 1/100 of the distance between adjacent partial areas of the plurality of partial areas. The method according to claim 11, wherein the fixing is performed at such a density.