CN103603057A - Antibody screening method based on heavy chain library/light chain library of infectable virus particle type antibody and preparation method of heavy chain library/light chain library - Google Patents
Antibody screening method based on heavy chain library/light chain library of infectable virus particle type antibody and preparation method of heavy chain library/light chain library Download PDFInfo
- Publication number
- CN103603057A CN103603057A CN201310277207.9A CN201310277207A CN103603057A CN 103603057 A CN103603057 A CN 103603057A CN 201310277207 A CN201310277207 A CN 201310277207A CN 103603057 A CN103603057 A CN 103603057A
- Authority
- CN
- China
- Prior art keywords
- antibody
- variable region
- sequence
- light chain
- heavy chain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Peptides Or Proteins (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
本发明涉及新型抗体筛选方法，该方法包括抗体库构建步骤：构建可感染病毒颗粒型抗体重链库和可感染病毒颗粒型抗体轻链库；第一筛选步骤：利用可感染病毒颗粒型抗体重链库和可感染病毒颗粒型抗体轻链库感染动物细胞，采用特异性抗原筛选被感染的、具有特异性抗体表达能力的动物细胞，获得阳性细胞株；第二筛选步骤：从阳性细胞株获取特异性抗体的重链可变区cDNA序列和轻链可变区cDNA序列，拼接引入特异性抗体的轻链信号肽序列，连接插入表达载体，构建特异性抗体的表达细胞株，以表达制备特异性抗体。本发明的筛选方法工艺简单高效、切实可行；筛选获得的基因工程抗体具有高特异性和高亲和性。
The present invention relates to a novel antibody screening method, which comprises the steps of constructing an antibody library: constructing an antibody heavy chain library of an infectable virus particle type antibody and an antibody light chain library of an infectious virus particle type antibody; Infect animal cells with the chain library and the light chain library of infectious virus particle antibodies, and use specific antigens to screen the infected animal cells with specific antibody expression ability to obtain positive cell lines; the second screening step: to obtain positive cell lines The cDNA sequence of the heavy chain variable region and the cDNA sequence of the light chain variable region of the specific antibody are spliced to introduce the light chain signal peptide sequence of the specific antibody, connected and inserted into the expression vector, and the expression cell line of the specific antibody is constructed to express and prepare the specific antibody. Sexual antibodies. The screening method of the invention is simple, efficient, and practical; the genetic engineering antibody obtained by screening has high specificity and high affinity.
Description
Technical field
The invention belongs to biological pharmacy technical field, and relate to antibody screening technical field.More specifically, the present invention relates to a kind of foundation and novel antibody screening method of can infecting virus particle type heavy chain of antibody storehouse and can infecting virus particle type light chain of antibody storehouse.
Background technology
Antibody molecule is biomedical sector purposes protein molecular the most widely, and therapeutic antibodies and diagnosis have wide application prospect and huge marketable value especially with antibody.Since first genetic engineering antibody people-mouse chimeric antibody in 1984 is born, novel gene engineered antibody is as the constantly appearance such as humanized antibody, unit price small molecular antibody, multivalence small molecular antibody.
Hybridoma cell technology has been brought revolutionary variation to monoclonal antibody production technique, greatly promoted the application process of antibody, but because this technology must be through the complicated very long technical process such as animal immune, cytogamy, colony screening, and have and can not prepare for defects such as the antibody of private antigen and human antibodies, thereby limited genetic engineering antibody, apply more widely.Antibody library makes people find the effective way addressing these problems, and wherein phage antibody library is to apply the most general Antibody library.Adopt this technology screening antibody without carrying out animal immune, there is no the processes such as cytogamy, colony screening, for each antibody-like of comprehensive preparation provides shortcut.Ribosomal display Antibody library is development in recent years another antibody screening technology out, whole procedure body carries out outward, without intestinal bacteria step of converting, be easy to screen high-affinity antibody and adopt in vitro method antagonist character to transform, but this technology is easy to carry out to immune animal, the screening of human antibody is not had to an advantage; And further investigation shows: a large amount of screenings that utilize various antibody libraries to carry out, and its product is usually because, poor specificity low to antigen avidity etc. are former thereby do not reach expection requirement; The people such as Roger R.Beerli have set up a kind of novel antibody screening method recently---and mammalian cell is shown method, but the method still cannot be broken away from the constraint of single-chain antibody.
Summary of the invention
The technical problem to be solved in the present invention is, for hybridoma cell technology in existing antibody screening technology, can not prepare for defects such as the specific antibody of private antigen and humanized antibodies; For the various defects such as the antigen avidity that antibody screening technology on antibody library basis occur is low, poor specificity that are based upon; The defect that can not prepare single-chain antibody for mammalian cell display technique, set up respectively a kind of can infecting virus particle type heavy chain of antibody storehouse and a kind of can infecting virus particle type light chain of antibody storehouse, utilize described antibody library to set up a kind of novel antibody screening method, screening approaches humanized antibody condition in body, high specific and high-affinity more.
The technical problem to be solved in the present invention is achieved by the following technical programs: a kind of antibody screening method is provided, comprises: antibody library construction step: structure can infecting virus particle type heavy chain of antibody storehouse and can infecting virus particle type light chain of antibody storehouse; The first screening step: utilize described can infecting virus particle type heavy chain of antibody storehouse and can infecting virus particle type light chain of antibody storehouse infection animal cell, adopt the zooblast that specific antigens screening is infected, have specific antibody ability to express, obtain positive cell strain; The second screening step: variable region of heavy chain cDNA sequence and the variable region of light chain cDNA sequence of obtaining described specific antibody from described positive cell strain, the light chain signal peptide sequence of described specific antibody is introduced in splicing, connect and insert expression vector, build the expression cell line of described specific antibody, to express the described specific antibody of preparation.
The present invention also provides a kind of construction process of can infecting virus particle type heavy chain of antibody storehouse and can infecting virus particle type light chain of antibody storehouse, comprises; Obtain respectively the mutant nucleotide sequence of the hypervariable region at human IgG variable region of heavy chain many combination series gene order, human IgG variable region of light chain many combination series gene order ,IgG family's variable region gene sequence and antigen binding site place, human IgG variable region; The shuttle vectors that structure contains T-A catenation sequence, signal peptide sequence and cross-film sequence; The mutant nucleotide sequence of described human IgG variable region of heavy chain many combination series gene order of obtaining, described human IgG variable region of light chain many combination series gene order, described IgG family's variable region gene sequence and the hypervariable region at antigen binding site place, described human IgG variable region is connected and inserts described shuttle vectors, build and obtain recombinant shuttle plasmid storehouse; Utilize recombinant shuttle plasmid storehouse and adenovirus recombination system build described can infecting virus particle type heavy chain of antibody storehouse and described can infecting virus particle type light chain of antibody storehouse.Implement technical scheme of the present invention, can obtain following technique effect: the present invention sets up a kind of can infecting virus particle type heavy chain of antibody storehouse and can in mammalian cell, by mode of infection, breed amplification, prolonged preservation in infecting virus particle type light chain of antibody storehouse; Comprise complete as far as possible IgG variable region of heavy chain and light chain variable region sequence ,IgG family's variable region sequences and the random mutation except nonsense mutation according to the hypervariable region at antigen binding site place, human IgG variable region, assurance can infecting virus particle type heavy chain of antibody storehouse and maximum abundance that can infecting virus particle type light chain of antibody storehouse, guarantees the capacity of antibody library; Guarantee that antibody molecule expression, at cell surface, presents more natural antibody form, be convenient to the foundation of follow-up novel antibody screening method; Specific antibody heavy chain, the secreted type of light chain are expressed in extracellular, have guaranteed the native state of engineered antibody, are convenient to follow-up purifying and produce.Can be for screening multiple specific antigens---as corresponding, the high specific of tumour antigen, virus antigen etc., high-affinity, approximate natural antibody.
The novel antibody screening method that the present invention sets up, be based upon can infecting virus particle type heavy chain of antibody storehouse and can the basis in infecting virus particle type light chain of antibody storehouse on, utilize antigen, antibody to mutually combine, use flow cytometer to carry out the sortings of many wheels; The positive cell strain obtaining is again combined with specific antigens after mono-clonal is cultivated again, again sorting obtain can with the antibody of the high affinity of specific antigens, high specific; The specific antibody heavy chain of screening and light chain cdna while double expression(DE) are in an eukaryotic vector respectively, guarantee that heavy chain, light chain form natural antibody structure, and by introducing signal peptide sequence, make antibody molecule be able to secretion type expression in extracellular, be convenient to later stage purifying.The screening method of setting up by described invention can screen and obtain high specific, high-affinity, natural structure, secretor type, humanized antibody, be used for the screening of the therapeutic antibodies of virus disease and tumour, or the screening for virus disease prevention with antibody, thereby effectively prevent and treat viral infectious and tumour generation.
Accompanying drawing explanation
Fig. 1 is the PAGE electrophorogram of the specific antibody for preparing.
Embodiment
In order to make technical scheme of the present invention and advantage clearer, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not intended to limit the present invention.
The present invention can infecting virus particle type heavy chain of antibody storehouse and can infecting virus particle type light chain of antibody storehouse by building, and set up novel antibody screening method on described antibody library basis, screening approaches humanized antibody condition in body, high specific, high-affinity more.
Structure can infecting virus particle type heavy chain of antibody storehouse and concrete steps that can infecting virus particle type light chain of antibody storehouse comprise: the fragment sequence of the random mutation of the hypervariable region that A. bioinformatic analysis technology is obtained human IgG weight, variable region of light chain many combination series gene order ,IgG family variable region gene sequence, antigen binding site place, human IgG variable region except nonsense mutation, the cDNA of different sources of take carries out pcr amplification acquisition as template; B. build the shuttle vectors that contains T-A catenation sequence, signal peptide sequence (SP, N-end) and cross-film sequence (TM, C-end); C. the fragment of full gene described in steps A connects the recombinant shuttle plasmid storehouse that insertion shuttle vectors structure contains variable region of heavy chain, variable region of light chain many combination series gene fragment; D. utilize recombinant shuttle plasmid storehouse and adenovirus recombination system build described can infecting virus particle type heavy chain of antibody storehouse and described can infecting virus particle type light chain of antibody storehouse.
The concrete steps of setting up described novel antibody screening method comprise E. utilize specific antigen protein (as the S antigen of HBV) with as described in can infecting virus particle type heavy chain of antibody storehouse and can be combined in infecting virus particle type light chain of antibody storehouse, identification, screening specific antibody express cell, twice sorting positive cell of flow cytometer high-throughput; F. obtain positive cell strain and cultivate through mono-clonal, screening, determines the specific antibody express cell that affinity is high again; Specific antibody variable region of heavy chain cDNA and variable region of light chain cDNA sequence are obtained in G.PCR amplification, by PCR splicing, introduce respectively light chain of antibody signal peptide, connect and insert double expression(DE) eukaryotic vector, build specific antibody heavy chain and stable expression cell strain light chain, that can secrete; H. separated, purification specificity antibody.
Further, above-mentioned steps A specifically comprises the following steps:
A1: human IgG is heavy, the obtaining of variable region of light chain many combination series gene order:
Along with completing of the Human Genome Project, Human genome library information completes substantially, and IgG gene is very various.In the databases such as the state-run bioinformation of U.S. center (NCBI), European bioinformation center (EBI), French immunoglobulin gene sequence library (IMGT), can find numerous human IgG gene fragments, utilize bioinformatic analysis technology, according to similarity score value, their variable region of heavy chain and variable region of light chain are divided into groups to compare, and according to its consequence devised, go out the universal primer of this group.For different echelon design, organize degenerated primer more.
A2: the obtaining of human IgG family protein molecular gene variable region sequences:
IgG family is the protein molecular that many and IgG antibody have analog structure, comprise: φt cell receptor, B-cell receptor, MHC, cell adhesion molecule, NK cell receptor, lymphocyte receptor, cytokine receptor, growth factor receptors etc., they all have important biological function, as: cell adhesion function, identification antigen, identification sepcific ligands, signal conduction etc., especially in immunity, identify, immuno-stimulating, in the transmission of immunity system signal, there is important biological function, these specific IgG family members also can be used in the specific antibody treatments such as tumour and virus disease.In the databases such as the state-run bioinformation of U.S. center (NCBI), European bioinformation center (EBI), French immunoglobulin gene sequence library (IMGT), can inquire a large amount of IgG family gene fragments, utilize bioinformatic analysis technology to divide into groups to compare to their variable region, for the many groups of different grouping design degenerated primers.
A3: the obtaining of human IgG variable region antigen binding site place hypervariable region random mutation sequence:
Human IgG variable region of light chain and variable region of heavy chain are comprised of 110 left and right amino acid respectively, wherein the amino-acid residue in some region changes larger compared with other parts of variable region, as 31st～35,50～65,95～102 of 24th～34,50～56,89～97 of light chains and heavy chains, these regions become hypervariable region, are the positions that antibody directly contacts with epitope.In order to increase the abundance of antibody library gene order information, random mutation by the hypervariable region at antigen binding site place, human IgG variable region except nonsense mutation, the fragment of acquisition is spliced the synthetic gene fragment that obtains variable region of light chain and variable region of heavy chain by PCR.
A4: the pcr amplification of variable region of heavy chain and chain variable region gene and PCR splicing:
From the fresh blood sample of different people of originating, extract total RNA and mRNA, reverse transcription behaviour cDNA, take people cDNA as template, with many group degenerated primers described in A1, A2, increases respectively, obtains separately the gene fragment storehouse of about 330bp left and right.Selected gene sequencing analysis confirms that they are needed goal gene.
The random mutation fragment in 24th～34,50～56,89～97, hypermutation region in the synthetic random mutation fragment for 31st～35,50～65,95～102, hypermutation region in variable region of heavy chain and variable region of light chain, wherein removes nonsense mutation in order to avoid causes the imperfection of antibody variable region sequence respectively.By PCR splicing, build variable region of heavy chain and the variable region of light chain of containing these random mutation fragments, in order to increase the abundance in heavy chain of antibody storehouse and light chain of antibody storehouse, guarantee the maximum gene information amount of antibody library.
Further, above-mentioned steps B specifically comprises the following steps:
B1: the obtaining of signal peptide sequence in shuttle vectors (SP, N-end):
Utilize shuttle vectors pShuttle-CMV in adenovirus system or pShuttle-IRES-hrGFP-2 for maternal carrier, transform accordingly.
In the database of the state-run bioinformation of U.S. center (NCBI), can find the whole genome sequence of variable region of light chain, they all contain similar signal peptide sequence, can guide antibody molecule to pass cytolemma due to cell surface, cutly in ripe antibody molecule fall.In order to guarantee that in antibody library, specific antibody can successfully also be expressed at cell surface through cytolemma effectively, need in shuttle vectors, introduce signal peptide sequence.In order to obtain the signal peptide sequence of human antibody light chain, the present invention extracts total RNA and mRNA from the fresh blood sample of people, and reverse transcription behaviour cDNA be take people cDNA as template, designs following primer:
Primer sequence the 1:5 '-ATGGACATGATGGTCCCCGC-3 ' of upstream primer;
Primer sequence the 2:5 '-CTGAGATGCCCGGCAAGTGA-3 ' of downstream primer
B2: the obtaining of cross-film sequence in shuttle vectors (TM, C-end):
In the database of the state-run bioinformation of U.S. center (NCBI), can find the whole genome sequence of IgG family member CD4, wherein contain cross-film sequence, i.e. TM sequence; TM sequence can be embedded in cytolemma and guarantee that molecule is positioned on cytolemma.In order to guarantee that in antibody library, specific antibody can be expressed at cell surface effectively, and be positioned at surface of cell membrane, need in shuttle vectors, introduce cross-film sequence.The present invention adopts the cross-film sequence of IgG family member CD4.In order to obtain the cross-film sequence of IgG family member CD4, the present invention extracts total RNA and mRNA from the fresh blood sample of people, and reverse transcription behaviour cDNA be take people cDNA as template, designs following primer:
Primer sequence the 3:5 '-GGCGTCGCCGGCCTCCTGCT-3 ' of upstream primer;
Primer sequence the 4:5 '-TCAAATGGGGCTACATGTCT-3 ' of downstream primer
B3: the pcr amplification of signal peptide sequence (SP, N-end) and cross-film sequence (TM, C-end):
From the fresh blood sample of people, extract total RNA and mRNA, reverse transcription behaviour cDNA, take people cDNA as template, with signal peptide sequence (SP, N-end) and upstream primer separately, the downstream primer of cross-film sequence (TM, C-end) increase respectively, obtain separately the cross-film sequence (TM of about 168bp, C-end) and 147bp signal peptide sequence (SP, N-end).Gene sequencing analysis confirms that they are needed object fragment.
B4: the structure of shuttle vectors:
Utilize shuttle vectors pShuttle-IRES-hrGFP-2 in adenovirus system for maternal carrier, transform accordingly.Cross-film sequence, signal peptide sequence are inserted respectively in pShuttle-IRES-hrGFP-2 carrier, introduce EcoRV restriction enzyme site.With restriction enzyme EcoRV enzyme, cut digestion, obtain flush end linear carrier, then utilize terminal phosphate transferring enzyme to introduce base T at 3 ' end, build and contain T-A catenation sequence, signal peptide sequence (SP, N-end) and the shuttle vectors of cross-film sequence (TM, C-end).
The fragment of full gene described in steps A (fragment sequence of the random mutation of the hypervariable region that comprises human IgG weight, variable region of light chain many combination series gene order ,IgG family variable region gene sequence, antigen binding site place, human IgG variable region except nonsense mutation) is connected and is inserted shuttle vectors and build recombinant shuttle plasmid storehouse by T-A sequence, utilize subsequently recombinant shuttle plasmid storehouse and adenovirus recombination system set up described can infecting virus particle type heavy chain of antibody storehouse and described can infecting virus particle type light chain of antibody storehouse.Above-mentioned heavy chain storehouse and light chain storehouse can be in eukaryotic cell (in HEK293 cell) be packaged into and there is infective virion storehouse, then by infecting multiple mammalian cell, as Chinese hamster ovary celI, VERO cell etc. infects amplification, prolonged preservation.Can infecting virus particle type heavy chain of antibody storehouse and can infecting virus particle type light chain of antibody storehouse in, antibody molecule is entered cell surface, by cross-film sequence, is positioned on cytolemma through cytolemma by the signal peptide guiding on shuttle vectors, guaranteed antibody expression and be positioned surface of cell membrane, so that follow-up application screening.
Can infecting virus particle type heavy chain of antibody storehouse and can infecting virus particle type light chain of antibody storehouse mammalian cell-infecting collecting cell, utilize specific antigen protein (as the S antigen of HBV, tumor marker molecule P53 etc.) respectively with infected Cell binding, utilize flow cytometer high-throughput sorting positive cell, the positive cell obtaining at 96 orifice plates after mono-clonal is cultivated and is amplified, binding specificity antigen protein again, and then carry out selected by flow cytometry apoptosis, finally determine positive cell strain.
Further, above-mentioned steps G specifically comprises the following steps:
G1: respectively from positive cell strain amplification antibody chain variable region cDNA and antibody heavy chain variable region cDNA:
HEK293 cell amplification culture is infected respectively in the positive cell strain obtaining, extract total RNA and mRNA, reverse transcription cDNA, utilize order-checking upstream primer 1 and order-checking downstream primer 2 in shuttle vectors to carry out pcr amplification, obtain respectively specific antibody variable region of heavy chain cDNA sequence and variable region of light chain cDNA sequence, through sequencing, determined.
Primer sequence the 5:5 '-CTCACGGGGATTTCCAAGTC-3 ' of order-checking upstream primer 1
Primer sequence the 6:5 '-ATGCAGTCGTCGAGGAATTG-3 ' of order-checking downstream primer 2
G2: pcr amplification and the connection of specificity variable region of heavy chain (containing signal peptide) sequence, variable region of light chain (containing signal peptide) sequence
Extract the mRNA of the positive cell of preserving, reverse transcription cDNA, utilize signal peptide (SP in shuttle vectors, downstream primer or the variable region of light chain downstream primer of the specific antibody variable region of heavy chain that N-end) in the primer sequence 1 of upstream primer and G1, sequencing obtains carry out pcr amplification, obtain respectively specific antibody variable region of heavy chain (containing signal peptide) sequence, variable region of light chain (containing signal peptide) sequence.By round pcr, splice, be connected to become the two sequence fragments in-IRES-variable region of heavy chain, variable region of light chain (containing signal peptide) (containing signal peptide) with IRES sequence (internal ribosome entry site).
G3: the secretor type double expression(DE) restructuring structure of eucaryon plasmid and the foundation of stable expression cell strain
For ease of feasibility and the practicality of the specific antibody that screens in multianalysis the present invention, the present invention adopts following carrier for expression of eukaryon as pIRES2-EGFP, pIRES DsRed-Express2 etc.Two sequence fragments described in step G2 are inserted to carrier for expression of eukaryon as structure secretor type double expression(DE) restructuring eucaryon plasmids such as pIRES2-EGFP, pIRES DsRed-Express2 by digestion with restriction enzyme, connection.
Because the double expression(DE) level of antibody is mainly determined jointly by expression vector and host cell (expression cell line), therefore, carrier for expression of eukaryon and mammalian cell expression system are best selections.In mammalian cell, have antibody to be folded to form the necessary molecular chaperones of correct space structure and folding enzymes, its endoplasmic reticulum for antibody molecule correctly in folding and chain and the formation of interchain disulfide bond favourable redox environment is provided; Mammalian cell has after complete transcribing, translation and posttranslational modification, the shearing of signal peptide and the advantages such as location of transmembrane protein, making to express antibody can be secreted into outside born of the same parents, space structure is approached more natural, be conducive to bring into play its biological function, improve the curative effect of therapeutic antibodies.
Utilize genetic engineering means the present invention can select HEK293 cell or Chinese hamster ovary cell CHO, and preferably use 293 cells as the host cell of expression vector, and set up stable expression cell line and be used for specific antibody heavy chain and the light chain of expression screening.
It is specific antigens that following examples be take HBV S antigen, describes the novel antibody screening method of how setting up based on can infecting virus particle type heavy chain of antibody storehouse/light chain storehouse in detail.
Utilize bioinformatic analysis to obtain human IgG weight, chain variable region gene sequence ,IgG family variable region gene sequence, design random degenerated primer; Take hbv antibody positive Peripheral Blood liquid, normal people's peripheral blood etc. is sample, and conventional test kit extracts mRNA, and reverse transcription is prepared human peripheral cDNA; The above-mentioned cDNA of take carries out pcr amplification as template and obtains a large amount of PCR products.In order to increase the abundance of antibody library gene order information, in IgG weight, variable region of light chain, hypervariable region (31～35,50～65,95～102 of 24th～34,50～56,89～97 of light chains and heavy chains) carries out random mutation, splicing obtains random mutation gene fragment storehouse, hypervariable region.
Take upstream primer (primer sequence 1:5 '-ATGGACATGATGGTCCCCGC-3 ') and downstream primer (primer sequence 2:5 '-CTGAGATGCCCGGCAAGTGA-3 ') is primer, human peripheral cDNA is template, pcr amplification obtains the signal peptide sequence of 147bp human antibody light chain, through sequencing, determine that sequence is the nucleotide sequence shown in the SEQ ID No.1 in sequence table (corresponding aminoacid sequence is shown in SEQ ID No.2), and introduce respectively Nhe I and two restriction enzyme sites of EcoR V.Take upstream primer (primer sequence 3:5 '-GGCGTCGCCGGCCTCCTGCT-3 ') and downstream primer (primer sequence 4:5 '-TCAAATGGGGCTACATGTCT-3 ') is primer, human peripheral cDNA is template, pcr amplification obtains the cross-film sequence of 168bp human IgG family member CD4, it is TM sequence, through sequencing, determine that sequence is the nucleotide sequence shown in the SEQ ID No.3 in sequence table (corresponding aminoacid sequence is shown in SEQ ID No.4), and introduce respectively EcoR V and two restriction enzyme sites of Sal I.
Take adenovirus system carrier pShuttle-IRES-hrGFP-2 as maternal carrier, utilize respectively Nhe I, EcoR V with EcoR V, Sal I, above-mentioned signal peptide to be connected with carrier with cross-film district, build shuttle vectors (containing human antibody light chain signal peptide HeCD4 cross-film district).With restriction enzyme EcoRV enzyme, cut digestion, obtain flush end linear carrier, then utilize terminal phosphate transferring enzyme to introduce base T at 3 ' end, build the shuttle vectors that contains T-A catenation sequence, signal peptide sequence and cross-film sequence.
The full gene fragment obtaining (is comprised to human IgG weight, variable region of light chain many combination series gene order, IgG family variable region gene sequence, the fragment sequence of the random mutation of the hypervariable region at antigen binding site place, human IgG variable region except nonsense mutation) by T-A sequence, connect and insert shuttle vectors structure recombinant shuttle plasmid storehouse, utilize recombinant shuttle plasmid storehouse and adenovirus recombination system transfected with human embryonic kidney HEK293 cell to be packaged into and there is infective virion storehouse, get final product infecting virus particle type heavy chain of antibody storehouse/light chain storehouse, the abundance of virus base can utilize shuttle vectors characteristic forward primer and reverse primer to carry out PCR evaluation, by being uniformly distributed of agarose gel electrophoresis detected characteristics bands of a spectrum, determined.Amplification, liquid nitrogen prolonged preservation are infected at eukaryotic cell Chinese hamster ovary celI and VERO cell etc. in above-mentioned heavy chain storehouse/light chain storehouse.
Respectively can infecting virus particle type heavy chain of antibody storehouse and can infecting virus particle type light chain of antibody storehouse mammalian cell-infecting collecting cell, by the HBV S antigen of Yeast system expression and purification respectively with above-mentioned Cell binding, repetitive scrubbing, 800-1000rpm centrifugal collecting cell, removes unconjugated HBV S antigen.The fluorescence antibody that utilizes anti-HBV S is combined with HBV S antigen that thereby to make the cell of expression specificity antibody be labeled fluorescence screened, utilizes the sorting of flow cytometer high-throughput with the positive cell of fluorescence, through twice sorting, determines and obtains positive cell.Positive cell, is again combined with HBV S antigen protein at 96 orifice plates after mono-clonal cultivate to amplify, and utilizes conventional ELISA(enzyme-linked immunosorbent assay) color reaction determine the positive cell of high-affinity.And then carry out selected by flow cytometry apoptosis, finally determine the high-affinity positive cell strain of specific antibody.
HEK293 cell amplification culture is infected in the positive cell strain obtaining, extract mRNA reverse transcription cDNA, utilize the order-checking upstream and downstream primer in shuttle vectors to carry out pcr amplification, obtain respectively HBV S antigen-specific, high-affinity antibody variable region of heavy chain cDNA sequence and variable region of light chain cDNA sequence, sequencing is determined.The specificity positive cell cDNA of take is template, with shuttle vectors signal peptide (SP, N-end) upstream primer (primer sequence 1) is forward primer, the specific antibody variable region of heavy chain downstream primer that the screening of take obtains or variable region of light chain downstream primer, as downstream primer carries out pcr amplification, obtain respectively specific antibody variable region of heavy chain (containing signal peptide) sequence, variable region of light chain (containing signal peptide) sequence.By round pcr, splice, be connected to become the two sequence fragments in-IRES-variable region of heavy chain, variable region of light chain (containing signal peptide) (containing signal peptide) with IRES sequence (internal ribosome entry site), insert eukaryotic expression vector pIRES 2-EGFP and build secretor type double expression(DE) restructuring eucaryon plasmid.Transfection Chinese hamster ovary cell CHO, stable expression cell strain is set up in G418 screening.Culturing cell is collected supernatant liquor, utilize the specific antibody (variable region of heavy chain and variable region of light chain) of specificity S antigen purification, collection and concentrating secreted expression, specific antibody is carried out to protein polyacrylamide gel electrophoresis (PAGE) to be detected, target protein clearly as seen after coomassie brilliant blue staining, result is as shown in the characteristic spectrum belt of Fig. 1.In Fig. 1, the two row bands in left side are respectively the electrophoresis result of variable region of heavy chain, and right side two row bands are respectively the electrophoresis result of variable region of light chain.
Claims (9)
1. an antibody screening method, is characterized in that, comprising:
Antibody library construction step: structure can infecting virus particle type heavy chain of antibody storehouse and can infecting virus particle type light chain of antibody storehouse;
The first screening step: utilize described can infecting virus particle type heavy chain of antibody storehouse and can infecting virus particle type light chain of antibody storehouse infection animal cell, adopt the zooblast that specific antigens screening is infected, have specific antibody ability to express, obtain positive cell strain;
The second screening step: variable region of heavy chain cDNA sequence and the variable region of light chain cDNA sequence of obtaining described specific antibody from described positive cell strain, the light chain signal peptide sequence of described specific antibody is introduced in splicing, connect and insert expression vector, build the expression cell line of described specific antibody, to express the described specific antibody of preparation.
2. antibody screening method according to claim 1, is characterized in that, described antibody library construction step comprises:
Obtain respectively the mutant nucleotide sequence of the hypervariable region at human IgG variable region of heavy chain many combination series gene order, human IgG variable region of light chain many combination series gene order ,IgG family's variable region gene sequence and antigen binding site place, human IgG variable region;
The shuttle vectors that structure contains T-A catenation sequence, signal peptide sequence and cross-film sequence;
The mutant nucleotide sequence of described human IgG variable region of heavy chain many combination series gene order of obtaining, described human IgG variable region of light chain many combination series gene order, described IgG family's variable region gene sequence and the hypervariable region at antigen binding site place, described human IgG variable region is connected and inserts described shuttle vectors, build and obtain recombinant shuttle plasmid storehouse;
Utilize described recombinant shuttle plasmid storehouse and adenovirus recombination system build described can infecting virus particle type heavy chain of antibody storehouse and described can infecting virus particle type light chain of antibody storehouse.
3. antibody screening method according to claim 2, is characterized in that, the mutant nucleotide sequence of the hypervariable region at antigen binding site place, described human IgG variable region comprises the fragment sequence of the random mutation except nonsense mutation.
4. antibody screening method according to claim 2, it is characterized in that, described signal peptide sequence has the aminoacid sequence shown in the nucleotide sequence shown in the SEQ ID No.1 in sequence table and SEQ ID No.2, and described cross-film sequence has the aminoacid sequence shown in the nucleotide sequence shown in the SEQ ID No.3 in sequence table and SEQ ID No.4.
5. antibody screening method according to claim 1, is characterized in that, between described the first screening step and described the second screening step, described method also comprises:
Described positive cell strain is carried out to mono-clonal cultivation, to obtain affinity positive cell strain that improve, expression specificity antibody.
6. antibody screening method according to claim 1, is characterized in that, described light chain of antibody signal peptide sequence has the aminoacid sequence shown in the nucleotide sequence shown in SEQ ID No.1 and SEQ ID No.2 in sequence table.
7. antibody screening method according to claim 1, is characterized in that, in described the second screening step, described expression vector is pIRES2-EGFP or pIRES DsRed-Express2.
8. antibody screening method according to claim 1, is characterized in that, in described the second screening step, described expression cell line is HEK293 cell or Chinese hamster ovary cell.
9. the construction process of can infecting virus particle type heavy chain of antibody storehouse and can infecting virus particle type light chain of antibody storehouse, is characterized in that, comprises;
Obtain respectively the mutant nucleotide sequence of the hypervariable region at human IgG variable region of heavy chain many combination series gene order, human IgG variable region of light chain many combination series gene order ,IgG family's variable region gene sequence and antigen binding site place, human IgG variable region;
The shuttle vectors that structure contains T-A catenation sequence, signal peptide sequence and cross-film sequence;
The mutant nucleotide sequence of described human IgG variable region of heavy chain many combination series gene order of obtaining, described human IgG variable region of light chain many combination series gene order, described IgG family's variable region gene sequence and the hypervariable region at antigen binding site place, described human IgG variable region is connected and inserts described shuttle vectors, build and obtain recombinant shuttle plasmid storehouse;
Utilize recombinant shuttle plasmid storehouse and adenovirus recombination system build described can infecting virus particle type heavy chain of antibody storehouse and described can infecting virus particle type light chain of antibody storehouse.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310277207.9A CN103603057B (en) | 2013-07-03 | 2013-07-03 | Based on can the antibody screening in infecting virus particle type heavy chain of antibody storehouse/light chain storehouse and preparation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310277207.9A CN103603057B (en) | 2013-07-03 | 2013-07-03 | Based on can the antibody screening in infecting virus particle type heavy chain of antibody storehouse/light chain storehouse and preparation method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103603057A true CN103603057A (en) | 2014-02-26 |
| CN103603057B CN103603057B (en) | 2016-01-06 |
Family
ID=50121317
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310277207.9A Active CN103603057B (en) | 2013-07-03 | 2013-07-03 | Based on can the antibody screening in infecting virus particle type heavy chain of antibody storehouse/light chain storehouse and preparation method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103603057B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107058235A (en) * | 2017-06-05 | 2017-08-18 | 深圳大学 | A kind of B cell screening technique and its application in monoclonal antibody preparation |
| CN108456682A (en) * | 2017-02-17 | 2018-08-28 | 苏州金唯智生物科技有限公司 | A kind of screening technique of monoclonal antibody and its application |
| WO2019036842A1 (en) * | 2017-08-21 | 2019-02-28 | Adagene Inc. | Dynamic human heavy chain antibody libraries |
| US11585014B2 (en) | 2017-08-21 | 2023-02-21 | Adagene Inc. | Dynamic human antibody light chain libraries |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101331223A (en) * | 2005-10-14 | 2008-12-24 | 米迪缪尼有限公司 | Cell display of antibody libraries |
| CN102690792A (en) * | 2012-05-25 | 2012-09-26 | 深圳大学 | Anti-human immunodeficiency virus (HIV) gene engineering recombinant virus and preparation method thereof, and anti-HIV gene engineering medicament |
-
2013
- 2013-07-03 CN CN201310277207.9A patent/CN103603057B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101331223A (en) * | 2005-10-14 | 2008-12-24 | 米迪缪尼有限公司 | Cell display of antibody libraries |
| CN102690792A (en) * | 2012-05-25 | 2012-09-26 | 深圳大学 | Anti-human immunodeficiency virus (HIV) gene engineering recombinant virus and preparation method thereof, and anti-HIV gene engineering medicament |
Non-Patent Citations (2)
| Title |
|---|
| 梁中锟: "全长人源VEGF抗体库的构建及展示", 《中国博士学位论文全文数据库医药卫生科技辑》 * |
| 郭明高等: "腺病毒介导的全抗体基因治疗卵巢癌的实验研究", 《中华医学杂志》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108456682A (en) * | 2017-02-17 | 2018-08-28 | 苏州金唯智生物科技有限公司 | A kind of screening technique of monoclonal antibody and its application |
| CN107058235A (en) * | 2017-06-05 | 2017-08-18 | 深圳大学 | A kind of B cell screening technique and its application in monoclonal antibody preparation |
| CN107058235B (en) * | 2017-06-05 | 2020-08-11 | 深圳大学 | A B cell screening method and its application in the preparation of monoclonal antibodies |
| WO2019036842A1 (en) * | 2017-08-21 | 2019-02-28 | Adagene Inc. | Dynamic human heavy chain antibody libraries |
| US11578426B2 (en) | 2017-08-21 | 2023-02-14 | Adagene Inc. | Dynamic human heavy chain antibody libraries |
| US11585014B2 (en) | 2017-08-21 | 2023-02-21 | Adagene Inc. | Dynamic human antibody light chain libraries |
| US12385162B2 (en) | 2017-08-21 | 2025-08-12 | Adagene Inc. | Dynamic human heavy chain antibody libraries |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103603057B (en) | 2016-01-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110041433A (en) | A kind of Chimeric antigen receptor and its application targeting BCMA | |
| CN106243219B (en) | A kind of pig-derived anti-porcine epidemic diarrhea virus single-chain antibody and its preparation method | |
| CN1328605A (en) | Methods for making recombinant cells | |
| CN107058235B (en) | A B cell screening method and its application in the preparation of monoclonal antibodies | |
| CN110964111A (en) | anti-PD-L1 monoclonal antibody, antigen binding fragment thereof and application thereof | |
| CN103603057A (en) | Antibody screening method based on heavy chain library/light chain library of infectable virus particle type antibody and preparation method of heavy chain library/light chain library | |
| CN113699118A (en) | Hybridoma cell strain, antibody produced by hybridoma cell strain and application of hybridoma cell strain | |
| CN106414717A (en) | human antibody-producing cells | |
| WO2023217072A1 (en) | Therapeutic agent comprising multispecific antibody and use thereof in tumor therapy | |
| CN112661841A (en) | Fully human monoclonal antibody 17-2 for neutralizing neoepitope of new coronavirus and application thereof | |
| CN109306008B (en) | Single-chain antibody of swine-origin anti-classical swine fever virus and preparation method thereof | |
| CN111647081A (en) | Recombinant mouse anti-human interleukin 19 monoclonal antibody, preparation method and application | |
| CN113150122B (en) | Preparation method of high-throughput whole rabbit source monoclonal antibody | |
| CN117106082B (en) | Humanized nanometer antibody against TGF beta | |
| CN108484770B (en) | Recombinant rat anti-mouse CD4 monoclonal antibody, preparation method and application | |
| CN112342198A (en) | PAT/PAT monoclonal antibody hybridoma cell strain, antibody produced by same and preparation method thereof | |
| CN116813765B (en) | Specific antibody for Rab8 protein and preparation method and application thereof | |
| CN117683726A (en) | Method for constructing a CHO cell line that stably exocrine expresses porcine epidemic diarrhea virus S protein | |
| CN119661701B (en) | Foot-and-mouth disease virus anti-idiotype antibody and application thereof | |
| CN114044819B (en) | High-flux preparation method of whole sheep-derived monoclonal antibody | |
| CN111647083B (en) | Recombinant mouse anti-human hemojulin monoclonal antibody, preparation method and application | |
| CN119462939B (en) | An anti-CD147 single-domain antibody and its application | |
| CN120365421B (en) | Fully human antibody of specific anti-SEZ 6 target spot and preparation method and application thereof | |
| CN1363682A (en) | A group of high-efficiency expression vectors for human antibodies in mammalian cells | |
| CN102021242A (en) | Method for analyzing epitope of protein |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |