WO2002077238A1 - Gene encoding lectin protein originating in gorse, protein encoded by the gene and process for producing the same - Google Patents

Abstract

(1) A gene (UEL1 gene) encoding a novel lectin showing an anti-H coagulation activity which is isolated from genomic DNA of gorse (Ulex europaeus) and a gene having a base sequence substantially equivalent thereto; (2) a novel lectin protein encoded by these genes; (3) a recombinant vector having the gene integrated thereinto; and (4) a process for producing a protein showing an anti-H coagulation activity which comprises transferring the recombinant vector into host cells to thereby transform the host, culturing the resultant transformant, and then collecting a polypeptide showing the anti-H coagulation activity from the culture.

Description

 Specification

TECHNICAL FIELD The present invention relates to a gene encoding a lectin protein derived from a gorse, a protein encoded by the gene, and a method for producing the same.

 TECHNICAL FIELD The present invention relates to a gene encoding a lectin derived from a gorse (Ulex europaeus), a novel lectin protein encoded by the gene, a recombinant vector incorporating the gene, and a method for producing the protein. Background art

 Lectins are known as substances that have an affinity for specific sugar structures and bind to the sugar chains of glycoconjugates (glycoproteins or glycolipids) in cell membranes to exert effects such as cell aggregation. Among them, the lectin contained in the crude extract of P. fern has the property of specifically reacting with human H antigen (antigen present on O-type blood cell membranes and the like) (anti-H aggregation activity). Utilizing this property, the lectin in seeds of Pseudomonas aeruginosa is used as O-type hemagglutinin in the determination of ABO blood type.

Up to now, three types of proteins (UEA-I, UEA-II, UEA-III) have been isolated and purified as a lectin obtained from the seeds of the gorse by Abinitik mouth chromatography. Among them, the entire amino acid sequence has been reported for UEA-I and UEA-II (J. Biochem. 109 (1991) p 650-658), and the partial amino acid sequence for UEA-III has been reported (Bio 1 Chera. Hoppe-Seyler 372 (1991) p95-102). However, it is not known whether the gorse contains a lectin different from the above three types. — 1, The gene sequences encoding UEA-II and UEA-III have not been known so far.

 Accordingly, an object of the present invention is to provide a method for producing a lectin protein by genetic engineering by exploring a previously unknown lectin derived from P. chinensis and elucidating the gene sequence encoding P. ponin lectin. . '' Disclosure of the Invention

 In order to solve the above problems, the present inventors isolated a novel lectin-encoding gene (UEL1 gene) from genomic DNA of P. fern and determined its base sequence. Further, the present inventors have integrated the UEL1 gene into a vector, transformed a host cell with this vector, and developed a method for producing a novel lectin using the vector, thereby completing the present invention.

 That is, the present invention provides a gene encoding a lectin shown below, a lectin protein encoded by the gene, and a method for producing a protein having anti-H aggregation activity by a genetic engineering method using the gene. is there.

 (1) A lectin protein exhibiting anti-H aggregation activity, comprising the nucleotide sequence of SEQ ID NO: 1, a complementary sequence thereof, or a nucleotide sequence in which some of the codons constituting these sequences are deleted or substituted. Gene to encode.

 (2) Among the nucleotide sequences described in SEQ ID NO: 1, base sequences 31 to 858, their complementary sequences, or other sequences so that the amino acid sequence does not differ for one or more codons of these sequences. 2. The gene according to the above 1, which comprises a sequence replaced with a codon.

(3) an anti-H aggregating activity comprising the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence in which one or more amino acids constituting the sequence are deleted or substituted. Protein.

 (4) A protein having the amino acid sequence of SEQ ID NO: 2.

 (5) A recombinant vector incorporating the gene according to 1 or 2 above.

 (6) Transformation by introducing the recombinant vector according to 5 above into a host cell, culturing the transformant, and collecting an anti-H agglutinating activity from the culture to obtain an anti-H agglutinating activity. A method for producing the indicated protein.

 (7) The thread-recombinant vector is a plasmid, which is transformed by introducing plasmid into agrobacterium, infecting plant cells with the transformed agrobacterium, and exhibiting anti-H aggregation activity from infected plant cells. 7. The method according to the above 6, wherein the protein is collected.

 (8) The method according to the above (7), wherein the plant cells are cultured tobacco cells. Detailed description of the invention

 (A) Lectin-encoding gene

 The lectin-encoding gene of the present invention is designed by designing an oligonucleotide probe based on the amino acid sequence of a known gorse lectin (UEA-I), applying this to the genomic DNA of gorse, and regenerating the internal sequence of the gene by PCR. This is the gene of SEQ ID NO: 1 determined as the full-length nucleotide sequence of the gene by designing new primers based on the results, and a gene consisting of a nucleotide sequence substantially equivalent thereto.

 Here, the gene of SEQ ID NO: 1 and a nucleotide sequence substantially equivalent thereto are:

(1) the nucleotide sequence of SEQ ID NO: 1

 (2) the complementary sequence of the base sequence described in SEQ ID NO: 1,

 (3) a base sequence in which a part of the codon constituting the sequence of (1) or (2) is deleted or substituted, and

(4) Base sequence obtained by further adding codons to (1) to (3) above including. However, the deletion or substitution of codons in (3) and the addition of codons in (4) are within the range in which the protein encoded by the nucleotide sequence after deletion, substitution or addition exhibits anti-H aggregation activity. .

 Such a base sequence of (3) or (4) includes a sequence whose homology to (1) or (2) is usually 80% or more, preferably 90% or more, and more specifically. Is

(a) a nucleotide sequence in which codons are substituted so that amino acid sequences do not differ; and

(b) It contains a DNA base sequence that hybridizes to the above sequence under stringent conditions. Here, the stringent conditions are, for example, in terms of temperature conditions, about 5 ° C. to about 30 ° C., preferably about 10 ° C. to about 25 ° C. below the melting temperature (Tm) of the complete hybrid. When hybridization occurs at a temperature lower by ° C.

 In the nucleotide sequence of SEQ ID NO: 1, the nucleotide sequence of Nos. 31 to 858 is a region encoding the lectin protein (polypeptide) of the present invention. Therefore, (i) the nucleotide sequence of Nos. 31 to 858 of SEQ ID NO: 1,

(ii) the complementary sequence of (i),

 (iii) a base sequence obtained by substituting codons so that the amino acid sequence of the sequence (i) or (ii) does not differ; and

 (iv) An anti-H agglutinating activity is observed in a product of a gene containing a DNA base sequence that hybridizes to the above sequence under stringent conditions.

In the present invention, primers are designed based on the amino acid sequence, and a method of amplifying genomic DNA by combining an appropriate PCR method is employed. DNA encoding a protein exhibiting the following can be obtained. For example, (1) a cDNA library is synthesized from poly (A) + RNA extracted from a Pseudomonas fern seed, and an oligonucleotide probe or a corresponding oligonucleotide probe synthesized based on an amino acid sequence of a known lectin (UEA-I) or the like. Scan cDNA library with antibody (2) Design primers based on the known amino acid sequence, amplify the DNA or cDNA of P. chinensis by an appropriate PCR method, and use the resulting PCR product as a probe in a cDNA library or P. It can be obtained by screening a genomic library. The nucleotide sequence of the DNA thus obtained can be analyzed by the dideoxy chain terminator method (proc. Natl. Acad. Sci. USA 74, 5463-5467 (1977) Sanger, F. et al.). it can.

 (B) Lectin protein

 The lectin protein of the present invention is a protein comprising the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence in which one or several amino acids constituting the sequence are deleted or substituted, and exhibits anti-H aggregation activity. .

 Deletion, substitution and addition of an amino acid sequence correspond to deletion, substitution and addition of a codon in a base sequence.

When the amino acid sequence of SEQ ID NO: 2 is compared with the amino acid sequence of a known gorse lectin, the sequences considered to be related to anti-H agglutinin activity are from positions 128 to 137 in the UEA-I lectin. The 10 amino acid `` Asp Thrlie Gly Ser Pro Val Asn Phe Trp '' located at, is thought to be a sugar-binding site, and this site is relatively conserved in other legume lectins. an array (J.Biochem. 111 (1992) p.436- 43 9).

 In UEL 1 of the present invention, this portion corresponds to the 154th to 163rd 10-amino acids “Asp Thrlie Gly Ser Pro Val Asn Ser Trp”, and is the same as UEA-I in 9 out of 10 amino acids. In the UEL 1 base sequence, this corresponds to 490 to 519 bases.

 (C) Method for producing lectin

The lectin of the present invention can be synthesized on the basis of the above amino acid sequence, but can be produced by genetic engineering based on the base sequence obtained above. Can be Typically, it can be produced by introducing the gene of the present invention described in the above (A) into a host cell via an appropriate vector, and expressing the gene. Expression may be performed directly using the DNA according to the gene of the present invention, or may be expressed as a fusion protein with another protein. In addition, the full-length gene described in (A) above may be expressed, or a part thereof may be expressed.

 Conventional host-vector systems used for protein production can be used. For example, an E. coli expression system can be used, and a pET vector system (Novagen), PinPoin tXa, pGEMEX, p Examples thereof include ET-5 (promega) and pGEX-5X (pharmacia).

Further, for example, E. coli-yeast shuttle vector a is _P PIC3K (In'vi Toro Zhen (Invitrogen) Co., Ltd.) and yeast such as Pichia pastoris is a yeast expression system with the host Pichia Expression Kit (Invitrogen Co.), an animal cell expression system BacVector system (Novagen), a cell-free expression system, RTS500 (Rapid Translation System RTS500) (Roche), and the like can be used. In addition, lectin is expressed by integrating it into a vector such as ρ Β Ι Ι Ι Ο Ι or pBIl21 (Clonetech) and introducing it into a plant such as tobacco using a transformation method using a gug mouth pacterium. It can also be done.

The host cell is cultured in a suitable medium to produce and accumulate lectin, and the cells are separated and disrupted by a conventional method to extract the recombinant protein in the cells. The lectin of the present invention can be produced by separating and collecting the extracted recombinant protein by means such as electrophoresis using a denaturing SDS-polyacrylamide gel. In addition, the lectin of the present invention can be produced by using an affinity chromatography method using a carrier to which sugar (fucose) is bound in addition to the ordinary protein separation method described above.

BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is a photograph showing an SDS-PAGE image of recombinant lectin (Fucose agarose gel fractions el to e6) according to the present invention and a conventionally known gorse lectin (UEA-I).

 FIG. 2 shows the recombinant lectin of the present invention (E. coli UE L1) obtained from E. coli, the recombinant lectin of the present invention (BY-2 UEL 1) obtained from tobacco BY-2 cells, and a conventionally known gorse FIG. 2 is a set of photographs showing the results of non-denaturing PAGE (N in the figure) and denaturing SDS-PAGE (R in the figure) of lectin (UEA-I). BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to the following description. Example 1: Extraction of genomic DNA from gorse

The above-ground part (about 50 mg) of the young plant of Harrier-fern is frozen in liquid nitrogen, crushed using a mortar and pestle, and 0.6 ml of a C TAB buffer (2 ° / ₀ cetyltrimethylammoniumpromi). , Tris-hydrochloric acid (0.1 M, pH 8), EDTA (0.02 Μ,; Η8), sodium chloride (1.4 M)) were added, stirred, and heated at 65 ° C for 30 minutes. Thereafter, a mixed solution of 0.7 ml of chloroform / isoamyl alcohol (2) was added, and the mixture was further stirred, and the resulting solution was centrifuged. Centrifugation

Replacement forms (rules) 0.9 ml of CTAB buffer for precipitation (1% cetyltrimethylammonium bromide, tris-HCl (0.05 M, pH8), EDTA (0.01 M, pH8)) was added to the supernatant obtained in, and stirred. Centrifuged. The precipitate obtained by centrifugation was dissolved in a buffer solution (0.01 M Tris-HCl, EDTA (ImM, pH8)), followed by ethanol precipitation to obtain purified genomic DNA. Example 2: Synthesis of degenerate primers and amplification of DNA by PCR method

 Based on the amino acid sequence of the known UEA-I lectin, degenerate primers UEA-F3 (SEQ ID NO: 3) and UEA-R4 (SEQ ID NO: 4) were synthesized. In the sequence listing, y represents c or t, n represents a or c or g or t, r represents a or g, w represents a or t, and s represents c or g.

 A PCR reaction was performed using primers (UEA-F3 and UEA-R4) using the genus DNA of P. niger obtained in Example 1 as type III. The reaction conditions consisted of denaturation (94, 1 minute), annealing (51 ° C, 1 minute), and extension (72 ° C, 1.5 minutes), which were repeated 30 times. Example 3: Base sequence determination of PCR product

 The PCR product obtained in Example 2 was ligated to the vector using the pGEM-TE asy vector system (manufactured by Promega), and the vector was ligated to a competent E. coli XL1-B1ue strain (Stratagene). After transfection, the cells were cultured on LB plates containing ampicillin (50 gz / ml). The Escherichia coli grown on the medium was cultured, and the plasmid was extracted and purified from the cultured cells using the Alkyrie SDS method.

The DNA sequence inserted into the purification plasmid is Ml3 forward, repurse primer (Futsubane Gene), Big Dye Terminator 1 cycle The nucleotide sequence was determined using a sequencing reaction kit (Big Dye Terminator Cycle Sequencing reaction kit) (manufactured by ABI) and ABI PRIZM310 Genetic Analyzer (manufactured by ABI). . The amino acid sequence deduced from this DNA sequence showed extremely high homology with the amino acid sequence of UE A-I protein. Therefore, the DNA sequence is considered to be a partial sequence inside the gene encoding lectin. Example 4: Extension of DNA sequence

 To obtain the full-length DNA sequence encoding the lectin protein, the partial sequence was extended by the tail-PCR method. Based on the partial sequence obtained in Example 3, a primer designed to amplify the upstream side (UEA11R (SEQ ID NO: 5)) and a primer designed to amplify the downstream side (UEA11R

44F (SEQ ID NO: 6)) were synthesized, respectively. Using these synthetic primers and a commercially available random primer, DNA oligomer (12) Set A (manufactured by Futatsu Gene Co.), the tai 1-PCR method (Shujunsha, PCR test protocol for plants, p83-89) A PCR reaction was performed according to the procedure. The obtained PCR product was clawed in the same manner as in Example 3, and then its nucleotide sequence was analyzed.

 As a result of the above analysis, the PCR product obtained with the primer designed to amplify the upstream side shows that the amino acid sequence deduced from its base sequence has extremely high homology with the N-terminal portion of the UEA-I protein. The amino acid sequence corresponding to the downstream showed extremely high homology with the portion including the C-terminus of the UEA-I protein. Therefore, these PCR products are thought to encode upstream and downstream of the lectin protein. Example 5: Synthesis of full-length gene encoding lectin protein

Partial sequence inside the gene obtained in Example 3, upstream and downstream thereof obtained in Example 4. Primers (UEA-F (SEQ ID NO: 7), UEA-R (SEQ ID NO: 8)) based on the sequence obtained in Example 4 to synthesize a full-length gene containing both Was synthesized and the genomic DNA of P. aeruginosa was amplified by PCR. When the nucleotide sequence of the obtained PCR product was analyzed, a DNA sequence represented by SEQ ID NO: 1 was obtained. The gene having this DNA sequence was designated as UEL1 gene.

 The UEL1 gene has a start codon ATG at bases 31 to 33 and a stop codon TGA at bases 856 to 858. The amino acid sequence encoded by this region is shown in SEQ ID NO: 2.

 The protein encoded by the UEL1 gene consists of 275 amino acids and has an estimated molecular weight of 30138 Da. This protein showed 90% homology with the UEA-I lectin protein. When this amino acid sequence was homologously searched using the database of the National Center for Biotechnology Information (USA) on the Internet, Cytisus sessilifolius lectin, Maackia murensis lectin, etc. It has homology (50% -70%) with many legume lectins and is considered to be a novel lectin. Example 6 Method for Producing New Lectin Protein Using Transformed Escherichia coli Expression of recombinant UEL1 lectin in Escherichia coli was performed using an Escherichia coli expression system pET-32Xa / LIC vector system (Novagen).

A primer containing the start codon of the UEL1 gene (UEL lZL IC-F (SEQ ID NO: 9)) and a primer containing the stop codon (UEL1 / LICR (SEQ ID NO: 10)) were designed and synthesized according to the kit protocol. . Using this, the UEL1 gene was synthesized by PCR, incorporated into the pET32 vector, and the vector DNA was transformed into a competent E. coli (Novablue Singles (Novagen)) and cultured on an LB plate containing 50 g / m.1 ampicillin. The grown Escherichia coli was cultured, and the plasmid DNA extracted and purified from the cultured cells was introduced into a competent Escherichia coli BL21 (DE3) strain for expression to transform Escherichia coli.

 The transformed Escherichia coli for expression was cultured overnight at 30 ° C in an LB medium (1% batatotryptone, 0.5% pactoist extratato, 0.5% sodium chloride) containing 1M isopropyl monosaccharide; S-D galactoside. . The obtained cells were centrifuged, the cells were disrupted by sonication, and the recombinant protein in the cells was extracted and purified. When the purified recombinant protein was subjected to electrophoresis by denaturing SDS-polyacrylamide gel (SDS-PAGE), a protein having a molecular weight of about 50 kDa was obtained.

 The recombinant protein was about 20 kDa larger than the expected molecular weight of the UEL1 protein, probably because it was expressed as a fusion protein with thioredoxin encoded by the vector ^ ". The lectin in the protein was treated with protease using Factor Xa (Boehringer Mannheim), cleaved thioredoxin, purified and separated.The molecular weight of the lectin after cleavage was about 30 kD In Example a, the estimated molecular weight of UEL 1 protein was the same as in Example 7. Example 7: Expression of UEL 1 protein by cultured tobacco cells

(1) Plasmid Construction and Transformation of Agrobacterium Terminus UEL 1 / Xba—F Primer for the full-length coding region of UEL 1 gene: tgcaaatctagaaagtgatgtc (G column number 11) and UELZ S ac—R primer: PCR was performed using aagtagagctccaaatcatgcag (SEQ ID NO: 12), followed by digestion with restriction enzymes NdeI and XhoI, and vector pBI121 (Clontech). Digested with XbaI and SacI Then, plasmid DNA (pre-UEL1-pBI) was extracted from the transformant obtained by ligation into the competent E. coli XL-1B1ue strain. Agrobacterium C58 cl PMP90 strain was transformed with the pre-UEL1-pBI plasmid by the electoral porting method. The transformed agrobacterium is grown on an LB solid medium containing 5 Omg / L kanamycin, and a colony of the AEL protein carrying the UEL1 gene is grown in an LB liquid medium containing 5 Omg / L kanamycin. And then used to infect cultured tapaco cells.

 (2) Transformation of tobacco BY-2 cultured cells

 Transformation of cultured tobacco cells BY-2 was carried out using the procedure described in “Experimental protocol J for observing plant cells J (Hiroshi Fukuda et al., Shujunsha (1997), p. 125-129). Globacterium was used to infect cultured tobacco BY-2 cells, and transformed BY-2 calli were selected and grown on BY-2 solid medium supplemented with kanamycin and carpenicillin.

 Take a portion of each of the transgenic (transformed) BY-2 callus (35 clones) grown on the selective medium, add PBS buffer (phosphate buffered saline), and add to an eppendorf tube. After grinding in a medium and centrifuging at 15000 rp mX for 5 minutes (4 ° C), 0.01% (w / v) trypsin was added to a portion of the supernatant, and treated at 37 for 1 hour at 3% (v / v) Physiological saline suspension of fresh human O-type blood cells was added and mixed, and the presence or absence of agglutinating activity was confirmed.

As a result, 18 out of 35 clones showed strong (++ to +++) human O-type hemagglutination ability. In addition, 10 clones aggregated weakly (+) human O-type blood cells, indicating that active UEL1 protein was synthesized in these cells. Example 8: Purification of recombinant lectin from tobacco BY-2 cultured cells Recombinant UEL1 lectin (rUEL1) derived from cultured tobacco cells was purified by the following procedure MM.

 In Example 7, an 18-clonal transgenic canoleth exhibiting strong agglutinating activity against human O-type blood cells was selected, and was subjected to spin culture in a BY-2 liquid medium supplemented with kanamycin and force / lepenicillin for 2 weeks. The nearly saturated BY-2 cultured cells were centrifuged at 8000 rpm for 30 minutes (4 ° C) to separate the culture supernatant and the cell fraction. The cell fraction was frozen at 80 ° C, thawed at room temperature, and an equal volume of PBS buffer containing 1.5% (w / v) Triton X-100 was added and mixed to lyse the cells. Was. After dissolution, the supernatant obtained by centrifugation at 8000 rpm for 30 minutes (4 ° C) was placed in a dialysis tube, and dialyzed in distilled water for 2 days to remove the surfactant in the solution.

 The dialyzed cell supernatant is filtered through a filter paper (Toyo Roshi Kaisha No. 2) to remove solids, and then passed through a column packed with fucose-agarose (Seikagaku) 1 Om1 to remove the solid content. UEL 1 protein was adsorbed. After washing the column after binding with a PBS buffer, the adsorbed fucose-bound fraction was eluted using a 2 OmM diaminopropane (DAP) solution. A part of the eluted fraction (el to e6) is combined with cell supernatant (SUP), column flow-through (f-t), and purified UEA-I (Sigma) by SDS-PAGE (mercaptoethanol added). ). The results are shown in Figure 1.

 It will be understood that r UEL 1 appears around 31 kDa as in the refined UEA-1. As described in Example 9, both 1 "11 £ 1 ^ 1 and purified 11 £ A-I were denatured (reductive cleavage of disulfide bond) in mercaptoethanol-added cascade SDS-PAGE, resulting in 2 volumes. It is thought that the body was divided into two bands.

The eluate was dialyzed overnight in distilled water and then freeze-dried to obtain purified recombinant UEL1 protein crystals. 300 ml saturated BY—from 2 cell culture Ultimately about 3.6 mg of purified recombinant UEL 1 (r UEL l)

was gotten. Example 9: Analysis of recombinant UEL1 protein

 (1) Hemagglutination activity

 To measure the hemagglutination activity, UEL 1 protein purified from BY-2 and purified UEA-I (Sigma) were dissolved in PBS buffer to a concentration of 0.02 mg Ζπι 1, Two-fold serial dilution in PBS buffer 2

To 51, 2% human O-type blood cell suspension treated with an equal amount of trypsin was added. Twenty minutes after the blood cells were added and mixed, the presence or absence of aggregation was observed. The results are shown in Table 1. In the table, ++ indicates strong aggregation, ++ indicates moderate aggregation, + indicates aggregation, and-indicates aggregation negative.

 Table 1: Hemagglutination activity

 Note: 1x concentration of 20 g / m 1 for both rUELl and UEA-I

 As shown in the above results, recombinant UEL1 protein (rUEL1) derived from cultured tobacco cells was diluted 128-fold (approximately 0.16 // gZml) into human O-type blood cell suspension treated with trypsin. )), Exhibiting agglutinating activity up to the same level as the UE A-I protein known so far.

 (2) Sugar aggregation inhibition experiment

Based on the agglutinating activity measured in (1) above, the agglutinin titer of purified UE L 1 protein derived from BY-2 and commercially available UEA-I was increased by a factor of 4 (about 0.6 μg / m 1). Diluted with PBS buffer. Diluted lectin solution 2 5 ^ 1 Was added with 25 μl of an aqueous solution of sugar (fucose, galactose, manoletose, lactose, saccharose, salicin, and mannose) diluted to 0.2 M to 0.001 M, mixed, and treated with an equal amount of trypsin. 2% human Ο-type blood cell suspension was further added and mixed. Aggregation activity was determined 20 minutes after mixing the blood cells, and the minimum concentration of sugars that completely inhibited blood cell aggregation (“-” based on the criteria in (1) above) was determined. Table 2 shows the results. Table 2: Inhibition of aggregation by sugar

 As a result of the inhibition experiment with saccharide, neither recombinant UEL 1 nor purified UEA-I was inhibited by saccharides of less than galactose even at 0.2 M, and aggregation was inhibited only by fucose. The minimum concentration of sugar that caused aggregation inhibition was 0.001M for recombinant lectin (rUEL1) and 0.055M for purified lectin.

 (3) Estimation of molecular weight

To estimate the molecular weight, the band of SDS-PAGE under denaturing conditions In addition to estimating the molecular weight from the mobility, acrylamide gel electrophoresis was performed under non-denaturing conditions except for mercaptoethanol from the electrophoresis buffer, and the molecular weight was estimated from the mobility of the band under each electrophoresis condition. SDS-PAGE electrophoresis followed the method of Lae 脑 li (Nature 15, Vol. 227 (259), pp. 680-685 (1970)).

 The results are shown in FIG. In the figure, N indicates non-denaturing conditions (PAGE without mercaptoethanol), and R indicates denaturing conditions (SDS-PAGE with mercaptoethanol).

 In the recombinant UEL1 (E. coli rUELL) derived from Escherichia coli, a single band was detected at a position corresponding to about 30 kDa under both non-denaturing conditions and denaturing conditions. On the other hand, recombinant UEL1 protein (BY-2 r UEL1) derived from cultured tobacco cells has about 53 kDa under non-denaturing conditions and about 32 kDa and 33 kDa under denaturing conditions. Was detected. The purified UEA-I protein showed two bands at about 50 kDa under non-denaturing conditions, and at about 32 kDa and 33 kDa under denaturing conditions. From these results, the recombinant UEL1 protein derived from cultured tobacco BY-2 cells shows the same electrophoresis image as the purified UEA-I under denaturing conditions, and a slight difference in the apparent molecular weight under non-denaturing conditions is observed. However, it was suggested that it may form a dimer like UEA-I. In addition, due to the difference in behavior under non-denaturing conditions, UEL1 and UEA-I are expected to have substantially the same three-dimensional structure of the subunits, but differ in the interaction between subunits due to amino acid substitution. It is thought that it is. Industrial applicability

In this study, we obtained a recombinant UEL 1 lectin having the same activity as UE A-I lectin extracted from P. chinensis seeds. Detailed analysis of the structure of UEL-1 lectin and the mechanism of binding to sugar chains using the recombinant protein expression system, for example, research on the effect of modifying the amino acid sequence on the binding activity to sugar chains has become possible. It is expected to contribute to research on the reaction mechanism of anti-H lectin.

 The UEA-I protein is also widely used as a blood grouping marker and as a marker for diseases such as cancer and Crohn's disease. Research using recombinant lectin is not limited to analysis of plant lectins, for example, humans Synthesize lectins with modified sugar chain binding sites so that they have specificity for any blood group antigen, and create lectins that can determine blood-type subtypes, or create new tumor markers This makes it possible to study the expression and structure of carbohydrate antigens in humans.

 By expressing the lectin gene of the present invention in host cells, a lectin protein that reacts with human H antigen can be produced with high purity and efficiency. In addition, it is possible to induce a previously unknown lectin and to obtain a lectin with higher anti-H agglutinating activity than a known lectin. Blood type determination can also be expected.

Further, in the present invention, about 4 mg of purified UEL1 protein was obtained from 300 ml of the BY-2 cell culture solution. Approximately 44 mg of purified lectin can be obtained from 1 kg of seeds when extracted from gorse fern by affinity chromatography.However, extraction from seeds is hard, and crushing is required to break the seed coat. While BY-2 cells can be easily crushed by adding a surfactant, the yield and titer depend on the individual and the growth stage of seeds when extracting from living organisms. Although it is likely to change, BY-2 cells are a homogeneous cell population, so that both yield and titer can be obtained stably, and the production of recombinant anti-H lectin is an extremely useful method from the viewpoint of substance production. Is Conceivable.

Claims

The scope of the claims 1. A lectin protein exhibiting anti-H aggregating activity, comprising the nucleotide sequence of SEQ ID NO: 1, its complementary sequence, or the nucleotide sequence in which some of the codons constituting these sequences are deleted or substituted. Gene to encode. 2. Among the base sequences described in SEQ ID NO: 1, base sequences 31 to 858, their complementary sequences, or other sequences so that the amino acid sequence does not differ for one or more codons of these sequences. 2. The gene according to claim 1, comprising a sequence replaced with a codon. 3. A protein comprising the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence in which one or more amino acids constituting the sequence has been deleted or substituted, and has anti-H aggregation activity. 4. A protein having the amino acid sequence of SEQ ID NO: 2. 5. A recombinant vector incorporating the gene according to claim 1 or 2. 6. Transformation by introducing the recombinant vector according to claim 5 into a host cell, culturing the transformant, and collecting a protein exhibiting anti-H aggregation activity from the culture, exhibiting anti-H aggregation activity. A method for producing a protein. 7. The recombinant vector is a plasmid, which is transformed by introducing the plasmid into agrobacterium, infecting plant cells with the transformed agrobacterium, and exhibiting anti-H aggregation activity from the infected plant cells. Claims for collecting proteins The method described in Box 6. 8. The method according to claim 7, wherein the plant cells are cultured tobacco cells.