JPWO2002077238A1 - Gene encoding a lectin protein derived from gorse, protein encoded by the gene, and method for producing the same - Google Patents

Abstract

(1) a gene encoding a novel lectin exhibiting anti-H aggregating activity (UEL1 gene) isolated from the genomic DNA of P. fern and a gene consisting of a nucleotide sequence substantially equivalent thereto, (2) those genes (3) a recombinant vector containing the gene, and (4) the recombinant vector is introduced into a host cell for transformation, the transformant is cultured, and the transformant is cultured. A method for producing a protein exhibiting anti-H agglutinating activity by collecting a polypeptide exhibiting H agglutinating activity.

Description

上記の結果に示されるように、タバコ培養細胞由来のリコンビナントＵＥＬ１タンパク質（ｒＵＥＬ１）はトリプシンで処理したヒトＯ型血球浮遊液に対し、１２８倍希釈（約０．１６μｇ／ｍｌ）まで凝集活性を示し、その強さは従来既知のＵＥＡ−Ｉタンパク質と同程度またはそれ以上である。
（２）糖による凝集阻害実験
ＢＹ−２由来の精製ＵＥＬ１タンパク質、および市販の精製ＵＥＡ−Ｉを上記（１）で測定した凝集活性に基づいて凝集素価が４倍（約０．６μｇ／ｍｌ）となるようにＰＢＳ緩衝液で希釈した。希釈したレクチン溶液２５μｌに、０．２Ｍから０．００１Ｍになるように希釈した糖（フコース、ガラクトース、マルトース、ラクトース、サッカロース、サリシン、およびマンノース）水溶液２５μｌを加えて混合した後、等量のトリプシンで処理した２％ヒトＯ型血球浮遊液をさらに加えて混合した。血球を混合してから２０分後に凝集活性の判定を行い、血球の凝集を完全に阻害（上記（１）の基準で「−」）する糖の最小濃度を求めた。結果を表２に示す。

糖による阻害実験の結果、リコンビナントＵＥＬ１、精製ＵＥＡ−Ｉのいずれも、ガラクトース以下の糖では０．２Ｍでも阻害が認められず、フコースによってのみ凝集が阻害が認められた。凝集阻害が起こる糖の最小濃度は、リコンビナントレクチン（ｒＵＥＬ１）では０．００１Ｍ、精製レクチンでは０．００５Ｍであった。
（３）分子量の推定
分子量の推定には、変性条件下でのＳＤＳ−ＰＡＧＥ電気泳動のバンドの移動度から分子量を推定すると共に、泳動緩衝液からメルカプトエタノールを除いた非変性条件下でのアクリルアミドゲル電気泳動を行い、それぞれの泳動条件下でのバンドの移動度から分子量の推定を行った。ＳＤＳ−ＰＡＧＥ電気泳動はＬａｅｍｍｌｉの方法（Ｎａｔｕｒｅ １５，Ｖｏｌ．２２７（２５９），ｐｐ．６８０−６８５（１９７０））に従った。
結果を図２に示した。図中、Ｎは非変性条件（メルカプトエタノール非添加ＰＡＧＥ）、Ｒは変性条件（メルカプトエタノール添加ＳＤＳ−ＰＡＧＥ）を示す。
大腸菌由来のリコンビナントＵＥＬ１（Ｅ．Ｃｏｌｉ ｒＵＥＬ１）は非変性条件下、変性条件下のいずれでも約３０ｋＤａに相当する位置に単一のバンドが検出された。一方、タバコ培養細胞由来のリコンビナントＵＥＬ１タンパク質（ＢＹ−２ ｒＵＥＬ１）は非変性条件下では約５３ｋＤａ、変性条件下では約３２ｋＤａと３３ｋＤａに２本のバンドが検出された。精製ＵＥＡ−Ｉタンパク質は、非変性条件下では約５０ｋＤａ、変性条件下では約３２ｋＤａと３３ｋＤａに２本のバンドが見られた。これらの結果から、タバコＢＹ−２培養細胞由来のリコンビナントＵＥＬ１タンパク質は、変性条件下では精製ＵＥＡ−Ｉと同様の泳動像を示し、非変性条件下では、みかけの分子量において若干の違いは認められるが、ＵＥＡ−Ｉと同様に２量体を形成している可能性が示唆された。また、非変性条件下における挙動の違いから、ＵＥＬ１とＵＥＡ−Ｉとはサブユニットの立体構造はほぼ同じであると予想されるものの、アミノ酸の置換によって、サブユニット同士の相互作用において異なっていると考えられる。
産業上の利用可能性
本研究においてハリエニシダ種子から抽出されたＵＥＡ−Ｉレクチンと同様の活性を有するリコンビナントＵＥＬ１レクチンが得られたことにより、リコンビナントタンパク質の発現系を利用したＵＥＬ１レクチンの構造や糖鎖との結合機構の詳細な解析、例えばアミノ酸配列を改変して糖鎖との結合活性に及ぼす影響を調べる等の研究が可能になり、抗Ｈレクチンの反応機構に関する研究に寄与することが期待される。
また、ＵＥＡ−Ｉタンパク質は、血液型判定や、癌やクローン病などの病気のマーカーとしても広く用いられており、リコンビナントレクチンを用いた研究は、植物レクチンの解析に限らず、例えば、ヒトの任意の血液型抗原に特異性をもつように糖鎖結合部位の構造を改変したレクチンを合成して血液型の亜型も判定できるレクチンを作成したり、新たな腫瘍マーカーを作成する等の研究が可能になり、ヒトにおける糖鎖抗原の発現や構造の解析においても有用であると考えられる。
本発明のレクチン遺伝子を宿主細胞において発現させることによりヒトのＨ抗原に反応するレクチンタンパク質を高純度かつ効率的に製造することができる。また、従来未知のレクチンを誘導すること、既知のレクチンよりも抗Ｈ凝集活性の高いレクチンを得ることも可能となり、ひいてはＡＢＯ型血液におけるＯ型判定の感度の向上等を通じて微少量の試料での血液型判定も期待できる。
さらに、本発明では、３００ｍｌのＢＹ−２細胞培養液から約４ｍｇの精製ＵＥＬ１タンパク質が得られた。ハリエニシダ種子からアフィニティークロマトグラフィーによって抽出した場合には、１ｋｇの種子から約４４ｍｇの精製レクチンが得られるが、種子からの抽出は硬い種皮を壊すための粉砕等の機器、操作が必要であるのに対し、ＢＹ−２細胞は界面活性剤を加えることによって容易に破砕でき、また生体からの抽出には個体間、また種子の成長段階によって収量や力価が変わる可能性が高いが、ＢＹ−２細胞は均一な細胞集団であるため収量、力価とも安定して得られ、物質生産の側面から見てもリコンビナント抗Ｈレクチンの製造法はきわめて有用な方法であると考えられる。
【配列表】

【図面の簡単な説明】
図１は、本発明によるリコンビナントレクチン（フコースアガロースゲル画分ｅ１〜ｅ６）と従来既知のハリエニシダレクチン（ＵＥＡ−Ｉ）のＳＤＳ−ＰＡＧＥ像を示す写真である。
図２は、Ｅ．ｃｏｌｉから得た本発明のリコンビナントレクチン（Ｅ．ｃｏｌｉ ＵＥＬ１）、タバコＢＹ−２細胞から得た本発明のリコンビナントレクチン（ＢＹ−２ ＵＥＬ１）及び従来既知のハリエニシダレクチン（ＵＥＡ−Ｉ）の非変性ＰＡＧＥ（図中、Ｎ）と変性ＳＤＳ−ＰＡＧＥ（図中、Ｒ）の結果を対比して示した写真である。Technical field
The present invention relates to a gorse (scientific name: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 a specific sugar structure and bind to a sugar chain portion of a glycoconjugate (glycoprotein or glycolipid) in a cell membrane to exert an effect such as cell aggregation. Among them, the lectin contained in the crude extract of gorse fern has the property of specifically reacting with human H antigen (antigen present on O-type blood cell membrane and the like) (anti-H aggregation activity). Utilizing this property, the lectin in the seeds of Pseudomonas aeruginosa is used as an O-type hemagglutinin in the ABO blood type determination.
Up to now, three types of proteins (UEA-I, UEA-II, UEA-III) have been isolated and purified by affinity chromatography as lectins obtained from the gorse seed. Among them, the entire amino acid sequence has been reported for UEA-I and UEA-II (J. Biochem. 109 (1991) p650-658), and the partial amino acid sequence for UEA-III has been reported (Biol. Chem.). Hoppe-Seyler 372 (1991) p95-102). However, it is not known whether the gorse contains lectins different from the above three types, and the gene sequence encoding any of UEA-I, UEA-II and UEA-III has not been known. unknown.
Therefore, 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, elucidating a gene sequence encoding P. lectin.
Disclosure of the invention
In order to solve the above-mentioned problems, the present inventors have isolated a novel lectin-encoding gene (UEL1 gene) from the genomic DNA of P. chinensis and determined its nucleotide sequence. Further, the present invention has completed the present invention by incorporating a UEL1 gene into a vector, transforming a host cell with the vector, and using the vector to produce a novel lectin.
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 exhibiting anti-H aggregation activity by genetic engineering using the gene.
(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) Of the nucleotide sequence of SEQ ID NO: 1, nucleotides 31 to 858, their complementary sequences, or one or more codons of these sequences are substituted with other codons so that the amino acid sequence is not different. 2. The gene according to the above 1, which comprises a modified sequence.
(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 described in 1 or 2 above.
(6) A protein exhibiting anti-H agglutinating activity, wherein the recombinant vector according to the above (5) is introduced into a host cell for transformation, the transformant is cultured, and a protein exhibiting an anti-H agglutinating activity is collected from the culture. Manufacturing method.
(7) The recombinant vector is a plasmid, the plasmid is introduced into Agrobacterium for transformation, plant cells are infected with the transformed Agrobacterium, and a protein exhibiting anti-H aggregation activity is collected from the infected plant cells. 7. The method according to 6 above,
(8) The method according to the above item 7, wherein the plant cell is a cultured tobacco cell.
DETAILED DESCRIPTION OF THE INVENTION
(A) Lectin-encoding gene
The lectin-encoding gene of the present invention designs an oligonucleotide probe based on the amino acid sequence of a known gorse lectin (UEA-I), applies this to a gorse genomic DNA, and obtains the internal sequence of the gene by PCR, The gene of SEQ ID NO: 1 obtained by designing a new primer based on the result as a full-length nucleotide sequence 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) a complementary sequence of the base sequence of 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) a base sequence obtained by further adding a codon to the above (1) to (3)
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,
(A) a base sequence in which codons are substituted so that amino acid sequences are not different, 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, hybridization is performed at a temperature of about 5 ° C. to about 30 ° C., preferably about 10 ° C. to about 25 ° C. lower than the melting temperature (Tm) of the complete hybrid. It means when it happens.
In the nucleotide sequence of SEQ ID NO: 1, the nucleotide sequence of the 31st to 858th is a region encoding the lectin protein (polypeptide) of the present invention. Therefore,
(I) the nucleotide sequence of positions 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 gene product containing a DNA base sequence that hybridizes to the above sequence under stringent conditions.
In the present invention, a method of amplifying genomic DNA by designing primers based on the amino acid sequence and combining an appropriate PCR method is employed. Can be obtained. For example, (1) a cDNA library is synthesized from poly (A) + RNA extracted from Pseudomonas fern seeds, and an oligonucleotide probe or a corresponding antibody synthesized based on an amino acid sequence of a known lectin (UEA-I) or the like is used. A method for screening a cDNA library, (2) a primer is designed based on a known amino acid sequence, DNA or cDNA of P. chinensis is amplified by an appropriate PCR method, and the resulting PCR product is used as a probe for a cDNA library or P. genus It can be obtained by a method for screening a library or the like. The nucleotide sequence of the DNA thus obtained may be analyzed by the dideoxy chain terminator method (proc. Natl. Acad. Sci. USA 74, 5463-5467 (1977) Sanger, F. et al.) Or the like. Can be.
(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.
The deletion, substitution and addition of an amino acid sequence correspond to the 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 sequence considered to be related to anti-H agglutinin activity has a 10A-position which is located at positions 128 to 137 in UEA-I lectin. The amino acid "Asp Thr Ile Gly Ser Pro Val Asn Phe Trp" is considered to be a sugar-binding site, and this site is a sequence that is relatively conserved in other legume lectins (J. Biochem). .111 (1992) p.436-439).
In UEL1 of the present invention, this portion corresponds to the 154th to 163rd 10 amino acids “Asp Thr Ile Gly Ser Pro Val Asn Ser Trp”, and 9 amino acids out of 10 amino acids are the same as UEA-I. In the base sequence of UEL1, this portion corresponds to 490 to 519 bases.
(C) Method for producing lectin
The lectin of the present invention can be synthesized based on the above amino acid sequence, but can be produced by genetic engineering techniques based on the base sequence obtained above. 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 of the gene of the present invention, or may be expressed as a fusion protein with another protein. Further, the full length of the gene described in the above (A) may be expressed, or a part thereof may be expressed.
Conventional host-vector systems used for the production of proteins can be used. For example, an Escherichia coli expression system can be used, including a pET vector system (Novagen), PinPointXa, pGEMEX, pET-5 (Promega (E), which contains an Escherichia coli K12 strain or a mutant thereof and an expression vector. Promega), pGEX-5X (Pharmacia) and the like.
Further, for example, pPIC3K (manufactured by Invitrogen), which is an E. coli / yeast shuttle vector, is used.Pichia  pastorisPichia Expression Kit (Invitrogen) which is a yeast expression system using a yeast as a host, a BacVector system (Novagen) which is an animal cell expression system, and a rapid translation system RTS500 (Rapid Translation System RTS500) which is a cell-free expression system. ) (Roche) can be used. Lectin can also be expressed by incorporating it into a vector such as pBI101 or pBI121 (Clonetech) and introducing it into a plant such as tobacco using a transformation method using Agrobacterium.
The host cells are 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.
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 (approximately 50 mg) of the gorse young plant was frozen in liquid nitrogen, pulverized using a mortar and a pestle, and 0.6 ml of a CTAB buffer (2% cetyltrimethylammonium bromide, Tris-HCl (0.1 M , PH 8), EDTA (0.02 M, pH 8), sodium chloride (1.4 M)), stirred, and heated at 65 ° C. for 30 minutes. Thereafter, 0.7 ml of a mixed solution of chloroform / isoamyl alcohol (24/1) was added thereto, further stirred, and the resulting solution was centrifuged. To the supernatant obtained by centrifugation, 0.9 ml of a CTAB buffer solution for precipitation (1% cetyltrimethylammonium bromide, Tris-HCl (0.05 M, pH 8), EDTA (0.01 M, pH 8)) was added, followed by stirring. Centrifuged again. The precipitate obtained by centrifugation was dissolved in a buffer solution (0.01 M Tris-HCl, EDTA (1 mM, pH 8)), followed by ethanol precipitation to obtain purified genomic DNA.
Example 2: Synthesis of degenerate primers and amplification of DNA by PCR
Degenerate primers UEA-F3 (SEQ ID NO: 3) and UEA-R4 (SEQ ID NO: 4) were synthesized based on the known amino acid sequence of UEA-I lectin. 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 was carried out using primers (UEA-F3 and UEA-R4) with the gorse genomic DNA obtained in Example 1 as a template. The reaction conditions were denaturation (94 ° C., 1 minute), annealing (51 ° C., 1 minute), and elongation (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 a pGEM-T Easy vector system (manufactured by Promega), and the vector was introduced into competent Escherichia coli XL1-Blue strain (Stratagene). The cells were cultured overnight on LB plates containing ampicillin (50 μg / ml). Escherichia coli grown on the medium was cultured, and the plasmid was extracted and purified from the cultured cells using the alkali-SDS method.
The DNA sequence inserted into the purified plasmid was M13 forward, reverse primer (Nippon Gene), Big Dye Terminator Cycle Sequencing Reaction Kit (ABI), ABI PRIZM310 Genetic Analyzer, a Big Dye Terminator Cycle Sequencing reaction kit (ABI). The base sequence was determined using (Genetic Analyzer) (manufactured by ABI). The amino acid sequence deduced from this DNA sequence showed extremely high homology with the amino acid sequence of the UEA-I protein. Therefore, the DNA sequence is considered to be a partial sequence inside the gene encoding lectin.
Example 4: DNA sequence extension
To obtain a full-length DNA sequence encoding a lectin protein, the partial sequence was extended by tail-PCR. 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 (UEA44F (SEQ ID NO: 6) )). Using these synthetic primers and a commercially available random primer DNA oligomer (12) set A (manufactured by Nippon Gene Co., Ltd.), a PCR reaction was performed according to a tail-PCR method (Shujunsha, PCR experiment protocol for plants, p83-89). went. The obtained PCR product was cloned 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 an amino acid sequence deduced from its nucleotide sequence showing extremely high homology with the portion containing the N-terminus of the UEA-I protein. The amino acid sequence corresponding to the downstream side showed extremely high homology with the portion including the C-terminus of the UEA-I protein. Thus, these PCR products are thought to encode upstream and downstream of the lectin protein.
Example 5: Synthesis of full length gene encoding lectin protein
To synthesize a full-length gene containing both the partial sequence inside the gene obtained in Example 3 and the upstream and downstream sequences obtained in Example 4, the sequence obtained in Example 4 was used. Then, primers (UEA-F (SEQ ID NO: 7) and UEA-R (SEQ ID NO: 8)) were synthesized to amplify the genomic DNA of P. niger by PCR. When the nucleotide sequence of the obtained PCR product was analyzed, the DNA sequence represented by SEQ ID NO: 1 was obtained. The gene having this DNA sequence was named UEL1 gene.
The UEL1 gene has the start codon ATG at bases 31 to 33 and the 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-1 lectin protein. Further, when this amino acid sequence was homology-searched using a database of the National Center for Biotechnology Information (USA) on the Internet,Cytisus  sessilifoliusLectins,Maackia  amurensisIt has homology (50% -70%) with many legume lectins such as lectins and is considered to be a novel lectin.
Example 6: Method for producing a novel lectin protein by transformed Escherichia coli
Expression of the recombinant UEL1 lectin in E. coli was performed using the E. coli expression system pET-32 Xa / LIC vector system (Novagen).
A primer containing the start codon of the UEL1 gene (UEL1 / LIC-F (SEQ ID NO: 9)) and a primer containing a stop codon (UEL1 / LIC-R (SEQ ID NO: 10)) were designed and synthesized according to the kit protocol. Using this, the UEL1 gene was synthesized by the PCR method, incorporated into the pET32 vector, and the vector DNA was introduced into competent Escherichia coli (Novablue Singles (Novagen)), and the DNA was collected on an LB plate containing 50 μg / ml ampicillin. Cultured overnight. 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% bactotryptone, 0.5% bactoeast extract, 0.5% sodium chloride) containing 1M isopropyl-β-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 using a denaturing SDS-polyacrylamide gel (SDS-PAGE), a protein having a molecular weight of about 50 kDa was obtained.
The recombinant protein was approximately 20 kDa larger than the expected molecular weight of the UEL1 protein, presumably because it was expressed as a fusion protein with thioredoxin encoded by the vector. The lectin in the recombinant protein was subjected to protease treatment using Factor Xa (Boehringer Mannheim), and then purified and separated by cutting thioredoxin. The molecular weight of the lectin moiety after cleavage was about 30 kDa, which was the same as the estimated molecular weight of the UEL1 protein.
Example 7: Expression of UEL1 protein by cultured tobacco cells
(1) Plasmid construction and Agrobacterium transformation
PCR was performed on the full-length coding region of the UEL1 gene using the UEL1 / Xba-F primer: tgcaaattctagaaaagtgagtc (SEQ ID NO: 11) and the UEL / Sac-R primer: aagtagagctccaaatcatgcag (SEQ ID NO: 12), followed by restriction enzymes NdeI and Xh. And ligated to the vector pBI121 (Clontech) digested with XbaI and SacI, and introduced into competent Escherichia coli XL-1 Blue strain to obtain a plasmid DNA (pre-UEL1) from a transformant obtained. -PBI) was extracted. Agrobacterium C58c1PMP90 strain was transformed with the pre-UEL1-pBI plasmid by electroporation. The transformed Agrobacterium is grown on an LB solid medium containing 50 mg / L kanamycin, and an Agrobacterium colony having the UEL1 gene is cultured overnight in an LB liquid medium containing 50 mg / L kanamycin, tobacco culture. Used to infect cells.
(2) Transformation of tobacco BY-2 cultured cells
The transformation of the tobacco cultured cell BY-2 was carried out according to the method described in "Experimental protocol for observing plant cells" (Hiroyuki Fukuda et al., Shujunsha (1997), pp. 125-129). This was performed by infecting BY-2 cultured cells. Transformed BY-2 calli were selected and grown on a BY-2 solid medium supplemented with kanamycin and carbenicillin.
A portion of each of the transgenic (transformed) BY-2 calli (35 clones) grown on the selection medium was taken, added to a PBS buffer (phosphate-buffered saline), and ground in an Eppendorf tube at 15,000 rpm. After centrifugation at × 5 minutes (4 ° C.), 3% (v / v) fresh human treated by adding 0.01% (w / v) trypsin and treating at 37 ° C. for 1 hour using a part of the supernatant A physiological saline suspension of 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. Moreover, it was shown that 10 clones weakly aggregated (+) human O-type blood cells, and active UEL1 protein was synthesized in these cells.
Example 8: Purification of recombinant lectin from cultured tobacco BY-2 cells
The recombinant UEL1 lectin (rUEL1) derived from tobacco cultured cells was purified by the following procedure.
In Example 7, 18 clones of transgenic calli which exhibited strong agglutinating activity on human O-type blood cells were selected, swirled in a BY-2 liquid medium supplemented with kanamycin and carbenicillin for 2 weeks, and almost saturated. The reached BY-2 cultured cells were centrifuged at 8000 rpm × 30 minutes (4 ° C.) to separate a culture supernatant and a cell fraction. The cell fraction was frozen at −80 ° C., then 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. After dissolution, the supernatant obtained by centrifugation at 8,000 rpm × 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 cell supernatant after dialysis was filtered through a filter paper (Toyo Filter Paper No. 2) to remove solids, and then passed through a column packed with 10 ml of fucose-agarose (Seikagaku Corporation), and the UEL1 protein in the cell supernatant was removed. Was adsorbed. After washing the column after binding with a PBS buffer, the adsorbed fucose-bound fraction was eluted with a 20 mM diaminopropane (DAP) solution. A part of the eluted fractions (e1 to e6) is subjected to SDS-PAGE (addition of mercaptoethanol) together with cell supernatant (SUP), column passing solution (ft), and purified UEA-I (Sigma). Was. The results are shown in FIG.
It will be appreciated that rUEL1 appears around 31 kDa, similar to purified UEA-1. As described in Example 9, in both rUEL1 and purified UEA-I, the dimer is separated into two bands due to denaturation (reductive cleavage of disulfide bonds) in SDS-PAGE with addition of mercaptoethanol. It is considered.
The eluate was dialyzed overnight in distilled water and then freeze-dried to obtain purified recombinant UEL1 protein crystals. About 3.6 mg of purified recombinant UEL1 (rUEL1) protein was finally obtained from 300 ml of saturated BY-2 cell culture.
Example 9: Analysis of recombinant UEL1 protein
(1) Hemagglutination activity
In order to measure the hemagglutination activity, UEL1 protein purified from BY-2 and purified UEA-I (Sigma) were dissolved in a PBS buffer to a concentration of 0.02 mg / ml, and PBS was dissolved in PBS. To 25 μl of a two-fold serial dilution with a buffer, 2% human type O 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. Table 1 shows the results. In the table, +++ indicates strong aggregation, ++ indicates moderate aggregation, + indicates aggregation, and-indicates aggregation negative.

As shown in the above results, the recombinant UEL1 protein (rUEL1) derived from cultured tobacco cells showed agglutinating activity in human O-type blood cell suspension treated with trypsin up to 128-fold dilution (about 0.16 μg / ml). , Its strength is equal to or higher than that of the conventionally known UEA-I protein.
(2) Aggregation inhibition experiment with sugar
The PBS buffer solution was used so that the purified UEL1 protein derived from BY-2 and the commercially available purified UEA-I had an agglutination titer of 4 times (about 0.6 μg / ml) based on the agglutination activity measured in (1) above. Diluted. To 25 μl of the diluted lectin solution, 25 μl of a sugar (fucose, galactose, maltose, lactose, saccharose, salicin, and mannose) aqueous solution diluted to 0.2 M to 0.001 M was added, mixed, and then mixed with an equal amount of trypsin. Was further added and mixed. Twenty minutes after mixing the blood cells, the agglutinating activity was determined, and the minimum concentration of the saccharide which completely inhibited the blood cell aggregation ("-" based on the above (1)) was determined. Table 2 shows the results.

As a result of the inhibition experiment with the saccharide, neither the recombinant UEL1 nor the purified UEA-I was inhibited by the sugar of less than galactose even at 0.2 M, and the inhibition was observed only by the fucose. The minimum concentration of saccharide at which aggregation inhibition occurred was 0.001M for recombinant lectin (rUEL1) and 0.005M for purified lectin.
(3) Estimation of molecular weight
The molecular weight was estimated by estimating the molecular weight from the mobility of the band in SDS-PAGE electrophoresis under denaturing conditions, and performing acrylamide gel electrophoresis under non-denaturing conditions by removing mercaptoethanol from the electrophoresis buffer. The molecular weight was estimated from the mobility of the band under each electrophoresis condition. SDS-PAGE electrophoresis followed the method of Laemmli (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 addition of mercaptoethanol) and R indicates denaturation conditions (SDS-PAGE with addition of mercaptoethanol).
In E. coli-derived recombinant UEL1 (E. coli rUEL1), 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, in the recombinant UEL1 protein (BY-2 rUEL1) derived from cultured tobacco cells, two bands were detected at about 53 kDa under non-denaturing conditions and at about 32 kDa and 33 kDa under denaturing conditions. 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 a migration image similar to that of purified UEA-I under denaturing conditions, and a slight difference in the apparent molecular weight is observed under non-denaturing conditions. However, it was suggested that dimer may be formed similarly to UEA-I. Further, from the difference in behavior under non-denaturing conditions, UEL1 and UEA-I are expected to have substantially the same three-dimensional structure of subunits, but differ in the interaction between subunits due to amino acid substitution. it is conceivable that.
Industrial applicability
In this study, a recombinant UEL1 lectin having the same activity as that of UEA-I lectin extracted from Pseudomonas fern seeds was obtained, so that the details of the structure of UEL1 lectin and the binding mechanism to sugar chains using a recombinant protein expression system were obtained. It is possible to carry out a simple analysis, for example, to study the effect of modifying the amino acid sequence on the binding activity to a sugar chain, and to contribute to a study on the reaction mechanism of anti-H lectin.
UEA-I protein is also widely used as a blood type determination and as a marker for diseases such as cancer and Crohn's disease. Research using recombinant lectin is not limited to the analysis of plant lectins, for example, in humans. Research on the synthesis of lectins with modified sugar chain binding sites so as to have specificity for any blood group antigen to create lectins that can determine blood group subtypes, and to create new tumor markers This is considered to be useful in the analysis of the expression and structure of sugar chain 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 efficiently produced with high purity. In addition, it is possible to induce a previously unknown lectin and to obtain a lectin having a higher anti-H agglutinating activity than a known lectin. Blood type determination can also be expected.
Furthermore, in the present invention, about 4 mg of purified UEL1 protein was obtained from 300 ml of BY-2 cell culture solution. When 44 g of purified lectin is obtained from 1 kg of seeds when affinity chromatography is performed on a gorse seed, extraction from the seeds requires equipment and operations such as pulverization for breaking a hard seed coat. On the other hand, BY-2 cells can be easily crushed by adding a surfactant, and for extraction from a living body, the yield and titer are likely to vary between individuals and depending on the stage of seed growth. Since the cells are a uniform cell population, the yield and titer can be obtained stably, and the production method of the recombinant anti-H lectin is considered to be an extremely useful method from the viewpoint of substance production.
[Sequence list]

[Brief description of the drawings]
FIG. 1 is a photograph showing an SDS-PAGE image of recombinant lectin (fucose agarose gel fractions e1 to e6) according to the present invention and a conventionally known gorse lectin (UEA-I).
FIG. Non-denatured PAGE of the recombinant lectin of the present invention (E. coli UEL1) obtained from E. coli, the recombinant lectin of the present invention (BY-2 UEL1) obtained from tobacco BY-2 cells, and the conventionally known gorse lectin (UEA-I). It is the photograph which showed and compared the result of (N in a figure) and denaturation SDS-PAGE (R in a figure).

Claims (8)

A gene encoding 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. . A nucleotide sequence of nucleotides 31 to 858 of the nucleotide sequence of SEQ ID NO: 1, a complementary sequence thereof, or a sequence in which one or more codons of these sequences have been replaced with other codons so that the amino acid sequence is not different The gene according to claim 1, comprising the gene. 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 have been deleted or substituted, and has anti-H aggregation activity. A protein having the amino acid sequence of SEQ ID NO: 2. A recombinant vector incorporating the gene according to claim 1 or 2. A recombinant vector according to claim 5, which is introduced into a host cell for transformation, the transformant is cultured, and a protein exhibiting anti-H agglutinating activity is collected from the culture. Production method. The recombinant vector is a plasmid, the plasmid is introduced into Agrobacterium for transformation, plant cells are infected with the transformed Agrobacterium, and a protein exhibiting anti-H aggregation activity is collected from the infected plant cells. The method of range 6. The method according to claim 7, wherein the plant cells are cultured tobacco cells.

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