JP2000191695A - New lectin-like protein and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new lectin-like protein contained in a liquid of a fibrovascular bundle of a plant, having a specific lectin-like activity, suitable for an antimicrobial agent, etc. SOLUTION: This lectin-like protein is contained in a liquid of a fibrovascular bundle of a cucurbitaceous plant. The lectin-like protein is (A) a protein composed of an amino acid sequence of the formula or (B) a protein composed of an amino acid sequence in which one or plural amino acids are added, deleted or substituted in the amino acid sequence of the formula and which has hemagglutination activity. A gene encoding the lectin-like protein is inserted into a vector DNA to give a recombinant DNA. A transformant containing the recombinant DNA is prepared. The transformant is cultured in a medium and the lectin-like protein is collected from the culture product to preferably produce the lectin-like protein. Preferably an antimicrobial agent containing the lectin-like protein as an active ingredient is prepared.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel lectin-like protein, a gene for the protein, and a recombinant DN containing the gene.
A, a transformant containing the recombinant DNA and a method for producing a lectin-like protein using the transformant. Further, the present invention relates to an antibacterial agent comprising the protein as an active ingredient.

[0002]

2. Description of the Related Art Proteins that recognize and bind to a specific sugar chain structure are collectively called lectins. For the first time in 1888,
Lectin was discovered in castor bean as a protein that aggregates animal red blood cells. A sugar chain that determines the blood type is bound to the erythrocyte membrane surface, and the lectin binds to this to agglutinate the red blood cells. Lectins are found not only in plant fruits and seeds, but also in influenza virus surface antigens and animal livers, and play various physiological functions. The main physiological actions are (1) agglutination of red blood cells in a blood group-specific manner,
(2) specifically aggregating only certain types of cancer cells,
(3) It promotes lymphocyte division, (4) shows toxicity to cells, and the like, and its application has attracted attention in the pharmaceutical field. A large number of plant-derived lectin proteins are found not only in the seeds of leguminous plants, but also in wheat, pokeweed, potato, and datura, but more lectins will be discovered in the future. Is expected.

[0003]

An object of the present invention is to provide a novel lectin-like protein and an antibacterial agent containing the protein as an active ingredient. Furthermore, an object of the present invention is to provide a novel recombinant D prepared using genetic engineering techniques.
An object of the present invention is to provide a production method for efficiently producing the lectin-like protein using a transformant containing NA and the recombinant DNA.

[0004]

Means for Solving the Problems As a result of intensive studies for solving the above problems, the present inventor has found, for the first time, a protein having a new hemagglutinating activity from the vascular fluid of Cucurbitaceae plants. Then, the protein was isolated, and cloning of the protein gene and analysis of the nucleotide sequence were successful. Furthermore, a novel recombinant DNA for efficiently producing the protein and a transformant containing the recombinant DNA were prepared using genetic engineering techniques, thereby completing the present invention.

That is, the present invention provides (1) a lectin-like protein which is contained in a vascular fluid. (2) A lectin-like protein characterized in that the lectin-like protein according to (1) is a protein derived from a Cucurbitaceae plant. (3) The following lectin-like protein of (a) or (b). (A) a protein consisting of the amino acid sequence of SEQ ID NO: 1; (B) a protein having hemagglutinating activity consisting of an amino acid sequence in which one or more amino acids have been added, deleted or substituted in the amino acid sequence of SEQ ID NO: 1; (4) A gene encoding the lectin-like protein according to any one of (1) to (3). (5) A recombinant DNA obtained by inserting a gene encoding the lectin-like protein according to (4) into vector DNA. (6) A transformant comprising the recombinant DNA according to (5). (7) culturing the transformant according to (6) in a medium, and obtaining the lectin-like protein according to any one of (1) to (3) from the culture. A method for producing a characteristic lectin-like protein. (8) An antibacterial agent comprising the lectin-like protein according to any one of (1) to (3) as an active ingredient.

[0006]

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel lectin-like protein derived from the vascular fluid of a plant. The vascular bundle of a plant is mainly composed of xylem (conduit, pseudoconduit) and phloem (phloem), and transports inorganic ions and various nutrients to other organs by the vascular fluid contained therein. An organization that has the function of performing The vascular fluid of the present invention refers to a liquid contained in the vascular bundle,
"Lectin-like protein" refers to a protein having hemagglutinating activity.

[0007] The lectin-like protein of the present invention may be any protein as long as it is contained in the vascular fluid of a plant and has a hemagglutinating activity. For example, a lectin-like protein derived from the vascular fluid of a Cucurbitaceae plant ,In particular,
(A) a lectin-like protein consisting of the amino acid sequence of SEQ ID NO: 1, and (b) a lectin-like protein consisting of an amino acid sequence in which one or more amino acids have been added, deleted or substituted in the amino acid sequence of SEQ ID NO: 1. Examples include proteins.

[0008] The lectin-like protein of the present invention (hereinafter referred to as the present protein) can be obtained directly from the vascular fluid of a plant, or can be obtained based on the amino acid sequence of the protein of the present invention which has been revealed in the course of the present invention. Although it can be synthesized, it is preferably obtained by a genetic engineering technique using the protein gene of the present invention. That is, a recombinant DNA obtained by inserting a gene encoding the protein of the present invention into vector DNA is appropriately introduced into a host for transformation, the resulting transformant is cultured, and the produced protein of the present invention is cultured. It can be obtained easily and in large quantities from objects. Hereinafter, a method for isolating the protein of the present invention from the vascular fluid of a plant, a method for preparing the gene for the protein of the present invention, and a method for producing the protein of the present invention by genetic engineering techniques, and further, the protein of the present invention as an active ingredient The contained antibacterial agent will be described in detail. [1] Isolation of lectin-like protein from vascular fluid In order to isolate the protein of the present invention, first, a vascular fluid of a plant, for example, a cucurbit plant, specifically, a cucumber, a loofah, a pumpkin or the like is used. get. Any method can be used for obtaining the cucumber (Cucumis).
sativus cv. Shimoshirazu-jibai) can be cut at a distance of 15 cm from the aerial part so as not to include leaves, and the conduit fluid can be collected in a tube in ice to obtain.

Next, in order to purify and isolate the protein of the present invention from the obtained vascular fluid, ordinary protein purification,
Isolation methods can be used. For example, a fractionation method in which ammonium sulfate, alcohol, acetone or the like is added, a gel filtration method using Sephadex, ultragel, biogel, or the like, an adsorption elution method using an ion exchanger, an adsorption elution method using hydroxyapatite, An electrophoresis method using an acrylamide gel, an affinity-chromatography method, a fractionation method using a molecular sieve membrane or a hollow fiber membrane, or the like can be appropriately selected or used in combination. It can also be purified and isolated by FPLC or HPLC. In this case, a Poros HQ column (Nippon Par-
Septic Corporation), Superdex 75HR10 / 30 (Pharmacia) as a gel filtration column for FPLC, TSKgel as a gel filtration column for HPLC
SW2000 (manufactured by Tosoh Corporation), Capsule Pack C18SG300 (manufactured by Shiseido) as a reversed phase column for HPLC
And the like. For the detection of the protein of the present invention, agglutinating activity with various cells and sedimentation activity with polysaccharides and complex saccharides can be used. In the present invention, hemagglutinating activity is used. Thus, the protein of the present invention can be purified and isolated. [2] Preparation of lectin-like protein gene The lectin-like protein gene of the present invention (hereinafter referred to as the gene of the present invention) may be any gene that encodes the protein of the present invention contained in the vascular fluid of a plant. It may be a gene, specifically, for example, a gene containing a gene encoding the protein of the present invention derived from the vascular fluid of a Cucurbitaceae plant,
More specifically, (a) the protein of the present invention comprising the amino acid sequence of SEQ ID NO: 1, (b) one or more amino acids are added to the amino acid sequence of SEQ ID NO: 1,
A gene encoding a mutant protein of the present invention comprising a deleted or substituted amino acid sequence can be mentioned. The gene of the present invention includes not only the gene encoding the amino acid sequence of the protein of the present invention described above, but also all the genes encoding the minimum amino acid sequence necessary for the expression of hemagglutinating activity.

The gene of the present invention can be obtained by cloning a natural gene derived from the DNA or cDNA of a plant having a vascular bundle used in the present invention. Cloning of gene
Examples of the screening method include a method of synthesizing an appropriate probe DNA based on the amino acid sequence of the protein of the present invention, and using the synthesized DNA to screen a plant DNA or cDNA library, or a method of screening. Producing an appropriate primer-DNA based on the amino acid sequence of the invention protein,
Suitable polymer such as 5'RACE method and 3'RACE method
A method comprising amplifying a DNA containing a fragment of the gene of the present invention by the ze-chain reaction (hereinafter referred to as a PCR method) and linking these to obtain a DNA containing a full-length gene, and other known cDNA cloning techniques And the method of obtaining the information.

For example, in the present invention, the gene of the present invention can be obtained as follows. First, a plant, for example, Cucurbitaceae, specifically, mRNA is obtained from the roots of cucumber, luffa, squash, etc., and a double-stranded cDNA is synthesized using an oligo dT primer, and the obtained synthetic DNA is obtained.
Into the EcoRI site of λgt11 to prepare a cDNA library. On the other hand, a rat is immunized with the protein of the present invention to obtain an antiserum. As a result of screening the cDNA library using the obtained antibody, a plasmid containing a positive clone is obtained. The plasmid can be inserted into another plasmid as appropriate using restriction enzymes. Next, the entire nucleotide sequence of the clone obtained by the screening is determined.

Next, a primer is prepared based on the obtained base sequence.
Are synthesized and PCR is performed using these synthetic primers and the cDNA obtained above as a template to obtain DNA encoding a part of the gene of the present invention. Furthermore, the obtained P
PCR is performed by appropriately combining the primers with the CR reaction product as a template to obtain a target gene of the present invention.
The nucleotide sequence of the obtained gene of the present invention is determined as it is or by subcloning into an appropriate plasmid.
The nucleotide sequence can be analyzed using, for example, a 370 DNA sequence system (manufactured by PerkinElmer).

Furthermore, in order to produce a larger amount of the protein of the present invention than in the case of using the gene of the present invention obtained by the above method, a DNA containing the gene of the present invention can be chemically synthesized. For example, the gene is divided into a plurality of oligonucleotides, chemically synthesized, annealed, and ligated by DNA ligase. In this case, the codon to be replaced is a codon that does not affect the amino acid sequence of the protein of the present invention, and is preferably a codon frequently used in the host cell to be used.

As a method for obtaining a gene encoding the mutant type protein of the present invention, there are provided a drug which is a mutagen,
Specifically, hydroxylamine, nitrous acid, sulfurous acid, 5 '
-Bromouracil and the like. In addition, UV irradiation method, cassette mutation method, PCR
Methods such as site-directed mutagenesis using the method can be widely used. Furthermore, it is also possible to construct a gene encoding a mutated protein of the present invention having a mutation at a desired site by annealing chemically synthesized DNA. [3] Preparation of recombinant DNA and transformant The obtained gene of the present invention is usually introduced into a host in the form of a recombinant DNA. Recombinant DNA comprises the gene of the present invention and a vector capable of autonomous replication, and if the target gene is available, it can be prepared relatively easily by ordinary genetic engineering techniques. As a vector into which the gene of the present invention can be inserted, for example, pMAL-C2 (NE
W England Labs), pGEX-5X
-1 (manufactured by Pharmacia), pXal (manufactured by Behringer), pUC119 (manufactured by Takara Shuzo), pBR322 (manufactured by Takara Shuzo), pBluescript SK + (Stra)
plasmid DNA-DN
A, bacteriophage vector DNA such as λEMBL3 (manufactured by Stratagene). These autonomously replicable vectors usually contain a promoter, an enhancer, a replication origin, a transcription termination site, a splicing sequence and / or It suitably comprises a base sequence such as a selection sequence.

For inserting the gene of the present invention into the above-mentioned vector, generally known methods can be used. Specifically, in order to perform the insertion efficiently, first, an appropriate restriction enzyme such as E.
It is desirable to provide a recognition site such as coRI or SalI.

The thus obtained recombinant DNA is appropriately introduced into a host. Eukaryotic cells and prokaryotic cells can be used as host cells for producing the protein of the present invention. Eukaryotic cells include cells of animals, plants, insects, yeast, etc., and prokaryotic cells include E. coli,
Bacillus subtilis, actinomycetes and the like. When a plant or insect cell is used as a host cell, it does not need to be a cultured cell, but may be a plant or insect, specifically a tobacco plant or silkworm larva. In the present invention, as a host, for example, Escherichia coli JM109 (manufactured by Takara Shuzo), XL-
Blue (manufactured by Funakoshi Co., Ltd.) and the like, and in order to efficiently produce the protein of the present invention, it is preferable to use plant cells or plants.

The above-mentioned host is a recombinant DN containing the gene of the present invention.
In order to introduce A and obtain the transformant of the present invention, a general transformation method or a transduction method can be used. Examples of the transformation method include D.I. M. Morri
son method (Methodin Enzymology)
y, 68, 326-331, 1979) and the calcium chloride method (Gene, 6, 23, 1979). As a transduction method, for example, B. Hohn
Method (Method in Enzymology,
68, 299-309, 1979). [4] Production of Lectin-Like Protein Using Transformant The protein of the present invention can be obtained by obtaining a large amount of vascular fluids of plants used, for example, Cucurbitaceae plants, specifically cucumber, loofah, pumpkin and the like. However, by using the above-mentioned transformant of the present invention, it can be obtained more efficiently. In order to produce the protein of the present invention using the transformant, first, the transformant is cultured. For the culture, for example, a method for culturing the host cell used is generally used.
As a collection method, an ordinary protein isolation method can be used. That is, the transformant is destroyed by a lysis treatment using an enzyme such as lysozyme, an ultrasonic crushing treatment, a grinding treatment, or the like, and the protein of the present invention is excreted outside the body. Then, the protein of the present invention having a degree of purification according to the purpose and application is obtained by appropriately using a method similar to the above-mentioned method for isolating the protein of the present invention, that is, gel filtration, ion exchange resin, reverse phase HPLC, or the like. Can be. The protein of the present invention thus obtained by the genetic engineering technique has the same hemagglutinating activity as the protein of the present invention isolated from the vascular fluid of a plant. [5] Antibacterial Agent Containing Lectin-Like Protein as Active Ingredient The protein of the present invention is, for example, Bacillus subti which causes spoilage of rice, bread and the like.
lis), Staphylococcus aureus (St)
aphilococcus aureus, Escherichia coli, Bacillus, Staphylococcus (S
staphylococcus, Clostridium (C)
Lostium, Streptococcus (St)
It can exert antibacterial activity widely on Gram-positive bacteria such as reptococcus, gram-negative bacteria such as Escherichia and Pseudomonas, and the like.

The protein of the present invention is used as an antibacterial agent as it is or in the form of tablets, powders, wettable powders, emulsions, capsules and the like using a commonly used solid carrier, liquid carrier, emulsifying dispersant and the like. be able to. As the carrier, water, gelatin, starch, magnesium stearate,
Lactose, gum arabic, vegetable oil and the like. When the protein of the present invention is used as an antibacterial agent, a vascular solution of a plant containing the protein or a crude culture having antibacterial activity obtained by a genetic engineering technique can be used without purification. The antimicrobial agents mentioned above include food preservatives, medical antimicrobial agents, building materials / paint antiseptics, agricultural and horticultural antimicrobial agents, livestock feed preservatives, fish feed preservatives, etc. Can be used at The antibacterial agent of the present invention can be used by being appropriately mixed with other bactericides, pharmaceuticals, preservatives, food additives and the like.

The present invention will be described more specifically with reference to the following examples. However, the technical scope of the present invention is not limited by these examples. [Example 1] Isolation and structural analysis of the protein of the present invention Cucumber (Cucumis sativus cv. Shimos) 6 weeks after seeding
hirazu-jibai) from the aerial part 15 so as not to include leaves
Cut at 30 cm, discard the first few drops of conduit fluid, wash the cut surface with sterile water, connect the tube to the stem,
Conduit fluid was collected in tubes in ice.

After 500 ml of the obtained conduit fluid was precipitated with acetone, the precipitate was washed with 10 ml of 15 mM Tris-HCl-5 mM EDTA (pH 7.
5) Dissolved in buffer. After centrifugation, the supernatant was washed with Poros equilibrated with 15 mM Tris-HCl-5 mM EDTA (pH 7.5) buffer.
The sample was applied to an anion exchange column of HQ (manufactured by Nippon Peseptip). The adsorbed fraction was eluted with a sodium chloride concentration gradient. That is, the salt concentration was increased from 0 M to 1.0 M over 10 minutes. The flow rate was 10 ml / min, and the eluate was fractionated by 3 ml. After dialyzing each fraction in a physiological saline phosphate solution, hemagglutination activity was measured. The fraction retaining the hemagglutination activity was concentrated and subjected to SDS-PAGE, whereupon a 30 kDa protein band was observed. Hemagglutination activity was measured in 25 μl of sample and 25 μl of a physiological saline solution of phosphate (PBS) containing 2% of blood cells of sheep wool.
Was added, and what showed aggregation was judged as (+).

Further, the concentrated solution of the hemagglutinating activity fraction was added to 0.1%.
MRPC C2 / equilibrated with 05% trifluoroacetic acid
This was applied to a reverse phase column of C18 PC 3.2 / 3 (manufactured by Pharmacia), and further purified. Adsorption fraction is 0.05
Eluted with a gradient of acetonitrile containing 5% trifluoroacetic acid. That is, first, the concentration of acetonitrile was increased from 0% to 20% over 5 minutes, and then from 20% to 30% over another 60 minutes.
The elution was performed at a flow rate of 0.25 ml / min while monitoring the absorbance at 225 nm. 2 eluted per 60 minutes
Two peaks each were collected.

When SDS-PAGE was performed, a major protein band was confirmed at a position of about 30 kDa.
XSP30 named (x ylem s ap p rotein) . The XSP30 protein was excised from an acrylamide gel and eluted in an elution buffer (0.1 M sodium acetate, 0.1% SDS, pH 8.5). When the protein (XSP30) contained in this eluate was analyzed by N-terminal amino acid sequence using a protein sequencer 377A (ABI), the sequence was as shown in SEQ ID NO: 3. Example 2 Preparation of the Gene of the Protein of the Present Invention To elucidate the entire primary structure of XSP30, the following is shown.
The cDNA encoding XSP30 was cloned by PCR. After the cucumber root was pulverized in the presence of liquid nitrogen, mRNA was purified according to the SDS-phenol method (Rural Culture Company, Manual for Plant Biotechnology, 1989). cDNA synthesis is performed by using this purified mRNA as a template and RNA PCR kit
(AMV) Ver.2.1 (Takara Shuzo) was used. The Oligo dT adapter primer attached to the kit was used as a primer at this time.

A mixed primer, primer 1 (SEQ ID NO: 4) and primer 2 (SEQ ID NO: 5) designed based on the N-terminal amino acid sequence of XSP30 (SEQ ID NO: 3) revealed by the protein sequencer were synthesized. Using the cDNA obtained in the previous step as a template and the synthesized mix primer 1 (SEQ ID NO: 4) and the adapter primer attached to the kit, a PCR reaction (94 ° C for 1 minute, 45 ° C for 1 minute, 72 ° C for 2 minutes)
(1 cycle for 30 minutes). Further, the amplification product obtained in the pre-PCR step was diluted 100-fold and used as a template,
Using the synthesized mixed primer 2 (SEQ ID NO: 5) and the adapter primer attached to the kit, a PCR reaction (94
(1 cycle at 55 ° C for 1 minute and 2 minutes at 72 ° C for 30 cycles), an amplification product of about 800 bp was obtained. Ex Taq DNA polymerase (manufactured by Takara Shuzo Co., Ltd.) was used for the polymerase in the amplification reaction, and the attached buffer and dNTP substrate were used according to the instructions. The thermal cycler used was GENE AMP PCR SYSTEM 9600 manufactured by PerkinElmer. This product was ligated to a plasmid vector pT7Blue (manufactured by Novagen) using a ligation kit (manufactured by Takara Shuzo), the reaction solution was added to E. coli JM109 competent cells (manufactured by Toyobo), transformation was performed, and 100 μg / ampicillin / Plated on LB plate containing ml. After overnight culture at 37 ° C., plasmid DNA was prepared from the growing clone. Change the nucleotide sequence of the inserted DNA to Dye Te
rminator Cycle Sequencing FS Ready ReactionKit (
It was examined using a DNA sequencer (Model 373S, manufactured by Applied Biosystems) using Applied Biosystems. The amino acid sequence deduced from this nucleotide sequence corresponds to residues 16 to 23 of the known N-terminal amino acid sequence of XSP30, indicating that the fragment of the target gene has been amplified. Was.

Next, the 3 ′ downstream nucleotide sequence including the region revealed in the previous step was obtained by the 3 ′ RACE method as follows. At this time, a kit of 3'-Full RACE Core Set (Takara Shuzo) was used. First, as an upstream primer for PCR for this purpose, the following sequence was selected from the nucleotide sequences identified in the previous step and synthesized, and this was designated as primer 3 (SEQ ID NO: 6). Using mRNA extracted from cucumber root as a template,
Single-stranded cDNA was synthesized with reverse transcriptase using the OligodT-3sites Adapter primer included in the kit. Using the obtained single-stranded cDNA as a template, primer 3 and 3si
When a PCR reaction was performed using tes Adapter Primer,
An amplification product of about 700 bp was obtained. Using a ligation kit (Takara Shuzo), this product is
Ligation with pT7Blue (Novagen)
In addition to 109 competent cells (manufactured by Toyobo Co., Ltd.), transformation was performed, and the resultant was spread on an LB plate containing 100 μg / ml of ampicillin. After overnight culture at 37 ° C., plasmid DNA was prepared from the growing clone. Dye Terminator Cycle Sequencing FS Ready
Using a Reaction Kit (Applied Biosystems), a DNA sequencer (Model 373S, Applied Biosystems) was used. As a result, XSP30
It was found that the base sequence at the 3 'end of the cDNA was amplified.

XSP30 cDNA from the sequence obtained up to the previous step
Was examined by the 5'-RACE method as follows. At this time, a kit of 5'-Full RACE Core Set (Takara Shuzo) was used. Primer 4 (SEQ ID NO: 7), Primer 5 (SEQ ID NO: 8), Primer 6 (SEQ ID NO: 9), and Primer 7 (SEQ ID NO: 10) were synthesized based on the sequences revealed in the analysis in the previous step. Using mRNA extracted from cucumber root as a template, primer 8 (SEQ ID NO: 1)
Using 1), single-stranded cDNA was synthesized by reverse transcriptase. Using the obtained single-stranded cDNA as a template, a two-step PCR reaction was performed. After performing PCR reaction by combining Primer 4 and Primer 5, Primer 6 and Primer 7
When the PCR reaction was performed in combination with
An amplification product of p was obtained. Using a ligation kit,
This product is ligated with the plasmid vector pT7Blue, and the reaction mixture is added to E. coli JM109 competent cells, transformed, and contains 100 μg / ml ampicillin.
Soaked on LB plate. After overnight culture at 37 ° C., plasmid DNA was prepared from the growing clone. The nucleotide sequence of the inserted DNA is Dye Terminator Cycle Sequencing FS Ready
The DNA sequencer was examined using a Reaction Kit, and the nucleotide sequence of the region including the 5 'end of XSP30 cDNA was obtained.

XSP30cDN revealed in the previous process
The following PCR was carried out to obtain partial sequences upstream and downstream of A as a continuous sequence, that is, a clone containing the full length of the cDNA of XSP30. XSP3 obtained
Primer-9 (SEQ ID NO: 1)
2) was synthesized. Next, Primer-10 (SEQ ID NO: 13) was synthesized based on the obtained downstream sequence of XSP30. Combining Primer-9 and Primer-10, cDN
When PCR was performed using A as a template, about 900 bp
Was obtained. This product was cloned into pT7Blue (manufactured by Novagen) and the nucleotide sequence was examined.
It was confirmed to be XSP30 cDNA containing the nucleotide sequence of SEQ ID NO: 2. The amino acid sequence of the protein encoded by the XSP30 cDNA was as shown in SEQ ID NO: 1. When the database was examined, homology with lectin derived from castor seed was observed. [Example 3] Preparation of transformant Cloned into pT7Blue (Novagen)
Escherichia coli is transformed with the recombinant DNA of XSP30, and the transformant is transformed.
E. coli XL1-Blue (pXSP3016) was obtained. Transformant, E. coli XL1-Blue
(pXSP3016) has been deposited as FERM P-17118 with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology.

Plasmid pUTE500K '(J. Biotechno) containing an expression control region such as a promoter, an operator and a ribosome binding site having an E. coli lactose operon and the like, and an NdeI cleavage site.
l. , 52, 11, 1996), to thereby obtain pUTEXSP30. Then D. M. Morrison's Method (Methods i
n Enzymology, 68, 326, 1979)
Transformant, E. coli XLI-B
lue (pUTEXSP30) was obtained. The obtained transformant was cultured in a TY medium (1% bactotryptone, 0.5% bactoist extract, 0.5% NaCl) containing 1 mM isopropyl-β-D-galactoside.
The cells were cultured with shaking at 15 ° C for 15 hours. The crude enzyme extract after sonication of the cultured cells showed hemagglutination activity.

[0027]

According to the present invention, a lectin-like protein derived from vascular fluid, a gene encoding the protein, a recombinant DNA obtained by inserting the gene into vector DNA, and the recombinant DNA A transformant and a method for producing a lectin-like protein are provided. The lectin-like protein obtained by the present invention has a specific lectin-like activity, and is not only suitable as an antibacterial agent but also industrially useful as a pharmaceutical.

[Sequence List] SEQUENCE LISTING <110> Noda Institute for Scientific Research <120> A novel lectinlike protein <130> SP223 <160> 13 <210> 1 <211> 272 <212> PRT <213> Cucumis sativus <400> 1 Val Pro Pro Asn Tyr Gly Tyr Gly Val Gly Tyr Gly Gly Val Pro 5 10 15 Gly Ala Thr His Leu Val Gly Arg Asp Gly Leu Cys Leu Glu Met 20 25 30 Ser Pro Trp Tyr Lys Pro Ala Gly Ile Asn Phe Pro Thr Arg Leu 35 40 45 Ser Pro Cys Asp Glu Lys Lys Gln Thr Gln Leu Trp Thr Ile Val 50 55 60 Gly Asp Gly Thr Ile Arg Pro Met Asn Asp Lys Phe Cys Leu Ala 65 70 75 Ala Glu Val Phe Tyr Gly Val Ile Asn Lys Ala Val Val Ser Glu 80 85 90 Cys Gly Lys Val Ser Asp Pro Asn Lys Lys Trp Thr Gln Lys Asn 95 100 105 Asp Gly Thr Ile Ala Leu Val Asp Ser Arg Met Val Leu Thr Gly 110 115 120 Asp Leu Asp Tyr Val Thr Leu Gln Ser Asn Lys Tyr Thr Pro Ser 125 130 135 Gln Ser Trp Glu Val Thr Glu Ser Leu Asn Ser Met Val Ala Asn 140 145 150 Ile Glu Trp Leu Asn Asn Leu Cys Leu Gln Ser Thr Asp Asp Ser 155 160 165 Ser His Val Gly Leu Asn Gly Cys Asn Thr Asp Asn Lys Tyr Gln 170 175 180 Arg Trp Ala Leu Tyr Ala Asp Gly Thr Ile Arg Gln His Val Asn 185 190 195 Lys Asn Tyr Cys Leu Thr Ser Asp Gln Asp Phe Gly Arg Phe Val 200 205 210 Val Val Ser Lys Cys Glu Asp Lys Pro Gln Gln Arg Trp Ser Leu 215 220 225 Asp Ala Lys Asp Tyr Thr Ile Asp His Pro Asn Thr Asp Met Val 230 235 240 Leu Asp Val Phe Ser Val Pro Asp Ser Thr Phe Pro Ser Val Leu 245 250 255 Val Thr Asn Arg Arg Asp Gly Ser Ala Ser Gln Arg Trp Thr Ile 260 265 270 Ile Asn 272 <210> 2 <211> 962 <212> DNA <213 > Cucumis sativus <400> 2 gtgcccccca actatggata tggagttgga tatggaggcg tccctggagc cacacatctt 60 gtgggtcgag atggattgtg tttagagatg tctccatggt acaaacctgc aggtattaat 120 tttccaactc gattatcacc atgtgacgag aagaaacaaa ctcaattatg gacgattgtc 180 ggagatggca caattcgacc catgaatgat aaattttgct tggctgctga agttttttat 240 ggggtcataa ataaagcagt agtaagtgag tgtggtaaag tatcagatcc taacaagaaa 300 tggacccaga agaatgacgg aaccata gcc ctcgtcgatt caagaatggt tctaacagga 360 gatttagact atgtgacatt gcaaagtaac aaatatacac catcacaaag ttgggaagtc 420 acggaaagtt taaactcaat ggttgcaaac atcgaatggc ttaacaactt gtgtttgcaa 480 tccacagacg attcaagtca tgtgggattg aatggatgta atacagacaa taagtaccaa 540 agatgggcat tgtatgcaga tggaaccatt cgacaacatg tgaacaaaaa ctattgcttg 600 acttctgacc aagattttgg tcgctttgta gttgtgtcta aatgtgaaga caaaccgcaa 660 caacgttgga gtcttgatgc taaagactat actattgacc atcccaacac tgacatggtc 720 ctagatgtgt ttagtgtgcc tgattctact tttccgtcag tactcgttac gaaccgtcgt 780 gatggaagtg ctagccaaag atggactatt attaactaat gaatcagata aataagatag 840 gggagatgtg aatccacacg aactcatgca tgcaaatgcc tttctacttc tttaactctc 900 tttctaatgc ttaatgtatg aacatcaata aattaataag ataagtgtgg atttatgtgt 960 tt 962 <210> 3 <211> 25 <212> PRT <213> Cucumis sativus <400> 3 Val Pro Pro Asn Tyr Gly Tyr Gly Val Gly Tyr Gly Gly Val Pro 5 10 15 Gly Ala Thr His Leu Val Gly Arg Asp Gly 20 25 <210> 4 <211> 23 <212> DNA <213> Artificial S equence <220> <223> Designed DNA (sense) degenerated from the N-terminal amino acid sequence for amplification of a part of cDNA. <400> 4 gtnccnggna aytayggnta ygg 23 <210> 5 <211> 26 <212> DNA < 213> Artificial Sequence <220> <223> Designed DNA (sense) degenerated from the N-terminal amino acid sequence for amplification of a part of cDNA. <400> 5 tayggngtng gntayggngg ngtncc 26 <210> 6 <211> 21 <212 > DNA <213> Artificial Sequence <220> <223> Designed DNA for amplification of a part of XSP30 cDNA in the 3 'RACE. <400> 6 cagaagaatg acggaaccat a 21 <210> 7 <211> 18 <212> DNA < 213> Artificial Sequence <220> <223> Designed DNA for amplification of a part of XSP30 cDNA in the 1st step of 5'RACE. <400> 7 aaaaacttca gcagccaa 18 <210> 8 <211> 18 <212> DNA < 213> Artificial Sequence <220> <223> Designed DNA for amplification of a part of XSP30 cDNA in the 1st step of 5'RACE. <400> 8 ggggtcataa ataaagca 18 <210> 9 <211> 18 <212> DNA <213> Artificial Sequence <223> Designed DNA for amplification of a part of XSP30 cDNA in the 2nd step of 5'RACE. <400> 9 agcaaaattt atcattca 18 <210> 10 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Designed DNA for amplification of a part of XSP30 cDNA in the 2nd step of 5'RACE. <400> 10 gtagtaagtg agtgtggt 18 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Designed DNA for reverse transcriptation of a part of XSP30 mRNA. <400> 11 aagtcaagca atagttttc 19 <210> 12 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Designed DNA for amplification of XSP30 cDNA. <400> 12 gaattcatag tcgaattaaa gtaatcaaag gatgaaagaa at 42 <210> 13 <211> 28 <212> DNA < 213> Artificial Sequence <220> <223> Designed DNA for amplification of XSP30 cDNA. <400> 13 aagaattcta tcttat ttat ctgattca 28

[Sequence List Free Text]

SEQ ID NO: 4: DNA designed based on the N-terminal amino acid sequence of XSP30. SEQ ID NO: 5: DNA designed based on N-terminal amino acid sequence of XSP30. SEQ ID NO: 6: DNA designed to amplify a part of cDNA of XSP30 by 3′RACE method. SEQ ID NO: 7: XSP30 by 5 ′ RACE method (1st step)
DNA designed to amplify a portion of the cDNA. SEQ ID NO: 8: XSP30 by 5 ′ RACE method (1st step)
DNA designed to amplify a portion of the cDNA. SEQ ID NO: 9: XSP30 by 5 ′ RACE method (2nd step)
DNA designed to amplify a portion of the cDNA. SEQ ID NO: 10: XSP30 by 5 ′ RACE method (2nd step)
DNA designed to amplify a portion of the cDNA. SEQ ID NO: 11: DNA designed to reverse transcribe a part of XSP30 mRNA into cDNA by 5 ′ RACE method. SEQ ID NO: 12: DNA designed to amplify full length of XSP30 cDNA. SEQ ID NO: 13: DNA designed to amplify full length of cDNA of XSP30.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 35/78 A61K 35/78 S 4C057 38/00 C07H 21/04 B 4C084 C07H 21/04 C12N 1/00 U 4C088 C12N 1/00 C12P 21/02 C 4H011 5/10 A61K 37/02 4H045 15/09 ZNA C12N 5/00 C C12P 21/02 15/00 ZNAA // (C12P 21/02 C12R 1:19) F Term (reference) 2B030 AA02 AB03 AD05 AD08 CA17 CB03 4B021 MC01 MK01 MK02 MK05 MK23 MP01 4B024 AA01 AA05 AA07 AA10 AA20 BA80 CA04 DA06 EA04 FA04 FA08 GA11 HA01 4B064 AG01 CA02 CA11 CA19 CC24 DA01X44A04 ACA4 DD01 MM04 4C084 AA01 AA02 AA03 AA07 BA22 CA13 CA53 MA01 NA14 ZB352 4C088 AB19 AC20 BA06 BA35 CA14 CA16 CA17 MA01 MA11 NA14 ZB35 4H011 AA01 AA02 DA02 DA03 DA05 DA15 DA16 4H045 AA10 AA20 AA30 BA10 CA30 DA80 EA01 EA07 EA29 FA72 FA74

Claims (8)

[Claims] 1. A lectin-like protein which is contained in a vascular fluid. 2. A lectin-like protein according to claim 1, wherein the lectin-like protein is a protein derived from a Cucurbitaceae plant. 3. A lectin-like protein of the following (a) or (b): (A) a protein consisting of the amino acid sequence of SEQ ID NO: 1; (B) a protein having hemagglutinating activity consisting of an amino acid sequence in which one or more amino acids have been added, deleted or substituted in the amino acid sequence of SEQ ID NO: 1; A gene encoding the lectin-like protein according to any one of claims 1 to 3. 5. A lectin-like protein according to claim 4,
A recombinant DNA obtained by inserting a gene to be inserted into a vector DNA. 6. A transformant comprising the recombinant DNA according to claim 5. 7. A lectin-like protein, wherein the transformant according to claim 6 is cultured in a medium, and the lectin-like protein according to any one of claims 1 to 3 is obtained from the culture. Manufacturing method. 8. An antibacterial agent comprising the lectin-like protein according to claim 1 as an active ingredient.