CN106008684B

CN106008684B - Recombinant alpha protein for inhibiting clostridium perfringens infection and preparation method and application thereof

Info

Publication number: CN106008684B
Application number: CN201610304595.9A
Authority: CN
Inventors: 宋晓晖; 孙雨; 翟新验; 胡冬梅
Original assignee: CHINA ANIMAL BLIGHT PREVENTION AND CONTROL CENTER
Current assignee: CHINA ANIMAL BLIGHT PREVENTION AND CONTROL CENTER
Priority date: 2016-05-09
Filing date: 2016-05-09
Publication date: 2020-01-14
Anticipated expiration: 2036-05-09
Also published as: CN106008684A

Abstract

The invention discloses a recombinant alpha protein for inhibiting clostridium perfringens infection and a preparation method and application thereof. The recombinant alpha protein is a) or b) or c): a) a protein consisting of the amino acid sequence of SEQ ID No. 2; b) a protein consisting of the amino acid sequence shown in SEQ ID No.2 at positions 51-353; c) a fusion protein obtained by carboxyl-terminal or/and amino-terminal fusion protein labels of the protein shown in a) or b). After the animal is immunized by the recombinant alpha protein, the animal can generate higher serum antibody level and can resist the attack of clostridium perfringens. The recombinant alpha protein has good solubility and simple purification, and can be used as a diagnostic antigen, prepared into a monoclonal antibody or used for further researching the function and conformation relation of the protein.

Description

Recombinant alpha protein for inhibiting clostridium perfringens infection and preparation method and application thereof

Technical Field

The invention relates to a recombinant alpha protein for inhibiting clostridium perfringens infection in the field of biotechnology, and a preparation method and application thereof.

Background

Clostridium Perfringens (Clostridium Perfringens), also called Clostridium welchii, is an important zoonosis, and the pathogen is one of the main pathogens of traumatic gas gangrene, human food poisoning, sheep plague, lamb dysentery, cattle and sheep necrotic enteritis, cattle and sheep enterotoxemia, and causes huge economic loss to the animal husbandry. The main pathogenic factor of clostridium perfringens is its secreted exotoxin, and the species of clostridium perfringens is up to 13, of which α, β and e are the most predominant exotoxins, and clostridium perfringens can be classified into A, B, C, D, E, five serotypes according to the species producing exotoxin. The control of infectious diseases of animals caused by alpha toxin of clostridium perfringens is one of the main problems which plague the control of epidemic diseases of animals at present. The traditional vaccine has certain effect on the aspect of treating and preventing the clostridium perfringens diseases of animals. However, these vaccines still suffer from drawbacks during use, such as local inflammation and toxic reaction of animals caused by traditional vaccine immunization. The development of a genetic engineering vaccine which can express the alpha exotoxin antigen protein, does not destroy the immunogenicity of the alpha exotoxin antigen protein and plays a role in preventing and controlling epidemic diseases caused by the alpha exotoxin of clostridium perfringens is a technical problem which needs to be solved urgently.

The method is characterized in that the Lena (Lena. separation and identification of Clostridium perfringens of sheep origin, and research on prokaryotic expression and immunogenicity of alpha toxin. Master academic thesis of Stone river university.2013) performs separation and identification on the Clostridium perfringens of sheep origin and researches on prokaryotic expression and immunogenicity of alpha toxin. The subject group designs a primer aiming at an alpha toxin mature peptide sequence aiming at a whole gene sequence of clostridium perfringens alpha toxin, amplifies the alpha toxin mature peptide sequence by adopting a PCR method, and inserts the alpha toxin mature peptide sequence into a plasmid pET-28b to construct a recombinant expression vector pET-28 b-cpa. The subject group uses the recombinant protein to prepare subunit vaccine to immunize mice, and the result shows that the recombinant alpha toxin can stimulate the generation of anti-alpha toxin antibody immune response reaction, the immune antibody reaches the highest level (1:6400) 28 days after immunization, and a certain immune protection effect can be provided for the immunized mice. However, the protein expressed by the recombinant plasmid is partially soluble protein, and the expression amount of the soluble protein accounts for 34.6 percent of the soluble protein of the bacterial cells.

In the prior art, the expression and purification method of main exotoxin proteins of clostridium perfringens is relatively complex, the expression products usually exist in the form of insoluble inclusion bodies, and the reports of soluble protein expression are very few at home and abroad. Since the expression product in inclusion bodies is biologically inactive, denaturation and renaturation treatments are required. The denaturation and renaturation of protein are a very complex process, the renaturation conditions of different proteins are different, and the renaturation rate is difficult to improve. This is the main limiting factor limiting its application. This problem is well overcome by using soluble expression. How to construct soluble expression vectors and optimize efficient expression methods of soluble proteins is a hot topic of research in the field for a long time.

Disclosure of Invention

One technical problem to be solved by the present invention is how to obtain a soluble protein vaccine that inhibits clostridium perfringens infection.

In order to solve the above technical problems, the present invention provides a method for preparing a recombinant α protein.

The method for preparing the recombinant alpha protein comprises the steps of expressing a coding gene of the recombinant alpha protein in an organism to obtain the recombinant alpha protein; the organism is a microorganism, a plant or a non-human animal;

the recombinant alpha protein is a) or b) or c) or d):

a) a protein consisting of the amino acid sequence of SEQ ID No. 2;

b) a protein consisting of the amino acid sequence shown in SEQ ID No.2 at positions 51-353;

c) a fusion protein obtained by carboxyl terminal or/and amino terminal fusion protein label of the protein shown in a) or b);

d) the soluble protein is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 2.

In the above method, the protein of a) is named α -his, and the protein of b) is named α -Y. SEQ ID No.2 consists of 353 amino acid residues.

In the above method, the protein tag refers to a polypeptide or protein that is expressed by fusion with a target protein by using a DNA in vitro recombination technology, so as to facilitate expression, detection, tracing, and/or purification of the target protein.

In the above method, the expressing the gene encoding the recombinant α protein in the organism comprises introducing the gene encoding the recombinant α protein into a recipient microorganism to obtain a recombinant microorganism expressing the recombinant α protein, and culturing the recombinant microorganism to express the recombinant microorganism to obtain the recombinant α protein.

In the above method, the recipient microorganism may be any one of C1) -C4):

C1) a prokaryotic microorganism;

C2) gram-negative bacteria;

C3) an Escherichia bacterium;

C4) escherichia coli BL21(DE 3).

In the above method, the gene encoding the protein is a gene represented by 1) or 2) or 3) or 4) below:

1) the coding sequence is a DNA molecule shown in SEQ ID No. 1;

2) the coding sequence is a DNA molecule shown in the 151 th-1062 th site of SEQ ID No. 1;

3) has more than 90% of identity with the DNA molecule defined in 1) or 2) and encodes the recombinant alpha protein.

Wherein, SEQ ID No.1 consists of 1068 nucleotides, the name is alpha-hisY gene, and the coded amino acid sequence is protein alpha-his of SEQ ID No. 2. The DNA molecule shown in position 151-1062 of SEQ ID No.1 is an alpha-Y gene encoding a protein alpha-Y consisting of the amino acid sequence shown in positions 51-353 of SEQ ID No. 2.

In the above method, the recombinant microorganism is a recombinant microorganism expressing a recombinant alpha protein whose amino acid sequence is SEQ ID No.2, which is obtained by introducing pET30 a-alpha-Y into Escherichia coli BL21(DE3), the recombinant microorganism is named BL21(DE3)/pET30 a-alpha-Y, and pET30 a-alpha-Y is a recombinant vector obtained by replacing the sequence between BamHI and XhoI sites of vector pET30a (+) with a DNA fragment shown in position 151-1062 of SEQ ID No. 1.

In the above method, the expression is induced expression by using 0.75mM IPTG at 16 ℃ for 13-16 hours or 13-24 hours or 13 hours or 16 hours.

Any of the following products is also within the scope of the present invention:

p1) a recombinant alpha protein being a) or b) or c) or d):

a) a protein consisting of the amino acid sequence of SEQ ID No. 2;

d) the soluble protein is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 2;

p2) a biological material related to the recombinant alpha protein, the biological material being any one of the following B1) to B16):

B1) a nucleic acid molecule encoding the recombinant alpha protein;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector comprising the nucleic acid molecule of B1);

B4) a recombinant vector comprising the expression cassette of B2);

B5) a recombinant microorganism comprising the nucleic acid molecule of B1);

B6) a recombinant microorganism comprising the expression cassette of B2);

B7) a recombinant microorganism containing the recombinant vector of B3);

B8) a recombinant microorganism containing the recombinant vector of B4);

B9) a transgenic animal cell line comprising the nucleic acid molecule of B1);

B10) a transgenic animal cell line comprising the expression cassette of B2);

B11) a transgenic animal cell line containing the recombinant vector of B3);

B12) a transgenic animal cell line containing the recombinant vector of B4);

B13) a transgenic plant cell line comprising the nucleic acid molecule of B1);

B14) a transgenic plant cell line comprising the expression cassette of B2);

B15) a transgenic plant cell line comprising the recombinant vector of B3);

B16) a transgenic plant cell line comprising the recombinant vector of B4);

p3) a vaccine for the prevention of clostridium perfringens infection in an animal comprising said recombinant alpha protein or said biological material.

In the above products, the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

In the above product, the recombinant α protein is produced according to any one of the methods described above;

the nucleic acid molecule is a gene shown in the following 1) or 2) or 3):

1) the coding sequence is a DNA molecule shown in SEQ ID No. 1;

3) a DNA molecule having 90% or more identity to the DNA molecule defined in 1) or 2) and encoding the protein;

the recombinant vector is the pET30 a-alpha-Y;

the recombinant microorganism is E1) or E2):

E1) the recombinant microorganism is a recombinant microorganism which is obtained by introducing the coding gene of the recombinant alpha protein into a receptor microorganism to express the recombinant alpha protein, and the receptor microorganism is any one of C1) -C4):

C1) a prokaryotic microorganism;

C2) gram-negative bacteria;

C3) an Escherichia bacterium;

C4) escherichia coli BL21(DE 3);

the recombinant microorganism is the BL21(DE3)/pET30 a-alpha-Y;

the active ingredient of the vaccine for preventing the clostridium perfringens infection of animals is the recombinant alpha protein or the biological material.

Any of the following applications also fall within the scope of the present invention:

y1) the use of the recombinant alpha protein for the preparation of a vaccine against Clostridium perfringens;

y2) the use of the biomaterial for the manufacture of a vaccine against Clostridium perfringens;

y3) for use in the manufacture of a vaccine against Clostridium perfringens;

y4) the recombinant alpha protein is used for preparing a clostridium perfringens disease diagnosis antigen;

y5) in the preparation of monoclonal antibodies. .

In the present invention, the clostridium perfringens can be any 5, any 4, any 3, any 2 or any 1 of the 5 types of clostridium perfringens, A, B, C, D and E.

In the invention, the anti-clostridium perfringens vaccine can be specifically the vaccine for preventing clostridium perfringens infection of animals.

In practical applications, the recombinant α protein of the present invention or its related biological material can be administered directly to a patient as a drug, or can be administered to a patient after mixing with a suitable carrier or excipient, for the purpose of treating clostridium perfringens infection. The carrier material herein includes, but is not limited to, water-soluble carrier materials (e.g., polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (e.g., ethyl cellulose, cholesterol stearate, etc.), enteric carrier materials (e.g., cellulose acetate phthalate, carboxymethyl cellulose, etc.). Among these, water-soluble carrier materials are preferred. The materials can be prepared into various dosage forms, including but not limited to tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, freeze-dried powder injections and the like. Can be common preparation, sustained release preparation, controlled release preparation and various microparticle drug delivery systems. In order to prepare the unit dosage form into tablets, various carriers well known in the art can be widely used. Examples of the carrier are, for example, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate and the like; wetting agents and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone and the like; disintegrating agents such as dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid ester, sodium dodecylsulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors such as sucrose, glyceryl tristearate, cacao butter, hydrogenated oil and the like; absorption accelerators such as quaternary ammonium salts, sodium lauryl sulfate and the like; lubricants, for example, talc, silica, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, and the like. The tablets may be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer and multi-layer tablets. In order to prepare the dosage form for unit administration into a pill, various carriers well known in the art can be widely used. Examples of the carrier are, for example, diluents and absorbents such as glucose, lactose, starch, cacao butter, hydrogenated vegetable oil, polyvinylpyrrolidone, Gelucire, kaolin, talc and the like; binders such as acacia, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or batter, etc.; disintegrating agents, such as agar powder, dried starch, alginate, sodium dodecylsulfate, methylcellulose, ethylcellulose, etc. In order to prepare the unit dosage form into suppositories, various carriers known in the art can be widely used. As examples of the carrier, there may be mentioned, for example, polyethylene glycol, lecithin, cacao butter, higher alcohols, esters of higher alcohols, gelatin, semisynthetic glycerides and the like. In order to prepare the unit dosage form into preparations for injection, such as solutions, emulsions, lyophilized powders and suspensions, all diluents commonly used in the art, for example, water, ethanol, polyethylene glycol, 1, 3-propanediol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitol fatty acid esters, etc., can be used. In addition, for the preparation of isotonic injection, sodium chloride, glucose or glycerol may be added in an appropriate amount to the preparation for injection, and conventional cosolvents, buffers, pH adjusters and the like may also be added. In addition, colorants, preservatives, flavors, flavorings, sweeteners or other materials may also be added to the pharmaceutical preparation, if desired. The preparation can be used for injection administration, including subcutaneous injection, intravenous injection, intramuscular injection, intracavity injection and the like; for luminal administration, such as rectally and vaginally; administration to the respiratory tract, e.g., nasally; administration to the mucosa. The above route of administration is preferably by injection.

The invention inserts DNA molecule shown in 151-position 1062 of SEQ ID No.1 into BamHI and XhoI sites of pET30a (+) to obtain recombinant expression vector pET30 a-alpha-Y for expressing recombinant protein alpha-his of SEQ ID No. 2. The recombinant expression vector pET30 a-alpha-Y is introduced into Escherichia coli BL21(DE3) to obtain the soluble target protein alpha-his. The invention optimizes the expression condition of alpha-his, further improves the expression quantity of alpha-his, and uses 0.75mM IPTG to induce for 13-24 hours at 16 ℃, the content of alpha-his reaches 90% of the total protein of the thallus, and the expressed target alpha-his protein is 94% soluble. Immunization of animals with alpha-his results in higher serum antibody levels in the animals and resistance to challenge by Clostridium perfringens. The immune protection rate of α -his against clostridium perfringens type a challenge was 100% within 7 days (20 all survived), all mice in the PBS control group died; the immune protection rate of alpha-his against clostridium perfringens type B challenge was 90% (18 survived, 2 died), all PBS control mice died; the immune protection rate of alpha-his against clostridium perfringens type C challenge was 85% (17 survived, 3 died), all PBS control mice died; the immune protection rate of alpha-his against challenge with clostridium perfringens type D is 90% (18 survived, 2 died), whereas all PBS control mice died. The antibody titer reaches the peak value 7-14 days after the third alpha-his immunization, and the highest antibody titer reaches 1: 128000. the alpha-his has good solubility and simple purification, and can be used as a diagnostic antigen, prepared into a monoclonal antibody or used for further researching the function and conformation relation of the protein.

Drawings

FIG. 1 is an SDS-PAGE electrophoresis of proteins expressed by each strain.

In the figure, M is Marker, 130kD, 95kD, 70kD, 62kD, 51kD, 40kD, 29kD, 1, receptor bacteria whole bacterial protein liquid for inducible expression, 2, BL21(DE3)/pET30 a-alpha-Y whole bacterial protein liquid for non-inducible expression, 3, BL21(DE3)/pET30 a-alpha-Y whole bacterial protein liquid for inducible expression, 4, BL21(DE3)/pET30 a-alpha-Y protein-containing supernatant for inducible expression, 5, BL21(DE3)/pET30 a-alpha-Y protein-containing precipitate for inducible expression, 6, BL 7378 (DE 3)/BL 30T a-alpha-W whole bacterial protein liquid for non-inducible expression, 7, BL21(DE3)/pET30 a-alpha-W protein liquid for inducible expression, BL 364642 (DE 4642)/pET 3 (DE a-alpha-W protein-containing supernatant for inducible expression, 9. induced expression of BL21(DE3)/pET30 a-alpha-W protein-containing precipitate, 10, non-induced expression of BL21(DE3)/pET30a-pm alpha-W whole bacterial protein liquid, 11, induced expression of BL21(DE3)/pET30a-pm alpha-W whole bacterial protein liquid, 12, induced expression of BL21(DE3)/pET30a-pm alpha-W protein-containing supernatant, 13, induced expression of BL21(DE3)/pET30a-pm alpha-W protein-containing precipitate.

FIG. 2 is a Western-blot spectrum.

In the figure, 1, receptor bacteria whole bacterial protein liquid for induced expression, 2, BL21(DE3)/pET30 a-alpha-Y whole bacterial protein liquid for non-induced expression, 3, BL21(DE3)/pET30 a-alpha-Y whole bacterial protein liquid for induced expression, 4, BL21(DE3)/pET30 a-alpha-Y protein-containing supernatant for induced expression, 5, BL21(DE3)/pET30 a-alpha-Y protein-containing precipitate for induced expression, 6, BL21(DE3)/pET30 a-alpha-W whole bacterial protein liquid for non-induced expression, 7, BL21(DE3)/pET30 a-alpha-W whole bacterial protein liquid for induced expression, 8, BL21(DE3)/pET30 a-alpha-W protein-containing supernatant for induced expression, BL21(DE3)/pET 69556 (DE 8253)/pET a-alpha-W protein-containing precipitate for induced expression, 10. uninduced expression BL21(DE3)/pET30a-pm alpha-W whole bacterial protein liquid, 11, induced expression BL21(DE3)/pET30a-pm alpha-W whole bacterial protein liquid, 12, induced expression BL21(DE3)/pET30a-pm alpha-W protein-containing supernatant, 13, induced expression BL21(DE3)/pET30a-pm alpha-W protein-containing precipitate.

FIG. 3 shows the AKTA purification identification of recombinant protein α -his. The arrow indicates the peak of the purified protein of interest.

FIG. 4 shows the molecular sieve purification identification of recombinant protein alpha-his. The arrow indicates the peak of the purified protein of interest.

FIG. 5 is an SDS-PAGE electrophoretogram of the purified target protein. The arrows indicate the destination strips.

Wherein M is Marker, and is 130kD, 95kD, 70kD, 62kD, 51kD, 40kD and 29kD respectively from top to bottom; 1 is BL21(DE3)/pET30 a-alpha-Y whole bacterial protein liquid for inducible expression; 2 is BL21(DE3)/pET30a-pm alpha-W whole bacterial protein liquid for induction expression; 3 is the whole bacterial protein liquid of BL21(DE3)/pET30 a-alpha-Y without induced expression; 4 is the supernatant containing the protein of BL21(DE3)/pET30 a-alpha-Y with inducible expression; 5 is the supernatant containing protein of BL21(DE3)/pET30a-pm alpha-W with inducible expression; 6 is the protein-containing precipitate of BL21(DE3)/pET30 a-alpha-Y with inducible expression; 7 is a molecular sieve purified alpha-his protein; 8 is the molecular sieve purified pm alpha-his protein.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

pET30a (+) is a product of Novagen. pET28a (+) is a product of Novagen.

The A-type clostridium perfringens virulent strain C57-10, the B-type clostridium perfringens virulent strain C58-5, the C-type clostridium perfringens virulent strain C59-4 and the D-type clostridium perfringens virulent strain C60-11 are all products of Chinese veterinary medicine supervision.

Example 1 soluble expression of alpha-hisY

1. Synthetic genes

The application designs 3 recombinant alpha genes, namely an alpha-hisY gene shown in SEQ ID No.1, an alpha-hisW gene shown in SEQ ID No.3 and a pm alpha-hisW gene shown in SEQ ID No. 4.

The alpha-hisY gene and the alpha-hisW gene both encode the protein alpha-his shown in SEQ ID No. 2. The pm alpha-hisW gene encodes the protein pm alpha-hisW shown in SEQ ID No. 5. Alpha-his is a protein obtained by deleting amino acid residues 52 to 146 of pm alpha-hisW.

The alpha-Y gene shown in the 151-position and 1062-position of SEQ ID No.1 (encoding the protein shown in the 51-353-position amino acid residues of SEQ ID No. 2), the alpha-W gene shown in the 151-position and 1062-position of SEQ ID No.3 (encoding the protein shown in the 51-353-position amino acid residues of SEQ ID No. 2), and the pm alpha-W gene shown in the 151-position and 1347-position of SEQ ID No.4 (encoding the pm alpha-W protein shown in the 51-448-position amino acid residues of SEQ ID No. 5) are synthesized by a chemical synthesis method.

2. Construction of recombinant expression vector and recombinant bacterium

The alpha-Y gene was used as a template, and an upstream primer F1 (sequence 5' -gg) was usedatccatgttttgggacccggacaccgac-3') and a downstream primer R1 (sequence 5-CTCGAGTTATTTGATGTTATAGGTGCTGT-3') was amplified by PCR, and BamHI sites (underlined sequences) and XhoI recognition sites (underlined sequences) were added to both ends of the α -Y gene to obtain a PCR product of the α -Y gene shown at positions 145-1068 of SEQ ID No. 1.

Using the α -W gene as a template, the forward primer F1 and the reverse primer R2 (sequence 5-CTCGAGTCATTTGATGTTGTAGGTGCTGT-3') and BamHI and XhoI recognition sites are added at both ends of the alpha-W gene to obtain the alpha-W gene PCR product shown at position 145-1068 of SEQ ID No. 3.

The pm alpha-W gene was used as a template, and an upstream primer F3 (sequence 5' -gg) was usedatccatgaaacgcaaaatctgcaaagccct-3') and a downstream primer R2, and BamHI and XhoI recognition sites are added at both ends of the pm alpha-W gene to obtain the pm alpha-W gene PCR product shown in 145-1353 of SEQ ID No. 4.

Digesting the PCR product of the alpha-Y gene by BamHI and XhoI, and recovering a target fragment (alpha-Y gene); meanwhile, the vector pET30a (+) is cut by BamHI and XhoI enzyme, and the large vector fragment is recovered; and connecting the recovered target fragment with the recovered vector large fragment to obtain the target plasmid. Coli BL21(DE3) competent cells were transformed with the plasmid of interest. This was spread evenly on LB plates containing kanamycin and cultured at 37 ℃ for 16 hours. The single colony is shake cultured overnight, the extracted plasmid is double-digested with BamHI and XhoI for identification, the plasmid with correct restriction enzyme is sequenced, the sequencing result shows that the sequence is a recombinant expression vector obtained by replacing the fragment between BamHI recognition sites and XhoI recognition sites of pET30a (+) with alpha-Y gene shown in 151-1062 site of SEQ ID No.1 and keeping the other sequence of pET30(+) unchanged, and the recombinant expression vector is named as pET30 a-alpha-Y. pET30 a-alpha-Y contains His-tagged alpha-hisY gene, the nucleotide sequence of the alpha-hisY gene is SEQ ID No.1, and the alpha-His protein shown in SEQ ID No.2 is coded. The recombinant E.coli containing pET30a- α -Y was named BL21(DE3)/pET30a- α -Y.

Digesting the PCR product of the alpha-W gene by BamHI and XhoI, and recovering a target fragment (alpha-W gene); meanwhile, the vector pET30a (+) is cut by BamHI and XhoI enzyme, and the large vector fragment is recovered; and connecting the recovered target fragment with the recovered vector large fragment to obtain the target plasmid. Coli BL21(DE3) competent cells were transformed with the plasmid of interest. This was spread evenly on LB plates containing kanamycin and cultured at 37 ℃ for 16 hours. The single colony is shake cultured overnight, the extracted plasmid is double-digested with BamHI and XhoI for identification, the plasmid with correct restriction enzyme is sequenced, the sequencing result shows that the plasmid is a recombinant expression vector which is obtained by replacing the fragment between BamHI recognition sites and XhoI recognition sites of pET30a (+) with alpha-W gene shown in 151-1062 site of SEQ ID No.3 and keeping other sequences of pET30(+) unchanged, and the recombinant expression vector is named as pET30 a-alpha-W. pET30 a-alpha-W contains His label fusion protein alpha-hisW gene, the nucleotide sequence of alpha-hisW gene is SEQ ID No.3, and the protein alpha-His shown in SEQ ID No.2 is coded. The recombinant E.coli containing pET30a- α -W was named BL21(DE3)/pET30a- α -W.

Digesting the PCR product of the pm alpha-W gene by BamHI and XhoI, and recovering a target fragment (pm alpha-W gene); meanwhile, the vector pET30a (+) is cut by BamHI and XhoI enzyme, and the large vector fragment is recovered; and connecting the recovered target fragment with the recovered vector large fragment to obtain the target plasmid. Coli BL21(DE3) competent cells were transformed with the plasmid of interest. This was spread evenly on LB plates containing kanamycin and cultured at 37 ℃ for 16 hours. The single colony is shake cultured overnight, the extracted plasmid is double-digested by BamHI and XhoI, the plasmid with correct restriction enzyme is sequenced, the sequencing result shows that the plasmid is a recombinant expression vector which is obtained by replacing the fragment between BamHI recognition sites and XhoI recognition sites of pET30a (+) by the pm alpha-W gene shown in the 151-th 1347 site of SEQ ID No.4 and keeping other sequences of pET30(+) unchanged, and the recombinant expression vector is named as pET30a-pm alpha-W. pET30a-pm alpha-W contains a pm alpha-hisW gene with a His tag, the nucleotide sequence of the pm alpha-hisW gene is SEQ ID No.4, and the pm alpha-hisW gene encodes a protein pm alpha-hisW shown in SEQ ID No. 5. The recombinant E.coli containing pET30a-pm alpha-W was named BL21(DE3)/pET30a-pm alpha-W.

3. Analysis and characterization of protein expression profiles

The four strains of BL21(DE3)/pET30 a-alpha-Y, BL21(DE3)/pET30 a-alpha-W, BL21(DE3)/pET30a-pm alpha-W and Escherichia coli BL21(DE3) (recipient bacteria for short) were individually inoculated into LB liquid medium containing 50. mu.g/ml kanamycin (medium obtained by adding kanamycin to the LB liquid medium to 50. mu.g/ml kanamycin), cultured at 37 ℃ with shaking to 0D with a Thermo MaxQ6000 type whole temperature shaker at 200rpm₆₀₀When the value (LB liquid medium containing 50. mu.g/mL kanamycin as a blank control) reached 0.6, one mL of the culture broth was taken out as a culture broth not induced to express (control), and isopropylthio-. beta. -D-galactoside (IPTG) was added to the remaining culture broth to induce expression. The four strains were induced at 16 ℃ for 13 hours with 0.75mM IPTG.

And taking the bacteria liquid with induced expression and the bacteria liquid without induced expression for analyzing the protein expression form. The specific steps are that 1mL of bacterial liquid is taken and placed in a 1.5mL centrifuge tube, the mark is made, the centrifugal separation is carried out for 30min at 8000rpm/min under the condition of 4 ℃, the supernatant is discarded, and the thalli sediment is collected. 1mL of PBS was added to resuspend the pellet, centrifuged at 8000rpm/min for 5min, and the supernatant was discarded. Adding 200 mu L PBS into the washed thallus precipitate, crushing thallus under high pressure, and cracking until the bacteria liquid is not sticky any more to obtain the whole mycoprotein liquid. The whole mycoprotein liquid is centrifuged for 30min at 16000rpm/min in a centrifuge at 4 ℃, supernatant (named as protein-containing supernatant) and sediment (named as protein-containing sediment) are collected respectively, and 50 mu L PBS is added into the protein-containing sediment to resuspend and wash the sediment. Adding 10 μ L of 5 xSDS-PAGE loading Buffer into the whole bacteria protein liquid, protein-containing supernatant and protein-containing precipitate, mixing, boiling in boiling water bath for 5min, cooling, and separating with a palm centrifuge. mu.L of the suspension was analyzed by SDS-PAGE electrophoresis, and the protein content was analyzed primarily in conjunction with protein gray scale analysis software. Transferring the gel after electrophoresis to an NC membrane, performing DAB coloration by taking a goat anti-mouse antibody of an anti-His label as a combined antibody, and performing Western-blot identification. The whole bacterial protein liquid and the protein-containing supernatant were filtered through a 0.22 μm filter and applied to a nickel column equilibrated in advance with solution 1 (solute and concentration: 20mM Tris, 150mM NaCl, solvent water, pH 8.0). The nickel column was loaded onto an AKTA machine, the impurity proteins in the nickel column were washed with 10 column volumes of

solution

1 and 10 column volumes of solution 2 (solutes and their concentrations are 20mM Tris, 150mM NaCl, 50mM imidazole, solvent is water, pH 8.0), respectively, and the protein peaks were monitored on the AKTA machine. The target protein suspended on the nickel column was washed with solution 3 (solute and its concentration are as follows: 20mM Tris, 150mM NaCl, 300mM imidazole, solvent is water, pH 8.0), and an eluted sample in which a peak of the target protein appeared was collected using AKTA, and this sample was referred to as a nickel column purified target protein sample.

The target protein sample purified by the nickel column was further purified by passing through a molecular sieve using Superdex200 gel column manufactured by GE. The mobile phase used solution 1. Removing a large amount of imidazole contained in the sample after the sample is purified by the molecular sieve, collecting an elution peak to obtain a target protein sample purified by the molecular sieve, and quantitatively analyzing the content of the protein (namely, soluble target protein) in the target protein sample purified by the molecular sieve by using a NanoDrop2000 ultramicro spectrophotometer (ND 2000). And measuring the protein content in the whole bacterial protein liquid by using a NanoDrop2000 ultramicro spectrophotometer (ND2000) to obtain the total protein content of the bacterial cells. After the protein-containing precipitate was dissolved in urea, the content of protein in the protein-containing precipitate was measured by a NanoDrop2000 ultramicro spectrophotometer (ND 2000).

The result shows that the whole mycoprotein liquid, the protein-containing supernatant and the protein-containing precipitate of the BL21(DE3)/pET30 a-alpha-Y with induced expression all contain the target protein alpha-his with the size of 41kD, and the whole mycoprotein liquid of the BL21(DE3)/pET30 a-alpha-Y with non-induced expression does not contain the target protein alpha-his with the size of 41 kD; the target protein alpha-his in the whole bacterial protein liquid of the induction-expressed BL21(DE3)/pET30 a-alpha-Y accounts for 90 percent of the total bacterial protein (the total bacterial protein), the target protein alpha-his in the protein-containing supernatant of the induction-expressed BL21(DE3)/pET30 a-alpha-Y accounts for 94 percent of the target protein alpha-his in the whole bacterial protein liquid of the induction-expressed BL21(DE3)/pET30 a-alpha-Y, and 94 percent of the target protein alpha-his is soluble protein; the target protein alpha-his in the protein-containing precipitate of the induction-expressed BL21(DE3)/pET30 a-alpha-Y accounts for 6 percent of the target protein alpha-his in the whole bacterial protein liquid of the induction-expressed BL21(DE3)/pET30 a-alpha-Y, and 6 percent of the target protein alpha-his is insoluble inclusion body protein; the result shows that the target protein alpha-his of BL21(DE3)/pET30 a-alpha-Y expressed by induction accounts for 90 percent of the total protein of the thallus, and that 94 percent of the target protein alpha-his expressed by BL21(DE3)/pET30 a-alpha-Y is soluble protein and 6 percent is insoluble inclusion body protein. The whole bacterial protein liquid of the BL21(DE3)/pET30 a-alpha-W with induced expression, the protein-containing supernatant and the protein-containing precipitate all contain target protein alpha-his with the size of 41kD, and the whole bacterial protein liquid of the BL21(DE3)/pET30 a-alpha-W with non-induced expression does not contain the target protein alpha-his with the size of 41 kD; the target protein alpha-his in the whole bacterial protein liquid of the BL21(DE3)/pET30 a-alpha-W subjected to induction expression accounts for 90 percent of the total bacterial protein, the target protein alpha-his in the protein-containing supernatant of BL21(DE3)/pET30 a-alpha-W subjected to induction expression accounts for 8 percent of the target protein alpha-his in the whole bacterial protein liquid of BL21(DE3)/pET30 a-alpha-W subjected to induction expression, and the 8 percent of the target protein alpha-his is soluble protein; the target protein alpha-his in the protein-containing precipitate of the induction-expressed BL21(DE3)/pET30 a-alpha-W accounts for 92 percent of the target protein alpha-his in the whole bacterial protein liquid of the induction-expressed BL21(DE3)/pET30 a-alpha-W, and 92 percent of the target protein alpha-his is insoluble inclusion body protein; the result shows that the target protein alpha-his of BL21(DE3)/pET30 a-alpha-W expressed by induction accounts for 90% of the total protein of the thallus, and 8% of the target protein alpha-his expressed by BL21(DE3)/pET30 a-alpha-W accounts for 92% of soluble protein and is insoluble inclusion body protein. The non-induction expression BL21(DE3)/pET30a-pm alpha-W whole bacterial protein liquid, the induction expression BL21(DE3)/pET30a-pm alpha-W whole bacterial protein liquid, the protein-containing supernatant and the protein-containing sediment do not contain the target protein pm alpha-his with the size of 51 kD; it shows that BL21(DE3)/pET30a-pm alpha-W does not express the target protein pm alpha-his. The whole bacterial protein liquid of the escherichia coli BL21(DE3) subjected to induction expression does not contain the target protein alpha-his with the size of 41 kD; it is shown that Escherichia coli BL21(DE3) does not express the desired protein alpha-his. The total protein mass of the thalli expressed by the same number of Colony Forming Units (CFU) and the induction expression of BL21(DE3)/pET30 a-alpha-Y and the induction expression of BL21(DE3)/pET30 a-alpha-W is the same (FIG. 1 and FIG. 2).

4. Purification of alpha-his

BL21(DE3)/pET30 a-. alpha. -Y was inoculated into LB liquid medium containing 50. mu.g/ml kanamycin (a medium obtained by adding kanamycin to LB liquid medium to 50. mu.g/ml kanamycin concentration), cultured at 37 ℃ with shaking to 0D using a ThermoMaxQ6000 type whole temperature shaker at 200rpm₆₀₀Value to 0D₆₀₀When the value (blank with LB liquid medium containing 50. mu.g/ml kanamycin) reached 0.6, isopropylthio-. beta. -D-galactoside (IPTG) was added for inducible expression. The induction of expression was carried out with 0.75mM IPTG for 13 hours at 16 ℃.

Collecting bacterial liquid after IPTG induced expression for 13h to collect bacterial precipitation. Adding PBS to resuspend the precipitate, centrifuging at 8000rpm/min for 5min, and discarding the supernatant. Adding PBS into the washed thallus precipitate, crushing thallus under high pressure, cracking until the thallus is not viscous, centrifuging at 16000rpm/min in a centrifuge at 4 ℃ for 30min, collecting supernatant (named as protein-containing supernatant), and discarding the precipitate. The protein-containing supernatant was filtered through a 0.22 μm filter and applied to a nickel column equilibrated in advance with solution 1 (a solution of a solute and its concentration shown below: 20mM Tris, 150mM NaCl, a solvent which is water, pH 8.0). The nickel column was loaded onto an AKTA machine, the impurity proteins in the nickel column were washed with 10 column volumes of

solution

1 and 10 column volumes of solution 2 (solutes and their concentrations are 20mM Tris, 150mM NaCl, 50mM imidazole, solvent is water, pH 8.0), respectively, and the protein peaks were monitored on the AKTA machine. The target protein suspended on the nickel column was washed with solution 3 (solute and its concentration are as follows: 20mM Tris, 150mM NaCl, 300mM imidazole, solvent is water, pH 8.0), and an eluted sample in which a peak of the target protein appeared was collected using AKTA and was referred to as nickel column purified α -his (FIG. 3).

The nickel column purified α -his was further purified by passing it through a molecular sieve using Superdex200 gel column manufactured by GE. The mobile phase used solution 1. After purification by molecular sieve, a large amount of imidazole contained in the sample was removed, and the elution peak was collected to obtain a molecular sieve-purified α -his protein (fig. 5), and the purity of the obtained protein was quantitatively analyzed by using a NanoDrop2000 ultramicro spectrophotometer (ND 2000).

The amino acid sequence of the purified alpha-his is subjected to mass spectrometry, and the result shows that the amino acid sequence of the alpha-his is shown as SEQID No. 2.

Three strains of BL21(DE3)/pET30 a-alpha-Y, BL21(DE3)/pET30a-pm alpha-W and Escherichia coli BL21(DE3) (recipient bacteria for short) were individually inoculated into LB liquid medium containing 50. mu.g/ml kanamycin (medium obtained by adding kanamycin to LB liquid medium to 50. mu.g/ml kanamycin), cultured at 37 ℃ with a ThermoMaxQ6000 type whole temperature shaker at 200rpm to 0D₆₀₀When the value (LB liquid medium containing 50. mu.g/mL kanamycin as a blank control) reached 0.6, one mL of the culture broth was taken out as a culture broth not induced to express (control), and isopropylthio-. beta. -D-galactoside (IPTG) was added to the remaining culture broth to induce expression. The three strains were induced at 16 ℃ for 13 hours with 0.75mM IPTG.

And taking the bacteria liquid with induced expression and the bacteria liquid without induced expression for analyzing the protein expression form. The specific steps are that 1mL of bacterial liquid is taken and placed in a 1.5mL centrifuge tube, the mark is made, the centrifugal separation is carried out for 30min at 8000rpm/min under the condition of 4 ℃, the supernatant is discarded, and the thalli sediment is collected. 1mL of PBS was added to resuspend the pellet, centrifuged at 8000rpm/min for 5min, and the supernatant was discarded. Adding 200 mu L PBS into the washed thallus precipitate, crushing thallus under high pressure, and cracking until the bacteria liquid is not sticky any more to obtain the whole mycoprotein liquid. The whole mycoprotein liquid is centrifuged for 30min at 16000rpm/min in a centrifuge at 4 ℃, supernatant (named as protein-containing supernatant) and sediment (named as protein-containing sediment) are collected respectively, and 50 mu L PBS is added into the protein-containing sediment to resuspend and wash the sediment. Adding 10 μ L of 5 xSDS-PAGE loading Buffer into the whole bacteria protein liquid, protein-containing supernatant and protein-containing precipitate, mixing, boiling in boiling water bath for 5min, cooling, and separating with a palm centrifuge. mu.L of the suspension was taken for SDS-PAGE analysis. The result shows that the target protein alpha-his expressed by BL21(DE3)/pET30 a-alpha-Y exists in the supernatant of the bacterial broken thallus in a soluble form, the band expression of the soluble protein is obvious, and the impurity in the supernatant is less. By optimizing the purification and elution conditions of the AKTA machine, a soluble target protein band with better purity can be obtained. After further purification by molecular sieves, a large amount of imidazole contained in the protein sample can be removed (fig. 4). The purified soluble protein passing through the molecular sieve can be used as a diagnostic antigen, prepared into a monoclonal antibody or further researched on the relationship between the protein function and conformation.

In addition, according to the above method, the sequence between the NheI and NotI sites of the restriction enzyme of pET28a (+) was replaced with the α -Y gene shown in the 151 th-1062 th site of SEQ ID No.1, and the other sequence of pET28a (+) was kept unchanged to obtain a recombinant expression vector containing the α -Y gene, which was named pET28a- α -Y. pET28 a-alpha-Y was transformed into competent cells of E.coli BL21(DE3), and the resulting recombinant E.coli was named BL21(DE3)/pET28 a-alpha-Y. BL21(DE3)/pET28 a-. alpha. -Y was inoculated into LB liquid medium containing 50. mu.g/ml kanamycin (a medium obtained by adding kanamycin to LB liquid medium to 50. mu.g/ml kanamycin concentration), cultured at 37 ℃ with shaking to 0D using Thermo MaxQ6000 type whole temperature shaker at 200rpm₆₀₀When the value (LB liquid medium containing 50. mu.g/ml kanamycin as a blank control) reached 0.6, lmL bacterial liquid was taken out as a bacterial liquid not induced to express (control), and isopropylthio-. beta. -D-galactoside (IPTG) was added to the remaining liquid to induce expression. The induction of expression was carried out with 0.75mM IPTG for 13 hours at 16 ℃. And (4) analyzing the protein expression form of the induced expression bacterial liquid and the non-induced expression bacterial liquid according to the method. The result shows that no target protein is expressed in the liquid of the BL21(DE3)/pET28 a-alpha-Y whole bacterial protein which is not induced to express, the liquid of the BL21(DE3)/pET28 a-alpha-Y whole bacterial protein which is induced to express, the supernatant of the BL21(DE3)/pET28 a-alpha-Y protein-containing liquid which is induced to express and the BL21(DE3)/pET28 a-alpha-Y protein-containing sediment which is induced to express. It can be seen that the same foreign target gene is used(alpha-Y gene), in different BL21(DE3) expression vectors-pET 28a (+) and pET30a (+), the expression situation of exogenous target genes is greatly different, the alpha-Y gene is introduced into escherichia coli BL21(DE3) through pET30a (+) to obtain high-efficiency soluble expression of the alpha-Y gene, and the alpha-Y gene is introduced into escherichia coli BL21(DE3) through pET28a (+) but is not expressed.

Example 2 animal immunoprotective assay of alpha-his

1. Preparation of vaccines against clostridium perfringens

The alpha-his protein purified by the molecular sieve in example 1 was dissolved in sterile PBS to obtain an alpha-his solution having an alpha-his concentration of 1000. mu.g/mL for immunization. Mixing the alpha-his solution and Freund's adjuvant at a volume of 1:1, emulsifying to prepare oil emulsion vaccine, and naming the oil emulsion vaccine as prime vaccine. Mixing the alpha-his solution and incomplete Freund's adjuvant at a volume of 1:1, emulsifying to prepare oil emulsion vaccine, and naming the oil emulsion vaccine as the diabrotic vaccine.

Taking out the virulent strain C57-10 of clostridium perfringens type A, the virulent strain C58-5 of clostridium perfringens type B, the virulent strain C59-4 of clostridium perfringens type C and the virulent strain C60-11 of clostridium perfringens type D, which are purchased from Chinese veterinary medicine supervision. Carefully wiping the outer wall of the ampoule bottle for storing the strains by using a 75% alcohol cotton ball in a super-clean workbench, then marking the upper third of the ampoule bottle by using a grinding wheel, and wrapping the ampoule bottle by using dry sterile gauze to break the ampoule bottle. Sucking 400 mu L of anaerobic liver broth liquid culture medium, repeatedly blowing and beating the strains in the ampoule, and dissolving the strain suspension by the freeze-dried strains. According to the following steps of 1: 100 proportion, inoculating the bacterial suspension into a test tube of anaerobic pork liver soup containing beef extract, and covering the upper layer of the test tube with 1-2cm of liquid paraffin to isolate air. And (3) placing the test tube inoculated with the bacteria into an anaerobic incubator, placing the anaerobic incubator in a constant-temperature incubator at 37 ℃, and observing the growth condition of the bacteria after culturing for 16-24 hours. The recovered strain is smeared, stained and examined under the microscope, and is used after 1-2 generations after no error is confirmed, and a part of the strain is stored in 30% glycerol saline at-80 ℃ in a refrigerator.

2. Clostridium perfringens type a challenge test

The resistance test of alpha-his to Clostridium perfringens type A virulent strain C57-10 was tested as follows:

30 female Kunming mice weighing 18-22g were randomly divided into 2 groups (a group of 20 challenge doses, and a PBS control group of 10 mice). In the challenge dose group, the first immunization, the second immunization and the third immunization are all immunized by adopting a subcutaneous injection method, the first immunization uses a first-immunity vaccine, the second immunization and the third immunization use a second-immunity vaccine, and the immunization dose is 0.2 mL/vaccine each time (the alpha-his immunization dose is 100 mu g/vaccine); each mouse in the PBS control group was immunized first, second, and third, subcutaneously with 0.2mL PBS. Before the first immunization, the mice are subjected to tail-cutting blood collection for one time, and serum is separated and used as negative control serum. After the first immunization, the second immunization was performed at 14d intervals, and the third immunization was performed 14d after the second immunization. Two weeks after the third immunization, mice in each challenge dose group and mice in each PBS control group were injected with 1.5X 10 intraperitoneal injections⁹A virulent strain C57-10 of clostridium perfringens type A of cfu is subjected to a challenge test. From the start of the immunization, mice were bled once a week for 5 mice per group, and sera were separated and stored in a refrigerator at-80 ℃ for antibody detection. The indirect ELISA is adopted to detect the antibody level of the immune animals, and the specific method is as follows:

1) coating: diluting the alpha-his purified by the molecular sieve in the example 1 by using 0.05mol/L of carbonate coating buffer solution of pH 9.0, then adding the diluted alpha-his into an ELISA plate one by one according to 100 mu L/hole, and putting the added ELISA plate in a refrigerator at 4 ℃ for overnight;

2) washing: the ELISA plates were removed from the 4 ℃ freezer, the liquid in the wells of the plates was discarded and patted dry on filter paper, 200. mu.L of PBST was added to each well and the plates were washed in a plate washer and repeated 4 times.

3) And (3) sealing: adding 100 μ L of PBST solution containing 5% skim milk to each well of the ELISA plate, and incubating in an incubator at 37 ℃ for 1 h;

4) washing: adding 200 mu L of PBST into each hole, placing the PBST into a plate washing machine for washing the plate, and repeating the steps for 4 times;

5) adding serum to be detected: diluting the serum to be detected with sterilized PBS according to a certain proportion, adding 100 μ L of the serum to each hole, setting negative control, positive control and blank control holes at the same time, and incubating for 1h in an incubator at 37 ℃;

6) washing: adding 200 mu L of PBST into each hole, placing the PBST into a plate washing machine for washing the plate, and repeating the steps for 4 times;

7) and (3) binding of an enzyme-labeled secondary antibody: HRP-labeled goat anti-mouse secondary antibodies were blocked with PBS blocking buffer containing 5% skim milk at a ratio of 1: diluting with 20000-1: 40000 concentration, adding 100 μ L into each well, and incubating at 37 deg.C for 1 h;

8) washing: adding 200 mu L of PBST into each hole, placing the PBST into a plate washing machine for washing the plate, and repeating the steps for 4 times;

9) and (3) color development reaction: adding a freshly prepared TMB color developing solution into each hole of the ELISA plate according to 100 mu L/hole, and placing the ELISA plate into a 37 ℃ incubator to be protected from light for color development for 15 min;

10) and (3) terminating the reaction: to 50. mu.L/well was added 2mol/L of concentrated H₂SO₄Stopping the reaction of the stop solution in an incubator at 37 deg.C for 5min to stop color development;

11) reading: placing the ELISA plate with the color development stopped in an enzyme-linked immunosorbent assay (ELISA) instrument for detecting the OD₄₅₀The value of (c).

12) Judging a detection result: and (5) judging the detection result by using the determined positive and negative critical values. Positive cut-off value-OD of negative sample₄₅₀Mean +3S (S is standard deviation). The titer of the serum to be detected is the corresponding serum dilution when the OD value of the serum to be detected is more than or equal to the positive critical value.

3. Clostridium perfringens type B challenge test

Except that the clostridium perfringens virulent strain C57-10 of type A is replaced by the clostridium perfringens virulent strain C58-5 of type B, the attacking dose is adjusted to be 2 multiplied by 10⁹The operation is exactly the same except for cfu.

4. Clostridium perfringens type C challenge test

Except that the clostridium perfringens type A virulent strain C57-10 is replaced by clostridium perfringens type C59-4, the attacking dose is adjusted to 1.5 multiplied by 10⁸The operation is exactly the same except for cfu.

5. Clostridium perfringens type D challenge test

Except that the clostridium perfringens virulent strain C57-10 of type A is replaced by the clostridium perfringens virulent strain C60-11 of type D, the attacking dose is adjusted to be 1.8 multiplied by 10⁹The operation is exactly the same except for cfu.

After three weeks of immunization, the mice were challenged with the doses of clostridium perfringens 1MLD 100 of each type, and the death of the mice was observed and recorded within one week. The result of the attacking shows that the attacking dose group (immune alpha-his) has certain immune protection effect on the attacking of various clostridium perfringens. The immune protection rate of the challenge dose group (immune α -his) against clostridium perfringens type a challenge within 7 days was 100% (20 survived, 0 died), all PBS control mice died; the immune protection rate against clostridium perfringens type B challenge in the challenge dose group (immune α -his) was 90% (18 survived, 2 died), all PBS control mice died; the immune protection rate against clostridium perfringens type C challenge was 85% (17 survived, 3 dead) in the challenge dose group (immune α -his), all mice in the PBS control group died; the immune protection rate against clostridium perfringens type D challenge was 90% (18 survived, 2 died) in the challenge dose group (immune α -his), whereas all PBS control mice died. The purified alpha-his is used as a diagnostic antigen to coat an enzyme label plate for detecting and detecting mouse immune antigen or serum antibody after challenge, and the detection method established by using the alpha-his as the diagnostic antigen is found to have very good sensitivity and specificity. The antibody titer levels in the serum of 0-6 weeks after the initial immunization of the mice in each experimental group are respectively detected, and the results show that the antibody titer of the alpha-his fusion toxin protein immune group is obviously improved, the antibody titer is rapidly increased after the second immunization, the antibody titer reaches the peak value 7-14 days after the third immunization, and the highest antibody titer reaches 1: 128000.

example 3 optimization of conditions for inducible expression of alpha-his

1. Optimization of induction temperature and time

BL21(DE3)/pET30 a-. alpha. -Y was inoculated into LB liquid medium containing 50. mu.g/ml kanamycin (a medium obtained by adding kanamycin to LB liquid medium to 50. mu.g/ml kanamycin concentration), cultured at 37 ℃ with shaking to 0D using a ThermoMaxQ6000 type whole temperature shaker at 200rpm₆₀₀When the value (LB liquid medium containing 50. mu.g/ml kanamycin as a blank) reached 0.6, isopropylthio-. beta. -D-galactoside (IPTG) was added theretoThe following 6 inducible expressions were performed. The first induced expression was induced with 0.75mM IPTG for 1 hour at 37 ℃. The second induced expression was induced with 0.75mM IPTG for 2 hours at 37 ℃. The third induced expression was induced with 0.75mM IPTG for 4 hours at 37 ℃. A fourth inducible expression was induced with 0.75mM IPTG for 5 hours at 37 ℃. The fifth inducible expression was induced with 0.75mM IPTG for 13 hours at 16 ℃. A sixth inducible expression is induced with 0.75mM IPTG for 24 hours at 16 ℃.

Placing 1mL of the induced recombinant bacteria liquid in a 1.5mL centrifuge tube, marking, centrifuging at 8000rpm/min at 4 ℃ for 30min, discarding the supernatant, and collecting the thallus precipitate. 1mL of PBS was added to resuspend the pellet, centrifuged at 8000rpm/min for 5min, and the supernatant was discarded. Adding 200 mu L PBS into the washed thallus precipitate, crushing thallus under high pressure, and cracking until the bacteria liquid is not sticky any more to obtain the whole mycoprotein liquid. Adding 10 μ L of 5 xSDS-PAGE loading Buffer into the whole bacterial protein liquid, mixing well, boiling in boiling water bath for 5min, cooling the sample, and separating instantly with a palm centrifuge. mu.L of the suspension was taken for SDS-PAGE analysis. The result shows that the expression level of the alpha-his protein gradually increases along with the time extension of BL21(DE3)/pET30 a-alpha-Y under the conditions that the induction temperature is 37 ℃ and the induction time is 1-4h, and the expression level of the protein is reduced under the induction condition of 5 h. However, the expression level of alpha-his is increased along with the reduction of the induction temperature, when the induction temperature is reduced to 16 ℃ and the induction time is 13 hours, the expression level of alpha-his reaches the maximum, and the target protein alpha-his accounts for 90 percent of the total protein of the whole bacteria. When the culture is continued for 24 hours, the expression level of alpha-his is slightly reduced, so that the optimal induction temperature of BL21(DE3)/pET30 a-alpha-Y is verified to be 16 ℃ through experiments, and the induction time is 13-24 hours.

2. Optimisation of IPTG concentration

BL21(DE3)/pET30 a-. alpha. -Y was inoculated into LB liquid medium containing 50. mu.g/ml kanamycin (a medium obtained by adding kanamycin to LB liquid medium to 50. mu.g/ml kanamycin concentration), cultured at 37 ℃ with shaking to 0D using a ThermoMaxQ6000 type whole temperature shaker at 200rpm₆₀₀When the value (LB liquid medium containing 50. mu.g/ml kanamycin as a blank) reached 0.6, the following 6 inducible expressions were carried out by adding isopropylthio-. beta. -D-galactoside (IPTG), respectively.The first induction of expression was induced with 0.1mM IPTG for 13 hours at 16 ℃. The second induced expression was induced with 0.3mM IPTG for 13 hours at 16 ℃. The third induced expression was induced with 0.5mM IPTG for 13 hours at 16 ℃. A fourth inducible expression is induced with 0.75mM IPTG for 13 hours at 16 ℃. The fifth inducible expression was induced with 1mM IPTG for 13 hours at 16 ℃. A sixth inducible expression is induced with 0mM IPTG for 13 hours at 16 ℃.

Placing 1mL of the induced recombinant bacteria liquid in a 1.5mL centrifuge tube, marking, centrifuging at 8000rpm/min at 4 ℃ for 30min, discarding the supernatant, and collecting the thallus precipitate. 1mL of PBS was added to resuspend the pellet, centrifuged at 8000rpm/min for 5min, and the supernatant was discarded. Adding 200 mu L PBS into the washed thallus precipitate, crushing thallus under high pressure, and cracking until the bacteria liquid is not sticky any more to obtain the whole mycoprotein liquid. Centrifuging the whole bacteria protein liquid in a centrifuge at 4 ℃ at 16000rpm/min for 30min, collecting the supernatant (named as protein-containing supernatant), adding 10 μ L of 5 xSDS-PAGE loading Buffer into the protein-containing supernatant, mixing well, boiling in a boiling water bath for 5min, cooling the sample, and performing flash separation by using a palm centrifuge. mu.L of the suspension was taken for SDS-PAGE analysis. The results show that the expression amount of the alpha-his is different under the induction of IPTG with different concentrations. Alpha-his expression was in an increasing relationship with the addition of IPTG at concentrations between 0.1 and 0.75 mM. When the IPTG induction concentration is 1mM, the protein expression amount is reduced, which may be related to the toxicity of IPTG itself. Therefore, an IPTG concentration of 0.75mM was chosen as the optimal induction concentration.

3. Optimization of Induction time

BL21(DE3)/pET30 a-. alpha. -Y was inoculated into LB liquid medium containing 50. mu.g/ml kanamycin (a medium obtained by adding kanamycin to LB liquid medium to 50. mu.g/ml kanamycin concentration), cultured at 37 ℃ with shaking to 0D using a ThermoMaxQ6000 type whole temperature shaker at 200rpm₆₀₀When the value (LB liquid medium containing 50. mu.g/ml kanamycin as a blank) reached 0.6, the following 2 inducible expressions were carried out by adding isopropylthio-. beta. -D-galactoside (IPTG), respectively. The first induced expression was induced with 0.75mM IPTG for 13 hours at 16 ℃. The second induced expression was induced with 0.75mM IPTG for 16 hours at 16 ℃.

Placing 1mL of the induced recombinant bacteria liquid in a 1.5mL centrifuge tube, marking, centrifuging at 8000rpm/min at 4 ℃ for 30min, discarding the supernatant, and collecting the thallus precipitate. 1mL of PBS was added to resuspend the pellet, centrifuged at 8000rpm/min for 5min, and the supernatant was discarded. Adding 200 mu L PBS into the washed thallus precipitate, crushing thallus under high pressure, and cracking until the bacteria liquid is not sticky any more to obtain the whole mycoprotein liquid.

Adding 10 μ L of 5 xSDS-PAGE loading Buffer into the whole bacterial protein liquid, mixing well, boiling in boiling water bath for 5min, cooling the sample, and separating instantly with a palm centrifuge. mu.L of the suspension was taken for SDS-PAGE analysis.

The result shows that the expression quantity of the target protein alpha-his is not greatly changed when BL21(DE3)/pET30 a-alpha-Y is induced at the temperature of 16 ℃ for 13h and 16h, the expressed protein is almost all soluble protein, and the insoluble inclusion body protein in the precipitate is hardly expressed. When the induction temperature is 16 ℃, the purity of the soluble target protein expressed under the condition of 13h and 16h is high, and the expression quantity is close. Therefore, through experimental verification, the optimal induction temperature of the recombinant bacteria is further determined to be 16 ℃, and the induction time is 13-16 h.

Claims

1. A method for producing a recombinant α protein, comprising the step of expressing a gene encoding a recombinant α protein in an organism to obtain the recombinant α protein; the recombinant alpha protein is a protein consisting of an amino acid sequence of SEQ ID No. 2; the expression of the coding gene of the recombinant alpha protein in organisms comprises the steps of introducing the coding gene of the recombinant alpha protein into a receptor microorganism to obtain a recombinant microorganism expressing the recombinant alpha protein, culturing the recombinant microorganism and expressing to obtain the recombinant alpha protein; the coding gene of the protein is a DNA molecule with a coding sequence shown in SEQ ID No. 1;

the recombinant microorganism is a recombinant microorganism which is obtained by introducing pET30 a-alpha-Y into Escherichia coli BL21(DE3) and expresses recombinant alpha protein with an amino acid sequence of SEQ ID No.2, the recombinant microorganism is named as BL21(DE3)/pET30 a-alpha-Y, the pET30 a-alpha-Y is a recombinant vector obtained by replacing a sequence between BamHI and XhoI sites of a vector pET30a (+) with a DNA fragment shown in 151-th 1062 site of SEQ ID No. 1; the expression is induced expression, which is induced with 0.75mM IPTG for 13-16 hours at 16 ℃.

2. The method of claim 1, wherein: the inducible expression was induced with 0.75mM IPTG for 13 hours at 16 ℃.

3. The method of claim 1, wherein: the inducible expression was induced with 0.75mM IPTG for 16 hours at 16 ℃.

4. A recombinant alpha protein produced according to the method of any one of claims 1-3.

5. A biomaterial related to the recombinant alpha protein, the biomaterial being any one of the following B1) to B8):

B1) a nucleic acid molecule encoding said recombinant alpha protein, said recombinant alpha protein being produced according to the method of any one of claims 1-3;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector comprising the nucleic acid molecule of B1);

B4) a recombinant vector comprising the expression cassette of B2);

B5) a recombinant microorganism comprising the nucleic acid molecule of B1);

B6) a recombinant microorganism comprising the expression cassette of B2);

B7) a recombinant microorganism containing the recombinant vector of B3);

B8) a recombinant microorganism containing the recombinant vector of B4);

the nucleic acid molecule is a DNA molecule with a coding sequence shown in SEQ ID No. 1;

the recombinant vector is pET30 a-alpha-Y described in claim 1;

the recombinant microorganism is BL21(DE3)/pET30 a-alpha-Y described in claim 1.

6. A vaccine for preventing Clostridium perfringens infection in an animal comprising the recombinant alpha protein of claim 4 or the biomaterial of claim 5.

7. The vaccine of claim 6, wherein: the active ingredient of the vaccine for preventing clostridium perfringens infection of animals is the recombinant alpha protein of claim 4 or the biological material of claim 5.

8. Any of the following applications:

y1) the use of the recombinant alpha protein of claim 4 for the preparation of a vaccine against Clostridium perfringens;

y2) use of the biomaterial of claim 5 for the manufacture of a vaccine against Clostridium perfringens;

y3) use of the process according to any one of claims 1 to 3 for the manufacture of a vaccine against clostridium perfringens.