CN106008719B

CN106008719B - Preparation method of fusion protein for inhibiting clostridium perfringens infection

Info

Publication number: CN106008719B
Application number: CN201610302606.XA
Authority: CN
Inventors: 宋晓晖; 孙雨; 翟新验; 董浩
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: CN106008719A

Abstract

The invention discloses a preparation method of a fusion protein for inhibiting clostridium perfringens infection. The method comprises the steps of expressing a gene encoding a protein in an organism to obtain the protein; the organism is a microorganism, a plant or a non-human animal; the 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 an amino acid sequence shown in the 51 st-937 th positions of SEQ ID No. 2; c) a fusion protein obtained by carboxyl-terminal or/and amino-terminal fusion protein labels of the protein shown in a) or b). The fusion protein prepared by the method 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

Preparation method of fusion protein for inhibiting clostridium perfringens infection

Technical Field

The invention relates to a preparation method of fusion protein for inhibiting clostridium perfringens infection in the field of biotechnology.

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. Currently, the immunity of clostridium perfringens is mainly realized by traditional inactivated monovalent vaccine, multiple vaccine or toxoid vaccine, although the traditional vaccines can induce animals to generate certain protective antibodies, the traditional vaccines have the defects of low safety, poor stability, large side reaction after immunization and the like, the application of the traditional vaccines is greatly limited, and the development of a genetic engineering subunit polyvalent vaccine aiming at multiple serotypes becomes a key research target of researchers in various countries in the world. Because the genetic engineering subunit multivalent vaccine contains the effective immune components necessary for generating protective immune response and removes the components irrelevant to immunity, the vaccine has better safety and stability compared with the traditional vaccine, simultaneously eliminates pyrogen, stressors, allergens and other reaction sources in the components of the traditional vaccine, and well solves the problems of side reactions, inflammatory stimulation reactions and the like of animals immunized by the traditional vaccine.

The expression and immunogenicity research of Gong Xue glu, etc. Clostridium perfringens alpha-beta 2-epsilon toxin fusion protein in colibacillus, China prevention veterinary academy report, No. 37, No.2, 2015 2, constructs a recombinant plasmid containing alpha, beta and epsilon fusion genes by utilizing a PCR technology, performs induced expression of the alpha-beta 2-epsilon fusion protein, provides a genetic material for further developing polyvalent genetic engineering subunit vaccine, and provides a new way for solving animal necrotic enteritis and enterotoxemia puzzling the animal husbandry in China. However, the alpha-beta 2-epsilon fusion protein expressed by the recombinant plasmid is of an inclusion body structure, and the expression quantity of the alpha-beta 2-epsilon fusion protein accounts for 18.4 percent of the total protein content of thalli.

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 technical problems, the invention provides a method for preparing soluble protein for inhibiting clostridium perfringens infection.

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

the 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 an amino acid sequence shown in the 51 st-937 th positions of SEQ ID No. 2;

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 - β 2- ε -his, and the protein of b) is named α - β 2- ε. SEQ ID No.2 consists of 937 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 expression of the gene encoding the protein in the organism comprises introducing the gene encoding the protein into a recipient microorganism to obtain a recombinant microorganism expressing the protein, and culturing the recombinant microorganism to express the 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) below:

1) the coding sequence is a DNA molecule (coding alpha-beta 2-epsilon-his) shown in SEQ ID No. 1;

2) the coding sequence is a DNA molecule (coding for alpha-beta 2-epsilon) shown in the 151-2814 position of SEQ ID No. 1;

3) a DNA molecule which has 90% or more identity with the DNA molecule defined in 1) or 2) and encodes the protein.

Wherein, SEQ ID No.1 consists of 2820 nucleotides, the name is alpha-beta 2-epsilon-hisY gene, and the coded amino acid sequence is protein alpha-beta 2-epsilon-his of SEQ ID No. 2. The DNA molecule shown in position 151-2814 of SEQ ID No.1 is an alpha-beta 2-epsilon-Y gene encoding a protein alpha-beta 2-epsilon consisting of the amino acid sequence shown in positions 51-937 of SEQ ID No. 2.

In the above-mentioned method, the recombinant microorganism is a recombinant microorganism expressing a protein whose amino acid sequence is SEQ ID No.2, which is obtained by introducing pET30 a-. alpha. -beta.2-. epsilon. -Y into E.coli BL21(DE3), and the recombinant microorganism is named BL21(DE3)/pET30 a-. alpha. -beta.2-. epsilon. -Y, and pET30 a-. alpha. -beta.2-. epsilon. -Y is a recombinant vector obtained by replacing the sequence between the BamHI and XhoI sites of vector pET30a (+) with a DNA fragment represented by position 151-2814 (encoding. alpha. -beta.2-. epsilon.) 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.

The application of the method in preparing the vaccine against clostridium perfringens also belongs to the protection scope of the invention.

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 protein of the present invention or the related biological material thereof can be administered to a patient as a drug directly, or can be administered to a patient after being mixed with a suitable carrier or excipient, so as to achieve 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 the DNA molecule shown in the 151-position 2814 of SEQ ID No.1 into BamHI and XhoI sites of pET30a (+) to obtain a recombinant expression vector pET30 a-alpha-beta 2-epsilon-Y for expressing the recombinant protein alpha-beta 2-epsilon-his of SEQ ID No. 2. The recombinant expression vector pET30 a-alpha-beta 2-epsilon-Y is introduced into Escherichia coli BL21(DE3) to obtain the soluble target protein alpha-beta 2-epsilon-his. The invention optimizes the expression condition of alpha-beta 2-epsilon-his and further improves the expression quantity of alpha-beta 2-epsilon-his, the content of alpha-beta 2-epsilon-his reaches 65 percent of the total protein of the thalli after the induction is carried out for 13 to 16 hours at 16 ℃ by 0.75mM IPTG, and the expressed alpha-beta 2-epsilon-his is soluble by 92 percent. Immunization of animals with α - β 2- ε -hisY results in higher serum antibody levels in the animals and is resistant to challenge by Clostridium perfringens. The immune protection rate of alpha-beta 2-epsilon-his against clostridium perfringens type a challenge within 7 days is 100%, and all mice in the PBS control group die; the immune protection rate of the alpha-beta 2-epsilon-his against challenge of clostridium perfringens type B is 100%, and all mice in a PBS control group die; the immune protection rate of the immune alpha-beta 2-epsilon-his against the challenge of clostridium perfringens type C is 90%, and all mice in a PBS control group die; the immune protection rate of alpha-beta 2-epsilon-his against challenge with clostridium perfringens type D is 100%, whereas all PBS control mice died. The antibody titer reaches the peak value 7-14 days after the third immunization of the alpha-beta 2-epsilon-his, and the highest antibody titer reaches 1: 128000. the alpha-beta 2-epsilon-hisY has good solubility and simple and easy purification, and can be used as a diagnostic antigen, prepared into a monoclonal antibody or used for further researching the relationship between the protein function and the conformation.

Drawings

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

In the figure, M is Marker and is respectively 180KD, 130KD, 95KD, 72KD, 55KD, 43KD, 34KD and 26KD from top to bottom; 1. receptor bacterium whole mycoprotein liquid for inducible expression, 2, BL21(DE3)/pET30 a-alpha-beta 2-epsilon-Y whole mycoprotein liquid for inducible expression, 3, BL21(DE3)/pET30 a-alpha-beta 2-epsilon-Y whole mycoprotein liquid for inducible expression, 4, BL21(DE3)/pET30 a-alpha-beta 2-epsilon-Y protein-containing supernatant for inducible expression, 5, BL21(DE3)/pET30 a-alpha-beta 2-epsilon-Y protein-containing precipitate for inducible expression, 6, BL21(DE3)/pET30 a-alpha-beta 2-epsilon-W whole mycoprotein liquid for inducible expression, 7, BL21(DE3)/pET30 a-alpha-beta 2-W whole mycoprotein liquid for inducible expression, 8, BL 3 (DE3)/pET 3-beta 2-W protein-containing supernatant for inducible expression Liquid, 9, BL21(DE3)/pET30 a-alpha-beta 2-epsilon-W protein-containing sediment for inducible expression, 10, BL21(DE3)/pET30a-pm alpha-beta 2-epsilon-W whole bacterial protein liquid for non-inducible expression, 11, BL21(DE3)/pET30a-pm alpha-beta 2-epsilon-W whole bacterial protein liquid for inducible expression, 12, BL21(DE3)/pET30a-pm alpha-beta 2-epsilon-W protein-containing supernatant for inducible expression, and 13, BL21(DE3)/pET30a-pm alpha-beta 2-epsilon-W protein-containing sediment for inducible expression.

FIG. 2 is a Western-blot spectrum.

In the figure, 1, receptor bacteria whole bacteria protein liquid for induced expression, 2, BL21(DE3)/pET30 a-alpha-beta 2-epsilon-Y whole bacteria protein liquid for non-induced expression, 3, BL21(DE3)/pET30 a-alpha-beta 2-epsilon-Y whole bacteria protein liquid for induced expression, 4, BL21(DE3)/pET30 a-alpha-beta 2-epsilon-Y protein-containing supernatant for induced expression, 5, BL21(DE3)/pET30 a-alpha-beta 2-epsilon-Y protein-containing precipitate for induced expression, 6, BL21(DE3)/pET30 a-alpha-beta 2-epsilon-W whole bacteria protein liquid for non-induced expression, 7, BL21(DE3)/pET30 a-alpha-beta 2-epsilon-W whole bacteria protein liquid for induced expression, 8, The method comprises the steps of performing inducible expression on BL21(DE3)/pET30 a-alpha-beta 2-epsilon-W protein-containing supernatant, 9, inducible expression on BL21(DE3)/pET30 a-alpha-beta 2-epsilon-W protein-containing precipitate, 10, non-inducible expression on BL21(DE3)/pET30a-pm alpha-beta 2-epsilon-W whole bacterial protein liquid, 11, inducible expression on BL21(DE3)/pET30a-pm alpha-beta 2-epsilon-W whole bacterial protein liquid, 12, inducible expression on BL21(DE3)/pET30a-pm alpha-beta 2-epsilon-W protein-containing supernatant, 13, inducible expression on BL21(DE3)/pET30a-pm alpha-beta 2-epsilon-W protein-containing precipitate.

FIG. 3 shows the AKTA purification identification of recombinant protein α - β 2- ε -his. The arrow indicates the peak of the purified protein of interest.

FIG. 4 shows the molecular sieve purification identification and the preliminary structure determination of recombinant protein α - β 2- ε -his. The arrow indicates the peak of the purified protein of interest.

FIG. 5 is an SDS-PAGE electrophoretogram of the purified target protein.

Wherein 1 is BL21(DE3)/pET30 a-alpha-beta 2-epsilon-Y protein-containing precipitate; 2 is BL21(DE3)/pET30 a-alpha-beta 2-epsilon-Y whole bacterial protein liquid; m is Marker, and is 180KD, 130KD, 95KD, 72KD, 55KD, 43KD, 34KD and 26KD from top to bottom respectively; 3 is receptor bacteria whole bacteria protein liquid; 4 is BL21(DE3)/pET30 a-alpha-beta 2-epsilon-Y protein-containing supernatant; 5, nickel column purifying target protein sample; 6 is purified alpha-beta 2-epsilon-his protein (molecular sieve purified protein sample of interest).

FIG. 6 is a comparative electrophoretogram of different induction temperatures versus time points for the grope BL21(DE3)/pET30 a-. alpha. -beta.2-. epsilon. -Y. The arrows indicate the destination strips.

Wherein M is Marker, and is 180KD, 130KD, 95KD, 72KD, 55KD, 43KD, 34KD and 26KD from top to bottom respectively; 1 is whole mycoprotein liquid after being induced for 1 hour at 37 ℃; 2 is whole mycoprotein liquid which is induced for 2 hours at 37 ℃; 3 is whole mycoprotein liquid which is induced for 4 hours at 37 ℃; 4 is whole mycoprotein liquid which is induced for 5 hours at 37 ℃; 5 is whole mycoprotein liquid which is induced for 13 hours at 16 ℃; 6 is whole mycoprotein liquid induced at 16 ℃ for 24 h. The arrows indicate the destination strips.

FIG. 7 is a comparative electrophoretogram of BL21(DE3)/pET30 a-alpha-beta 2-epsilon-Y under different IPTG concentration conditions. The arrows indicate the destination strips.

Wherein M is Marker, and is 180KD, 130KD, 95KD, 72KD, 55KD, 43KD, 34KD and 26KD (5 μ L) respectively from top to bottom; 1 is whole mycoprotein liquid after induction at 16 ℃ and 0.1 mM; 2 is whole mycoprotein liquid after being induced by 0.3mM at 16 ℃; 3 is whole mycoprotein liquid after induction at 16 ℃ and 0.5 mM; 4 is whole mycoprotein liquid after induction at 16 ℃ and 0.75 mM; 5 is whole mycoprotein liquid after 1mM induction at 16 ℃; 6 is a no inducer blank.

FIG. 8 is a comparative electrophoretogram of BL21(DE3)/pET30 a-alpha-beta 2-epsilon-Y at different induction temperatures.

Wherein M is Marker, and is 180KD, 130KD, 95KD, 72KD, 55KD, 43KD, 34KD and 26KD (5 μ L) respectively from top to bottom; 1 is protein-containing supernatant extracted after low-temperature induction at 16 ℃ for 13 h; 2 is the supernatant containing protein extracted after 16h of induction at low temperature of 16 ℃.

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-beta 2-epsilon-his

1. Synthetic genes

3 fusion genes are designed, namely an alpha-beta 2-epsilon-hisY gene shown in SEQ ID No.1, an alpha-beta 2-epsilon-hisW gene shown in SEQ ID No.3 and a pm alpha-beta 2-epsilon-hisW gene shown in SEQ ID No. 4.

The alpha-beta 2-epsilon-hisY gene and the alpha-beta 2-epsilon-hisW gene both encode the protein alpha-beta 2-epsilon-his shown in SEQ ID No. 2. The pm alpha-beta 2-epsilon-hisW gene encodes the protein pm alpha-beta 2-epsilon-hisW shown in SEQ ID No. 5. The α - β 2- ε -his is a protein obtained by deleting amino acid residues 52 to 146, amino acid residues 464-492 and amino acid residue 743-750 of pm α - β 2- ε -hisW.

The alpha-beta 2-epsilon-Y gene shown in the 151-position 2814 of the SEQ ID No.1 (encoding the protein shown in the 51-position 937-position amino acid residue of the SEQ ID No. 2), the alpha-beta 2-epsilon-W gene shown in the 151-position 2814 of the SEQ ID No.3 (encoding the protein shown in the 51-position 937-position amino acid residue of the SEQ ID No. 2) and the pm alpha-beta 2-epsilon-W gene shown in the 151-position 3210 of the SEQ ID No.4 (encoding the protein pm alpha-beta 2-epsilon-W shown in the 51-position 1069-position amino acid residue of the SEQ ID No. 5) are synthesized by a chemical synthesis method.

2. Construction of recombinant expression vector and recombinant bacterium

The alpha-beta 2-epsilon-Y gene was used as a template with the use of the upstream primer F1 (sequence 5-ggatccatgttttgggacccggacaccgac-3') and a downstream primer R1 (sequence 5-CTCGAGTCATTTGATGCCCGGTGCTTTGA-3') was subjected to PCR amplification to add BamHI sites (underlined sequence) and XhoI recognition sites (underlined sequence) at both ends of the. alpha. -beta.2-. epsilon. -Y gene to obtain the. alpha. -beta.represented by position 145 and 2820 of SEQ ID No.12-epsilon-Y gene PCR product.

The alpha-beta 2-epsilon-W gene was used as a template, and the upstream primer F1 and the downstream primer R2 (sequence 5' -CTCGAGTCATTTGATACCCGGCGCTTTGA-3') is subjected to PCR amplification, BamHI and XhoI recognition sites are added at both ends of the alpha-beta 2-epsilon-W gene to obtain the alpha-beta 2-epsilon-W gene PCR product shown in 145-2820 of SEQ ID No. 3.

The pm alpha-beta 2-epsilon-W gene was used as a template with the upstream primer F3 (sequence 5-ggatccatgaaacgcaaaatctgcaaagcc-3') and a downstream primer R2, and BamHI and XhoI recognition sites are added at both ends of the pm alpha-beta 2-epsilon-W gene to obtain the pm alpha-beta 2-epsilon-W gene PCR product shown in 145-3216 th position of SEQ ID No. 4.

Digesting the PCR product of the alpha-beta 2-epsilon-Y gene by using BamHI and XhoI, and recovering a target fragment (alpha-beta 2-epsilon-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 plasmid is a recombinant expression vector obtained by replacing the fragment between BamHI and XhoI recognition sites of pET30a (+) with alpha-beta 2-epsilon-Y gene shown in the 151-2814 position of SEQID No.1 and keeping other sequences of pET30(+) unchanged, and the recombinant expression vector is named as pET30 a-alpha-beta 2-epsilon-Y. pET30 a-alpha-beta 2-epsilon-Y contains alpha-beta 2-epsilon-hisY gene with His label, the nucleotide sequence of the alpha-beta 2-epsilon-hisY gene is SEQ ID No.1, and the protein alpha-beta 2-epsilon-His shown in SEQ ID No.2 is coded. The recombinant E.coli containing pET30 a-alpha-beta 2-epsilon-Y was named BL21(DE3)/pET30 a-alpha-beta 2-epsilon-Y.

Digesting the PCR product of the alpha-beta 2-epsilon-W gene by using BamHI and XhoI, and recovering a target fragment (alpha-beta 2-epsilon-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 identified by BamHI and XhoI through double digestion, the plasmid with correct digestion verification is sequenced, the sequencing result shows that the plasmid is a recombinant expression vector obtained by replacing a fragment between BamHI and XhoI recognition sites of pET30a (+) by alpha-beta 2-epsilon-W gene shown in the 151-2814 position of SEQID No.3 and keeping other sequences of pET30a (+) unchanged, and the recombinant expression vector is named as pET30 a-alpha-beta 2-epsilon-W. pET30 a-alpha-beta 2-epsilon-W contains His label fusion protein alpha-beta 2-epsilon-hisW gene, the nucleotide sequence of alpha-beta 2-epsilon-hisW gene is SEQ ID No.3, and the protein alpha-beta 2-epsilon-His shown in SEQ ID No.2 is coded. The recombinant E.coli containing pET30 a-alpha-beta 2-epsilon-W was named BL21(DE3)/pET30 a-alpha-beta 2-epsilon-W.

Digesting the pm alpha-beta 2-epsilon-W gene PCR product by using BamHI and XhoI, and recovering a target fragment (pm alpha-beta 2-epsilon-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. Shaking and culturing a single colony overnight, extracting a plasmid, carrying out double digestion identification by using BamHI and XhoI, sequencing the plasmid with correct digestion verification, replacing a fragment between BamHI recognition sites and XhoI recognition sites of pET30a (+) by using the pm alpha-beta 2-epsilon-W gene shown in the 151-H3210 site of SEQ ID No.4 according to a sequencing result, and keeping other sequences of pET30(+) unchanged to obtain a recombinant expression vector which is named as pET30a-pm alpha-beta 2-epsilon-W. pET30a-pm alpha-beta 2-epsilon-W contains a pm alpha-beta 2-epsilon-hisW gene with a His label, the nucleotide sequence of the pm alpha-beta 2-epsilon-hisW gene is SEQ ID No.4, and the nucleotide sequence encodes the protein pm alpha-beta 2-epsilon-hisW shown in SEQ ID No. 5. The recombinant E.coli containing pET30a-pm alpha-beta 2-epsilon-W was named BL21(DE3)/pET30a-pm alpha-beta 2-epsilon-W.

3. Analysis and characterization of protein expression profiles

The four strains of BL21(DE3)/pET30 a-alpha-beta 2-epsilon-Y, BL21(DE3)/pET30 a-alpha-beta 2-epsilon-W, BL21(DE3)/pET30a-pm alpha-beta 2-epsilon-W and Escherichia coli BL21(DE3) (recipient for short) are respectively and independently inoculated to a strain containing 50 mu g/ml of Carnacnospora canadensisLB liquid Medium (Medium obtained by adding kanamycin to LB liquid Medium to a kanamycin concentration of 50. mu.g/ml), 37 ℃ and shaking-culturing to 0D with a ThermoMaxQ6000 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 bacterial liquid without induced expression and the induced expression bacterial liquid for analyzing the protein expression form. The specific steps are that 1mL of bacterial liquid is taken and placed in a 1.5m L centrifugal tube, the mark is made, the centrifugal tube is centrifuged for 30min at 8000rpm/min under the condition of 4 ℃, the supernatant is discarded, and the bacterial precipitation is collected. Adding 1ml PBS to resuspend the precipitate, centrifuging at 8000rpm/min for 5min, and discarding the supernatant. 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 of the BL21(DE3)/pET30 a-alpha-beta 2-epsilon-Y with induced expression, the protein-containing supernatant and the protein-containing precipitate all contain the target protein alpha-beta 2-epsilon-his with the size of 105kD, and the whole mycoprotein liquid of the BL21(DE3)/pET30 a-alpha-beta 2-epsilon-Y with non-induced expression does not contain the target protein alpha-beta 2-epsilon-his with the size of 105 kD; the target protein alpha-beta 2-epsilon-his in the whole bacterial protein liquid of the BL21(DE3)/pET30 a-alpha-beta 2-epsilon-Y subjected to induction expression accounts for 65 percent of the total bacterial protein (the total bacterial protein), the target protein alpha-beta 2-epsilon-his in the protein-containing supernatant of BL21(DE3)/pET30 a-alpha-beta 2-epsilon-Y subjected to induction expression accounts for 92 percent of the target protein alpha-beta 2-epsilon-his in the whole bacterial protein liquid of BL21(DE3)/pET30 a-alpha-beta 2-epsilon-Y subjected to induction expression, and 92 percent of the target protein alpha-beta 2-epsilon-his is soluble protein; the target protein alpha-beta 2-epsilon-his in the protein-containing sediment of the induction-expressed BL21(DE3)/pET30 a-alpha-beta 2-epsilon-Y accounts for 8 percent of the target protein alpha-beta 2-epsilon-his in the whole-bacterium protein liquid of the induction-expressed BL21(DE3)/pET30 a-alpha-beta 2-epsilon-Y, and 8 percent of the target protein alpha-beta 2-epsilon-his is insoluble inclusion body protein; the result shows that the target protein alpha-beta 2-epsilon-his of BL21(DE3)/pET30 a-alpha-beta 2-epsilon-Y expressed by induction accounts for 65 percent of the total protein of the thallus, 92 percent of the target protein alpha-beta 2-epsilon-his expressed by BL21(DE3)/pET30 a-alpha-beta 2-epsilon-Y is soluble protein, and 8 percent is insoluble inclusion body protein. The whole bacterial protein liquid of the BL21(DE3)/pET30 a-alpha-beta 2-epsilon-W with induced expression, the protein-containing supernatant and the protein-containing precipitate all contain the target protein alpha-beta 2-epsilon-his with the size of 105kD, and the whole bacterial protein liquid of the BL21(DE3)/pET30 a-alpha-beta 2-epsilon-W with non-induced expression does not contain the target protein alpha-beta 2-epsilon-his with the size of 105 kD; the target protein alpha-beta 2-epsilon-his in the whole bacterial protein liquid of the induction-expressed BL21(DE3)/pET30 a-alpha-beta 2-epsilon-W accounts for 65 percent of the total bacterial protein, the target protein alpha-beta 2-epsilon-his in the protein-containing supernatant of the induction-expressed BL21(DE3)/pET30 a-alpha-beta 2-epsilon-W accounts for 8 percent of the target protein alpha-beta 2-epsilon-his in the whole bacterial protein liquid of the induction-expressed BL21(DE3)/pET30 a-alpha-beta 2-epsilon-W, and the 8 percent of the target protein alpha-beta 2-epsilon-his is soluble protein; the target protein alpha-beta 2-epsilon-his in the protein-containing sediment of the induction-expressed BL21(DE3)/pET30 a-alpha-beta 2-epsilon-W accounts for 92 percent of the target protein alpha-beta 2-epsilon-his in the whole-bacterium protein liquid of the induction-expressed BL21(DE3)/pET30 a-alpha-beta 2-epsilon-W, and 92 percent of the target protein alpha-beta 2-epsilon-his is insoluble inclusion body protein; the result shows that the target protein alpha-beta 2-epsilon-his of BL21(DE3)/pET30 a-alpha-beta 2-epsilon-W expressed by induction accounts for 65 percent of the total protein of the thallus, 8 percent of the target protein alpha-beta 2-epsilon-his expressed by BL21(DE3)/pET30 a-alpha-beta 2-epsilon-W is soluble protein, and 92 percent is insoluble inclusion body protein. The whole mycoprotein liquid of the BL21(DE3)/pET30a-pm alpha-beta 2-epsilon-W without induced expression and the whole mycoprotein liquid of the BL21(DE3)/pET30a-pm alpha-beta 2-epsilon-W with induced expression, the protein-containing supernatant and the protein-containing sediment do not contain the target protein pm alpha-beta 2-epsilon-his with the size of 120 KD; it shows that BL21(DE3)/pET30a-pm alpha-beta 2-epsilon-W does not express the target protein pm alpha-beta 2-epsilon-his. The whole bacterial protein liquid of the escherichia coli BL21(DE3) subjected to induction expression does not contain the target protein alpha-beta 2-epsilon-his with the size of 105 kD; it is shown that Escherichia coli BL21(DE3) does not express the target protein alpha-beta 2-epsilon-his. The same amount of Colony Forming Units (CFU) of induced expression BL21(DE3)/pET30 a-alpha-beta 2-epsilon-Y and induced expression BL21(DE3)/pET30 a-alpha-beta 2-epsilon-W expressed total protein of the thallus have the same mass (FIG. 1 and FIG. 2).

4. Purification of alpha-beta 2-epsilon-his

BL21(DE3)/pET30 a-. alpha. -beta.2-. epsilon. -Y was inoculated into LB liquid medium containing 50. mu.g/ml kanamycin (a medium obtained by adding kanamycin to LB liquid medium to a kanamycin concentration of 50. mu.g/ml), cultured at 37 ℃ with shaking to 0D with a Thermo MaxQ6000 type full temperature shaker at 200rpm₆₀₀When the value (blank with LB liquid medium containing 50. mu.g/ml kanamycin) reached 0.6, IPTG was added for inducible expression. The inducible expression was induced with 0.75mM IPTG for 13h 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 - β 2- ε -his protein (nickel column-purified target protein sample) (FIG. 3).

The nickel column purified α - β 2- ε -his protein was further purified by passing it through a molecular sieve using Superdex200 gel column from GE. The mobile phase used solution 1. After the sample is purified by a molecular sieve, a large amount of imidazole contained in the sample can be removed, and the structure of the alpha-beta 2-epsilon-his protein is a structure in which a monomer and a dimer coexist. And (3) collecting elution peaks of the monomer and dimer structures to obtain the alpha-beta 2-epsilon-his protein purified by the molecular sieve (a target protein sample purified by the molecular sieve), and quantitatively analyzing the purity of the obtained protein by using a NanoDrop2000 ultramicro spectrophotometer (ND 2000).

The amino acid sequence of the alpha-beta 2-epsilon-his protein purified by the molecular sieve is analyzed by mass spectrometry, and the result shows that the amino acid sequence of the alpha-beta 2-epsilon-his is shown as SEQ ID No. 2.

The two strains BL21(DE3)/pET30 a-. alpha. -beta.2-. epsilon. -Y and E.coli BL21(DE3) (recipient bacteria for short) were each separately 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 at 200rpm using a Thermo MaxQ6000 type whole temperature shaker until 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 expression of both strains was induced with 0.75mM IPTG for 13 hours at 16 ℃.

And (4) taking the bacterial liquid after IPTG induced expression for 13h for protein expression form analysis. The specific steps are that the recombinant bacterial liquid induced by 1m L is placed in a 1.5m L centrifugal tube, marked, centrifuged for 30min at 8000rpm/min at 4 ℃, the supernatant is discarded, and the bacterial precipitation is collected. Adding 1ml PBS to resuspend the precipitate, centrifuging at 8000rpm/min for 5min, and discarding the supernatant. 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. And simultaneously carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic analysis on the nickel column purified target protein sample and the purified alpha-beta 2-epsilon-his protein (molecular sieve purified target protein sample).

The result shows that the target protein alpha-beta 2-epsilon-his expressed by BL21(DE3)/pET30 a-alpha-beta 2-epsilon-Y exists in the supernatant of bacterial broken thalli in a soluble form, the expression of the band of the soluble protein is obvious, and the impurities in the supernatant are less. By optimizing the purification and elution conditions of the AKTA machine, a soluble target protein band with better purity can be obtained, a large amount of imidazole contained in a protein sample can be removed after further purification by a molecular sieve (figure 5), and the structure of the fusion recombinant protein is found to be a coexisting structure of a monomer and a dimer for the first time. 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 - β 2- ε -Y gene represented by position 151-2814 of SEQ ID No.1, and the other sequence of pET28a (+) was kept unchanged to obtain a recombinant expression vector containing the α - β 2- ε -Y gene, which was named pET28a- α - β 2- ε -Y. pET28 a-alpha-beta 2-epsilon-Y is transferred into competent cells of Escherichia coli BL21(DE3), and the obtained recombinant Escherichia coli is named as BL21(DE3)/pET28 a-alpha-beta 2-epsilon-Y. BL21(DE3)/pET28 a-. alpha. -beta.2-. epsilon. -Y was inoculated into LB liquid medium containing 50. mu.g/ml kanamycin (a medium obtained by adding kanamycin to LB liquid medium to a kanamycin concentration of 50. mu.g/ml), cultured at 37 ℃ with shaking to 0D with a Thermo MaxQ6000 type full 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 whole bacterial protein liquid of BL21(DE3)/pET28 a-alpha-beta 2-epsilon-Y which is not induced to express, the whole bacterial protein liquid of BL21(DE3)/pET28 a-alpha-beta 2-epsilon-Y which is induced to express, the protein-containing supernatant of BL21(DE3)/pET28 a-alpha-beta 2-epsilon-Y which is induced to express and the protein-containing precipitate of BL21(DE3)/pET28 a-alpha-beta 2-epsilon-Y which is induced to express. As can be seen, although the same exogenous target gene (alpha-beta 2-epsilon-Y gene) is adopted, the expression vector-pET 28a (+) is expressed in different BL21(DE3)) In pET30a (+), the expression of exogenous target gene is greatly different, the alpha-beta 2-epsilon-Y gene is transferred into Escherichia coli BL21(DE3) through pET30a (+), so that the high-efficiency soluble expression of the alpha-beta 2-epsilon-Y gene can be obtained, and the alpha-beta 2-epsilon-Y gene is transferred into Escherichia coli BL21(DE3) through pET28a (+), but the alpha-beta 2-epsilon-Y gene is not expressed.

Example 2 animal immunoprotection assay for alpha-beta 2-epsilon-his

1. Preparation of vaccines against clostridium perfringens

The α - β 2- ε -his protein purified by the molecular sieve in example 1 was dissolved in sterile PBS to obtain a solution of α - β 2- ε -his with a concentration of 1000. mu.g/mL for immunization. Mixing alpha-beta 2-epsilon-his solution and Freund's adjuvant in a volume of 1:1, emulsifying to prepare an oil emulsion vaccine, and naming the oil emulsion vaccine as a prime vaccine. Mixing alpha-beta 2-epsilon-his solution and incomplete Freund's adjuvant in the same volume ratio of 1:1, emulsifying to prepare oil emulsion vaccine, and naming the oil emulsion vaccine as the 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 α - β 2- ε -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 immunization dose of alpha-beta 2-epsilon-his 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-beta 2-epsilon-his protein 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 protein 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 10⁹Other operations than cfuAre identical.

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 challenge result shows that the challenge dose group (immune alpha-beta 2-epsilon-his) has a certain immune protection effect on the challenge of various clostridium perfringens. The immune protection rate of the challenge dose group (immunization alpha-beta 2-epsilon-his) against clostridium perfringens type a challenge within 7 days was 100% (20 mice were all alive), and PBS control mice were all dead; the immune protection rate against clostridium perfringens type B challenge in the challenge dose group (immune α - β 2-epsilon-his) was 100% (20 mice were all alive), all mice in the PBS control group died; the immune protection rate against clostridium perfringens type C challenge in the challenge dose group (immune α - β 2-epsilon-his) was 90% (18 survived, 2 died), all PBS control mice died; the challenge dose group (immunized α - β 2- ε -his) gave 100% immune protection against challenge with Clostridium perfringens type D (20 mice were all alive), whereas PBS control mice were all dead. The purified alpha-beta 2-epsilon-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-beta 2-epsilon-his as the diagnostic antigen has very good sensitivity and specificity. Respectively detecting the antibody titer level in serum of 0-6 weeks after the initial immunization of the mice in each experimental group, wherein the result shows that the antibody titer of the alpha-beta 2-epsilon-his fusion toxin protein immunity group is obviously increased, 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-beta 2-epsilon-his

1. Optimization of induction temperature and time

BL21(DE3)/pET30 a-. alpha. -beta.2-. epsilon. -Y was inoculated into LB liquid medium containing 50. mu.g/ml kanamycin (a medium obtained by adding kanamycin to LB liquid medium to a kanamycin concentration of 50. mu.g/ml), cultured at 37 ℃ with shaking to 0D with a Thermo MaxQ6000 type full temperature shaker at 200rpm₆₀₀Value (in the 50 u g/ml kanamycin LB liquid cultureBlank control group) reached 0.6, the following 6 inducible expressions were performed by adding isopropylthio- β -D-galactoside (IPTG). 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 the recombinant bacterial liquid induced by 1m L in a 1.5m L centrifuge tube, marking, centrifuging at 8000rpm/min at 4 ℃ for 30min, discarding the supernatant, and collecting the thallus precipitate. Adding 1ml PBS to resuspend the precipitate, centrifuging at 8000rpm/min for 5min, and discarding the supernatant. 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-beta 2-epsilon-his protein gradually increases along with the time extension under the conditions that the induction temperature of BL21(DE3)/pET30 a-alpha-beta 2-epsilon-Y 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. But the expression level of alpha-beta 2-epsilon-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-beta 2-epsilon-his reaches the highest level, and the target protein alpha-beta 2-epsilon-his accounts for 65 percent of the total protein of the whole bacteria. When the culture is continued for 24 hours, the expression level of alpha-beta 2-epsilon-his is slightly reduced, so that the optimal induction temperature of BL21(DE3)/pET30 a-alpha-beta 2-epsilon-Y is verified to be 16 ℃ through experiments, and the induction time is 13-24 hours (figure 6).

2. Optimisation of IPTG concentration

BL21(DE3)/pET30 a-. alpha. -beta.2-. epsilon. -Y was inoculated into LB liquid medium containing 50. mu.g/ml kanamycin (a medium obtained by adding kanamycin to LB liquid medium to a kanamycin concentration of 50. mu.g/ml), cultured at 37 ℃ with shaking to 0D with a Thermo MaxQ6000 type full temperature shaker at 200rpm₆₀₀Value (in terms ofLB liquid medium with 50. mu.g/ml kanamycin as a blank control) reached 0.6, the following 6 inducible expressions were performed, respectively, by adding isopropylthio-. beta. -D-galactoside (IPTG). 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 the recombinant bacterial liquid induced by 1m L in a 1.5m L centrifuge tube, marking, centrifuging at 8000rpm/min at 4 ℃ for 30min, discarding the supernatant, and collecting the thallus precipitate. Adding 1ml PBS to resuspend the precipitate, centrifuging at 8000rpm/min for 5min, and discarding the supernatant. 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 quantity of the alpha-beta 2-epsilon-his is different under the induction of IPTG with different concentrations. The expression level of alpha-beta 2-epsilon-his is in an increasing relationship with the addition of IPTG at concentrations of 0.1-0.75 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 (FIG. 7).

3. Optimization of Induction time

BL21(DE3)/pET30 a-. alpha. -beta.2-. epsilon. -Y was inoculated into LB liquid medium containing 50. mu.g/ml kanamycin (a medium obtained by adding kanamycin to LB liquid medium to a kanamycin concentration of 50. mu.g/ml), cultured at 37 ℃ with shaking to 0D with a Thermo MaxQ6000 type full 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 2 inducible expressions were performed. 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 the recombinant bacterial liquid induced by 1m L in a 1.5m L centrifuge tube, marking, centrifuging at 8000rpm/min at 4 ℃ for 30min, discarding the supernatant, and collecting the thallus precipitate. Adding 1ml PBS to resuspend the precipitate, centrifuging at 8000rpm/min for 5min, and discarding the supernatant. 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-beta 2-epsilon-his is not greatly changed when BL21(DE3)/pET30 a-alpha-beta 2-epsilon-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 sediment 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 determined to be 16 ℃, and the induction time is determined to be 13-16h (figure 8).

Claims

1. A method for producing a protein, comprising the step of expressing a gene encoding a protein in an organism to obtain the protein;

the protein is composed of an amino acid sequence of SEQ ID No. 2; the expression of a gene encoding a protein in an organism comprises introducing the gene encoding the protein into a recipient microorganism to obtain a recombinant microorganism expressing the protein, culturing the recombinant microorganism, and expressing the recombinant microorganism to obtain the 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-beta 2-epsilon-Y into Escherichia coli BL21(DE3) and expresses protein with an amino acid sequence of SEQ ID No.2, the recombinant microorganism is named as BL21(DE3)/pET30 a-alpha-beta 2-epsilon-Y, and pET30 a-alpha-beta 2-epsilon-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 the 151-2814 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 ℃.