An immunological composition, its preparation and use

Abstract

The application discloses an immunological composition, a preparation method and application thereof, and belongs to the field of animal epidemic disease prevention and control. The immunological composition comprises recombinant Clostridium perfringens alpha toxin protein, recombinant Clostridium perfringens beta1 toxin protein and recombinant Clostridium perfringens epsilon toxin protein; the preparation method comprises the following steps: respectively cloning coding genes of the recombinant Clostridium perfringens alpha toxin protein, the recombinant Clostridium perfringens beta1 toxin protein and the recombinant Clostridium perfringens epsilon toxin protein into eukaryotic expression vectors, respectively expressing the three kinds of recombinant proteins by CHO-K1 cells, and purifying the three kinds of recombinant proteins by ion exchange chromatography; compared with the prior art, the sheep clostridial disease triple four-prevention genetic engineering subunit vaccine prepared from the immunological composition produced according to the application has the following advantages: ① safe to use; ② stable quality and good immunogenicity; ③ high yield and low production cost; ④ simple process and reactor production can be realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of animal epidemic prevention and control, in particular to an immunological composition, a preparation method and application thereof. TECHNICAL BACKGROUND

[0002] Ovine clostridial disease is a general term for ovine clostridial infectious diseases caused by infection of various clostridial pathogens in Clostridium. It includes lamb dysentery, sudden death, enterotoxemia, fast epidemic, black epidemic, tetanus and botulinum toxin poisoning, etc. Among them, lamb dysentery is caused by infection of B-type Clostridium perfringens, sudden death of sheep is caused by infection of C-type Clostridium perfringens, enterotoxemia of sheep is caused by infection of D-type Clostridium perfringens; fast epidemic of sheep is caused by infection of Clostridium septicum; black epidemic of sheep is caused by infection of B-type Clostridium novyi; tetanus and botulinum toxin poisoning are caused by infection of Clostridium tetani and Clostridium botulinum, respectively.

[0003] Among the many clostridial diseases, fast epidemic, sudden death, lamb dysentery and enterotoxemia are the most common, and often mixed infection. Among them, sheep of 6-18 months old are most susceptible to fast epidemic, which is concentrated in high incidence in autumn and winter and early spring when the temperature changes dramatically; sheep of 6-24 months old are most susceptible to sudden death, which is mostly sporadic or endemic, mainly in low-lying or marshy pastures in early spring and autumn; lambs within 1 month are most susceptible to lamb dysentery, which is mostly high in the season of lambing, poor nutrition, severe cold or hot weather; sheep of 2-12 months are most susceptible to enterotoxemia, which is mostly sporadic and often occurs in spring, summer or autumn. Ovine clostridial disease has the characteristics of acute onset, short course, high mortality, and often death before treatment. Therefore, vaccine immunization is the most effective means of preventing the disease, and the majority of breeders attach great importance to the immunoprevention and control of such diseases.

[0004] The most widely used goat fast epidemic, sudden confrontation, lamb dysentery, enterotoxemia triple four preventive inactivated vaccine has good immunogenicity according to the exotoxin protein produced in the propagation process of clostridial pathogens, and is prepared by adding an adjuvant after detoxification of the culture of B or C type Clostridium perfringens, D type Clostridium perfringens and Clostridium septicum inactivated by formaldehyde solution. However, due to the fact that a large amount of culture medium is mixed in the vaccine, the effective antigen content is low, the production process is complicated, the toxin production is unstable between batches, and the immunization dose is large, the vaccine has the problems of large immune stress, poor protection effect and the like in the use process. The serum neutralization method is used to detect the goat triple four preventive vaccines produced from 2006 to 2016 by China Institute for Control of Animal Disease and Drug Administration, and the results show that the unqualified rates of B, C and D type Clostridium perfringens are 55.56% (15 / 27), 63.64% (21 / 33) and 39.39% (13 / 33) respectively, and the comprehensive unqualified rate is 72.73% (24 / 33); the unqualified rate of Clostridium septicum is 45.45% (15 / 33). It can be seen that the unqualified rate of the commercially available goat triple four preventive inactivated vaccine is high. Therefore, increasing the effective antigen content of the vaccine, or using the genetic engineering method to prepare the recombinant Clostridium perfringens alpha, beta and epsilon toxin proteins and Clostridium septicum alpha toxin protein without biological toxicity, and increasing the toxin production stability of the culture medium, optimizing the production process, developing a new type of adjuvant or improving the stability of the aluminum gel adjuvant, increasing the number of immunizations and the like will help to improve the immunization effect of the vaccine. SUMMARY

[0005] In order to make up for the deficiencies of the prior art, solve the problems of high unqualified rate, difficult to control the quality of the culture medium, low effective antigen content, large immunization dose and serious immune side reaction of the commercially available goat triple four preventive inactivated vaccine in the prior art, the application provides an immune composition, a preparation method and application thereof.

[0006] In order to achieve the above-mentioned purpose, the application provides the following solutions.

[0007] The application provides an immune composition, which comprises recombinant Clostridium septicum alpha toxin protein, recombinant Clostridium perfringens beta1 toxin protein and recombinant Clostridium perfringens epsilon toxin protein.

[0008] The amino acid sequence of the recombinant Clostridium septicum alpha toxin protein is shown as SEQ ID NO: 2.

[0009] The amino acid sequence of the recombinant Clostridium perfringens beta1 toxin protein is shown as SEQ ID NO: 4.

[0010] The amino acid sequence of the recombinant Clostridium perfringens epsilon toxin protein is shown as SEQ ID NO: 6.

[0011] Preferably, the mass ratio of the recombinant Clostridium perfringens alpha toxin protein, the recombinant Clostridium perfringens beta1 toxin protein and the recombinant Clostridium perfringens epsilon toxin protein in the immunological composition is 1:2:2.

[0012] Preferably, the immunological composition of the present application further comprises a pharmaceutically acceptable adjuvant, and the adjuvant is at least one of SP 01 adjuvant, MONTANIDE™ ISA201VG adjuvant and MONTANIDE™ ISA 206VG adjuvant, and the adjuvant is more preferably MONTANIDE™ ISA 206VG. TM

[0013] Preferably, the volume ratio of the adjuvant to the immunological composition is 1:1.

[0014] Preferably, the gene sequence encoding the recombinant Clostridium perfringens alpha toxin protein is shown in SEQ ID NO:1; the gene sequence encoding the recombinant Clostridium perfringens beta1 toxin protein is shown in SEQ ID NO:3; and the gene sequence encoding the recombinant Clostridium perfringens epsilon toxin protein is shown in SEQ ID NO:5.

[0015] The present application also provides a preparation method of the immunological composition, comprising:

[0016] 1) cloning each target gene into a eukaryotic expression vector to obtain a recombinant expression vector, wherein the target genes include a gene encoding a recombinant Clostridium perfringens alpha toxin protein, a gene encoding a recombinant Clostridium perfringens beta1 toxin protein and a gene encoding a recombinant Clostridium perfringens epsilon toxin protein;

[0017] 2) transfecting the recombinant vector obtained in step 1) into CHO cells respectively, and obtaining high-expression engineering cells by culturing and screening respectively;

[0018] 3) fermenting the engineering cells obtained in step 2) to obtain target proteins, wherein the target proteins are respectively a recombinant Clostridium perfringens alpha toxin protein, a recombinant Clostridium perfringens beta1 toxin protein and a recombinant Clostridium perfringens epsilon toxin protein;

[0019] 4) mixing the recombinant Clostridium perfringens alpha toxin protein, the recombinant Clostridium perfringens beta1 toxin protein and the recombinant Clostridium perfringens epsilon toxin protein obtained in step 3) in a certain proportion, and then emulsifying with an immunological adjuvant to obtain the immunological composition.

[0020] Preferably, the eukaryotic expression vector is at least one of pcDNA3.1, pEE 6.4, pEE 12.4 and pKS001.

[0021] ​Preferably, the host cell comprises at least one of DG44, DXB11, CHO-K1 and CHO-S cells.

[0022] The application also provides the use of the immunological composition in the preparation of a medicine for preventing Clostridium perfringens infection.

[0023] The application also provides the use of the immunological composition in the preparation of a Clostridium perfringens triple quadruple protection genetic engineering subunit vaccine.

[0024] The application discloses the following technical effects:

[0025] The wild type Clostridium perfringens CSA gene, Clostridium perfringens CPB gene and Clostridium perfringens ETX gene are subjected to site-directed mutation and codon optimization, so that the cytotoxicity and biological toxicity of the recombinant protein are completely inhibited, and the immunogenicity of the wild type toxin protein is retained, so that the recombinant protein has the same immunogenicity as the wild type Clostridium perfringens alpha toxin protein, Clostridium perfringens beta1 toxin protein or Clostridium perfringens epsilon toxin protein.

[0026] The three strains of engineering cells constructed and screened by the application have high expression yield of the target protein, are easy to purify and industrialize, and the expressed recombinant protein can induce test animals to produce high levels of neutralizing antibodies and has good immunogenicity. The Clostridium perfringens triple quadruple protection genetic engineering subunit vaccine produced by the application has the following advantages: ① safe to use; ② stable quality and good immunogenicity; ③ high yield and low production cost; and ④ simple process and reactor production.

[0027] The Clostridium perfringens triple quadruple protection genetic engineering subunit vaccine produced by the application is safe after being injected into test animals at an overdose (3.0 ml / dose for rabbits and 6.0 ml / dose for lambs), and the protection rates against 1 MLD of B, C and D type Clostridium perfringens and Clostridium perfringens toxin are all 100% 21 days after immunization (1.8 ml / dose for rabbits and 3.0 ml / dose for goats), and the corresponding neutralizing antibody titers of the immune serum are 2, 4, 4 and 4, respectively, and the immunoprotective efficacy is good.

[0028] In addition, the application also provides a method for producing recombinant protein in a serum-free, high-density and suspension culture mode in a bioreactor, which has the advantages of low production cost, high yield, simple process and good immunogenicity. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 is the double enzyme digestion test result of each recombinant plasmid; wherein, Figure 1 -A is the double enzyme digestion test result of the recombinant plasmid CSA-pKS001; Figure 1B is the double enzyme digestion test result of recombinant plasmid CPB-pKS001; Figure 1 C is the double enzyme digestion test result of recombinant plasmid ETX-pKS001.

[0030] Figure 2 is the SDS-PAGE detection result of 3L flask culture supernatant of each engineering cell; wherein, Figure 2 A is the supernatant result of CSA-pKS001-CHO strain cell culture; Figure 2 B is the supernatant result of CPB-pKS001-CHO strain cell culture; Figure 2 C is the supernatant result of ETX-pKS001-CHO strain cell culture.

[0031] Figure 3 is the Western Blot detection result of 3L flask culture supernatant of each engineering cell; wherein, Figure 3 A is the supernatant result of CSA-pKS001-CHO strain cell culture; Figure 3 B is the supernatant result of CPB-pKS001-CHO strain cell culture; Figure 3 C is the supernatant result of ETX-pKS001-CHO strain cell culture.

[0032] Figure 4 is the average body temperature monitoring result of experimental laboratory trial vaccine over-dose inoculation test animals; wherein, Figure 4 A is the body temperature monitoring result of trial vaccine over-dose inoculation of rabbits; Figure 4 B is the body temperature monitoring result of trial vaccine over-dose inoculation of lambs. DETAILED DESCRIPTION

[0033] The application provides an immune composition, comprising recombinant Clostridium perfringens alpha toxin protein, recombinant Clostridium perfringens beta1 toxin protein and recombinant Clostridium perfringens epsilon toxin protein.

[0034] The amino acid sequence of the recombinant Clostridium perfringens alpha toxin protein is shown as SEQ ID NO: 2;

[0035] The amino acid sequence of the recombinant Clostridium perfringens beta1 toxin protein is shown as SEQ ID NO: 4;

[0036] The amino acid sequence of the recombinant Clostridium perfringens epsilon toxin protein is shown as SEQ ID NO: 6.

[0037] In the application, the mass ratio of the recombinant Clostridium perfringens alpha toxin protein, the recombinant Clostridium perfringens beta1 toxin protein and the recombinant Clostridium perfringens epsilon toxin protein in the immune composition is 1:2:2.

[0038] The immunological composition of the present application also comprises a pharmaceutically acceptable immunological adjuvant. In the present application, the adjuvant is preferably at least one of the following: SPO1 adjuvant, MONTANIDE TM ISA201VG adjuvant and MONTANIDE TM ISA206VG adjuvant, and more preferably MONTANIDE TM ISA206VG adjuvant.

[0039] In the present application, the volume ratio of the adjuvant to the immunological composition is 1:1.

[0040] The present application preferably replaces the original signal peptide sequence (1-31) in the wild-type Clostridium perfringens alpha toxin protein (NCBI Accession No: AFB82980.2) with the CHO cell exosomal peptide sequence (1-25) through directed mutation technology, and also deletes the "KKRRGKR" sequence (392-398) rich in basic amino acids at the front end of the original "tail peptide" sequence.

[0041] The present application preferably obtains the gene sequence encoding the recombinant Clostridium perfringens alpha toxin protein as shown in SEQ ID NO: 1 by performing murine codon bias optimization on the wild-type Clostridium perfringens CSA gene (NCBI Accession No: JN793989.2) according to the mutant amino acid sequence of the wild-type Clostridium perfringens alpha toxin protein of the present application.

[0042] In the present application, the amino acid sequence encoded by the gene sequence of the recombinant Clostridium perfringens alpha toxin protein is as shown in SEQ ID NO: 2, and the recombinant Clostridium perfringens alpha toxin protein composed of this amino acid sequence has lost cytotoxicity and biological toxicity, but still retains the immunogenicity of the wild-type Clostridium perfringens alpha toxin protein.

[0043] The present application preferably replaces the original signal peptide sequence (1-27) in the wild-type Clostridium perfringens beta1 toxin protein (NCBI Accession No: AJI77135.1) with the CHO cell exosomal peptide sequence (1-25) through directed mutation technology (position 239: arginine to glutamic acid), and obtains the mutant amino acid sequence.

[0044] The present application preferably obtains the gene sequence encoding the recombinant Clostridium perfringens beta1 toxin protein as shown in SEQ ID NO: 3 by performing murine codon bias optimization on the wild-type Clostridium perfringens CPB gene (NCBI Accession No: KP064405.1) according to the mutant amino acid sequence of the wild-type Clostridium perfringens beta1 toxin protein of the present application.

[0045] In the present application, the amino acid sequence coded by the gene sequence of the recombinant Clostridium perfringens beta1 toxin protein is shown in SEQ ID NO: 4, and the recombinant Clostridium perfringens beta1 toxin protein composed of the amino acid sequence has lost cytotoxicity and biological toxicity, but still retains the immunogenicity of the wild-type Clostridium perfringens beta1 toxin protein.

[0046] In the present application, the original amino acid sequence of the wild-type Clostridium perfringens epsilon toxin protein (NCBI Accession No: AML79725.1) is mutated by site-directed mutation technology (33th: lysine -> alanine; 45th: lysine -> alanine) to obtain a mutated amino acid sequence, in which a repeated prepeptide sequence "AEISNTVSNEMSA" is connected in series before the prepeptide sequence (33th-45th), the signal peptide sequence (1st-32nd) in the original amino acid sequence is replaced with the CHO cell exogenous peptide sequence (1st-25th), and the "serine" at the 321th position in the original amino acid sequence is mutated to "tyrosine".

[0047] In the present application, the wild-type Clostridium perfringens ETX gene (NCBI Accession No: KU726256.1) is codon preference optimized for mice according to the mutated amino acid sequence of the wild-type Clostridium perfringens epsilon toxin protein to obtain a gene sequence coding for the recombinant Clostridium perfringens epsilon toxin protein, which is shown in SEQ ID NO: 5.

[0048] In the present application, the amino acid sequence coded by the gene sequence of the recombinant Clostridium perfringens epsilon toxin protein is shown in SEQ ID NO: 6, and the recombinant Clostridium perfringens epsilon toxin protein composed of the amino acid sequence has lost cytotoxicity and biological toxicity, but still retains the immunogenicity of the wild-type Clostridium perfringens epsilon toxin protein.

[0049] The present application also provides a preparation method of the immunological composition, comprising:

[0050] 1) cloning each target gene into a eukaryotic expression vector to obtain a recombinant expression vector, wherein the target genes are the coding gene of the recombinant Clostridium perfringens alpha toxin protein, the coding gene of the recombinant Clostridium perfringens beta1 toxin protein, and the coding gene of the recombinant Clostridium perfringens epsilon toxin protein;

[0051] 2) transfecting the recombinant plasmid obtained in step 1) into CHO cells, and then culturing and screening to obtain a high-expression engineering cell strain;

[0052] 3) fermenting and culturing the high-expression engineering cell obtained in step 2) to obtain the target protein, wherein the target protein is the recombinant Clostridium perfringens alpha toxin protein, the recombinant Clostridium perfringens beta1 toxin protein, and the recombinant Clostridium perfringens epsilon toxin protein.

[0053] 4) mixing the recombinant Clostridium perfringens alpha toxin protein, the recombinant Clostridium perfringens beta1 toxin protein and the recombinant Clostridium perfringens epsilon toxin protein obtained in step 3) in a proportion, and then emulsifying the mixture with an immune adjuvant to obtain the immune composition.

[0054] The present application clones each target gene into a eukaryotic expression vector to obtain a recombinant plasmid, wherein the target genes are a coding gene of the recombinant Clostridium perfringens alpha toxin protein, a coding gene of the recombinant Clostridium perfringens beta1 toxin protein and a coding gene of the recombinant Clostridium perfringens epsilon toxin protein.

[0055] In the present application, the expression vector is preferably at least one of pcDNA3.1, pEE 6.4, pEE 12.4 and pKS001, and more preferably pKS001.

[0056] After obtaining the recombinant plasmid, the present application transfects each recombinant plasmid into CHO cells respectively, and obtains a high-expression engineering cell strain after culturing and screening.

[0057] In the present application, the host cell preferably includes at least one of DG44, DXB11, CHO-K1 and CHO-S cells, and more preferably CHO-K1 strain. The CHO-K1 cell in the present application is a host cell that can be cultured in high density suspension and stably express heterologous proteins.

[0058] After obtaining the high-expression engineering cell, the present application ferments and harvests the target proteins from the obtained high-expression engineering cell, wherein the target proteins are the recombinant Clostridium perfringens alpha toxin protein, the recombinant Clostridium perfringens beta1 toxin protein and the recombinant Clostridium perfringens epsilon toxin protein.

[0059] The recombinant protein in the present application also includes purifying the obtained fermentation culture. In the present application, the purifying method preferably adopts ion exchange chromatography, and the ion exchange chromatography medium is preferably at least one of SP Sephrose FF chromatography medium and Q Sephrose FF chromatography medium.

[0060] After harvesting the target proteins, the present application mixes the recombinant Clostridium perfringens alpha toxin protein, the recombinant Clostridium perfringens beta1 toxin protein and the recombinant Clostridium perfringens epsilon toxin protein, and then emulsifies the mixture with an immune adjuvant to obtain the immune composition.

[0061] The present application also provides the use of the immune composition in the preparation of a medicine for preventing Clostridium perfringens infection in sheep.

[0062] The present application also provides the use of the immune composition in the preparation of a Clostridium perfringens triple quadruple prevention genetic engineering subunit vaccine for sheep.

[0063] The present application will be further described below in conjunction with the accompanying drawings and examples, and the examples of the present application are only used to describe the technical solutions of the present application, and are not limited to the present application. The suspension culture medium, cloning plasmid and reagents, drugs and the like used in the examples of the present application are all commercially available products.

[0064] The sources of the reagents and drugs of the present application are as follows:

[0065] CHO-K1 cells are from the China Institute of Cell Culture Preservation Committee Cell Library, Shanghai Institute of Life Sciences, Chinese Academy of Sciences;

[0066] The virulent bacteria for testing are purchased from the China Veterinary Drug Inspection Institute, including: Clostridium perfringens type B C58-2 strain, preservation number CVCC60082; Clostridium perfringens type C C59-2 strain, preservation number CVCC60102; Clostridium perfringens type D C60-2 strain, preservation number CVCC60201; Clostridium putrefaciens C55-1 strain, preservation number CVCC60021;

[0067] CHO CD04 medium and CHO Feed 02 medium are purchased from Zhongshan Kangtian Shenghe Biotechnology Co., Ltd.;

[0068] pKS001 eukaryotic expression vector is constructed by the Immunology Room of the Institute of Microbiology and Epidemiology of Military Medical Research Institute of Military Science Academy;

[0069] DPBS is purchased from Gibco Company; EBEL electroporation buffer is purchased from Suzhou Yida Biotechnology Co., Ltd.; L-Methioninesulfoximine (MSX) is purchased from Sigma Company;

[0070] DNA gel rapid purification kit and plasmid large extraction kit are both purchased from Beijing Quanshi Gold Biotechnology Co., Ltd.;

[0071] BCA protein concentration determination kit is purchased from Beijing Solabio Technology Co., Ltd.;

[0072] MONTANIDE TM ISA206VG adjuvant is purchased from SEPPIC (Shanghai), France.

[0073] Example 1

[0074] I. Gene synthesis

[0075] 1. Synthesis of recombinant Clostridium putrefaciens alpha toxin protein gene sequence

[0076] (1) The present application replaces the signal peptide sequence (1-25) of the original amino acid sequence of the wild-type Clostridium perfringens alpha toxin protein (NCBI Accession Number: AFB82980.2) with the CHO cell exogenous peptide sequence (1-25) through directional mutation technology, and deletes the "KKRRGKR" sequence rich in basic amino acids at the front end of the original amino acid sequence "tail peptide" (392-398), to obtain the mutated amino acid sequence;

[0077] (2) The wild-type Clostridium perfringens CSA gene (NCBI Accession Number: JN793989.2) is subjected to murine codon bias optimization according to the mutated amino acid sequence obtained in step (1), to obtain the gene sequence of the recombinant Clostridium perfringens alpha toxin protein as shown in SEQ ID NO: 1, and the amino acid sequence of the recombinant Clostridium perfringens alpha toxin protein encoded by the gene is as shown in SEQ ID NO: 2. The recombinant Clostridium perfringens alpha toxin protein composed of the amino acid sequence has lost cytotoxicity and biological toxicity, but still retains the immunogenicity of the wild-type Clostridium perfringens alpha toxin protein.

[0078] 2. Synthesis of the gene sequence of the recombinant Clostridium perfringens beta1 toxin protein

[0079] (1) The present application first replaces the signal peptide sequence (1-27) of the original amino acid sequence of the wild-type Clostridium perfringens beta1 toxin protein (NCBI Accession Number: AJI77135.1) with the CHO cell exogenous peptide sequence (1-25) through directional mutation technology, and obtains the mutated amino acid sequence;

[0080] (2) The wild-type Clostridium perfringens CPB gene (NCBI Accession Number: KP064405.1) is subjected to murine codon bias optimization according to the mutated amino acid sequence obtained in step (1), to obtain the gene sequence of the recombinant Clostridium perfringens beta1 toxin protein as shown in SEQ ID NO: 3, and the amino acid sequence of the recombinant Clostridium perfringens beta1 toxin protein encoded by the gene is as shown in SEQ ID NO: 4. The recombinant Clostridium perfringens beta1 toxin protein composed of the amino acid sequence has lost cytotoxicity and biological toxicity, but still retains the immunogenicity of the wild-type Clostridium perfringens beta1 toxin protein.

[0081] 3. Synthesis of the gene sequence of the recombinant Clostridium perfringens epsilon toxin protein

[0082] (1) The present application carries out site-directed mutation on the propeptide sequence in the original amino acid sequence (NCBI accession number: Am178725.1) of wild-type Clostridium perfringens epsilon toxin protein by site-directed mutation technology (position 33: lysine→alanine; position 45: lysine→alanine), and a repeated propeptide sequence "AEISNTVSNEMSA" is connected in series after the propeptide sequence (positions 33-45), the signal peptide sequence (positions 1-32) in the original amino acid is replaced with the CHO cell exogenous peptide sequence (positions 1-25), and the serine at position 321 in the original amino acid sequence is site-directed mutated to tyrosine, to obtain the mutated amino acid sequence;

[0083] (2) The wild-type Clostridium perfringens ETX gene (NCBI accession number: KU726256.1) is subjected to murine codon bias optimization according to the mutated amino acid sequence obtained in step (1), to obtain the gene sequence of the recombinant Clostridium perfringens epsilon toxin protein as shown in SEQ ID NO: 5, and the amino acid sequence of the recombinant Clostridium perfringens epsilon toxin protein encoded by the gene is as shown in SEQ ID NO: 6. The recombinant Clostridium perfringens epsilon toxin protein composed of the amino acid sequence has lost cytotoxicity and biological toxicity, but still retains the immunogenicity of the wild-type Clostridium perfringens epsilon toxin protein.

[0084] II. Construction of CSA-pKS001 / CPB-pKS001 / ETX-pKS001 recombinant plasmid

[0085] 1. Synthesis of CSA-pUC57 / CPB-pUC57 / ETX-pUC57 recombinant plasmid

[0086] The codon-optimized recombinant Clostridium perfringens alpha toxin protein encoding gene (as shown in SEQ ID NO: 1), the recombinant Clostridium perfringens beta1 toxin protein encoding gene (as shown in SEQ ID NO: 3), and the recombinant Clostridium perfringens epsilon toxin protein encoding gene (as shown in SEQ ID NO: 5) are introduced with a Hind III enzyme recognition site at the N terminus and a Not I enzyme recognition site at the C terminus, respectively, and are constructed on a pUC57 cloning vector to construct a CSA-pUC57 / CPB-pUC57 / ETX-pUC57 cloning plasmid, which is synthesized by Nanjing Kingsrui Biotechnology Co., Ltd.

[0087] 2. Transformation of CSA-pUC57 / CPB-pUC57 / ETX-pUC57 recombinant plasmid into Trans 5a competent cells, respectively

[0088] (1) Take 50 μl of ice-bath-melted competent cells, add 10 μl of recombinant plasmid, mix gently, and then place in ice bath for 30 min;

[0089] (2) Take out the sample tube, place in 42°C water bath for heat shock for 45s, then immediately transfer to ice bath and stand for 2 min, the process cannot shake the sample tube;

[0090] (3) Take out the sample tube, add 500μl of fresh LB medium (without antibiotics) to the sample tube in the super-clean table, gently suck and blow after placing in 37°C constant temperature incubator, 200rpm for 1 hour to make the bacteria recover;

[0091] (4) Take out the sample tube, centrifuge at 8000rpm for 1 min, suck and discard 300μl of supernatant, suck and blow the bacteria in the tube, and connect 50μl of bacteria liquid to Amp + LB agar plate, evenly spread the stick on the plate;

[0092] (5) After placing the plate in the 37°C incubator for 1 hour, continue to culture the inverted plate for 12-16 hours;

[0093] (6) Observe the single colony growth on the plate.

[0094] 3. Extraction of CSA-pUC57 / CPB-pUC57 / ETX-pUC57 recombinant plasmid

[0095] (1) Use a inoculating loop to pick the single colony grown on the plate and connect it to 5ml Amp + LB liquid medium, place in 37°C, 200rpm constant temperature incubator for overnight culture;

[0096] (2) Take 1ml of cultured bacteria liquid to the EP tube, centrifuge at 10000g for 1 min to collect the bacteria, suck and discard the supernatant, and add 250μl of RB solution (containing RNase A) to the tube to resuspend the bacteria;

[0097] (3) After mixing well, add 250μl of LB solution to the tube, gently mix up and down for 5-6 times within 5 min, so that the bacteria are fully lysed to form a blue transparent solution;

[0098] (4) Add 350μl of NB solution to the tube, gently mix 5-6 times until a compact yellow agglomeration is formed, stand at room temperature for 2 min, and centrifuge at 12000g for 5 min;

[0099] (5) Carefully suck and transfer the supernatant to the adsorption column, centrifuge at 12000g for 1 min, discard the effluent, and put the adsorption column back into the collection tube;

[0100] (6) Add 650μl of WB solution to the adsorption column, centrifuge at 12000g for 1 min, and discard the effluent;

[0101] (7) Put the adsorption column back into the collection tube, 12000g centrifugal for 2min, to remove the residual WB solution;

[0102] (8) Take out the adsorption column and put it into a clean centrifugal tube, and place it in the fume hood for natural drying for 3-5min. Then add 50μl EB solution preheated at 70℃ to the center of the membrane. After standing for 1min, centrifuge at 10000g for 1min to elute the plasmid. Repeat the elution once more. The eluted plasmid is stored at -20℃ for standby use.

[0103] 4. CSA-pUC57 / CPB-pUC57 / ETX-pUC57 recombinant plasmid, pKS001 empty double enzyme digestion

[0104] Each recombinant plasmid, pKS001 empty, was digested with Hind III enzyme and Not I enzyme at 37℃ water bath for 3 hours. The enzyme digestion reaction system is shown in Table 1.

[0105] Table 1. Enzyme digestion reaction system

[0106]

[0107] 5. Gel recovery and purification of enzyme digestion products

[0108] (1) After gel electrophoresis of the enzyme digestion products, cut the gel with the gel cutter and put the target DNA band in a clean EP tube.

[0109] (2) Add 3 times the volume of GSB solution to the above EP tube and place it in a 55℃ water bath for 6-10min. During this period, gently invert the EP tube 3-5 times to melt the gel completely. After melting, the gel color is the same as the GSB color.

[0110] (3) After the temperature drops to room temperature, add it all to the adsorption column, stand for 1min, centrifuge at 10000g for 1min, discard the liquid at the bottom of the tube, and put the adsorption column back into the collection tube.

[0111] (4) Add 650μl WB washing solution to the adsorption column, centrifuge at 10000g for 1min, discard the liquid at the bottom of the tube, and put the adsorption column back into the collection tube.

[0112] (5) Centrifuge the adsorption column at 10000g for 2min to remove the WB solution, and then dry the adsorption column completely.

[0113] (6) Put the adsorption column into a clean EP tube and add 50μl EB solution (preheated at 70℃) to the center of the membrane. Stand for 1min, centrifuge at 10000g for 1min, and recover the purified DNA fragment.

[0114] (7) Repeat elution 1 time, to obtain purified CSA / CPB / ETX gene fragment and purified linearized pKS001 empty vector, and store at 4°C for standby use.

[0115] 6. Ligation reaction

[0116] The purified linearized pKS001 empty vector of step 5 above is connected with the CSA / CPB / ETX gene fragment respectively using T4 ligase at 4°C overnight, and the ligation reaction system is shown in Table 2 below.

[0117] Table 2. Ligation reaction system

[0118]

[0119] 7. Transform CSA-pKS001 / CPB-pKS001 / ETX-pKS001 recombinant plasmid into Trans 5a respectively

[0120] The competent cells are prepared by the method of step 2.

[0121] 8. Large-scale extraction of CSA-pKS001 / CPB-pKS001 / ETX-pKS001 recombinant plasmid

[0122] (1) Use a swab to pick a single colony growing on the plate and inoculate into 5 ml Amp + LB liquid medium, and incubate overnight at 37°C, 200 rpm in a constant temperature shaker; then inoculate each activated bacterial strain into 250 ml Amp + LB liquid medium, and incubate overnight at 37°C, 200 rpm in a constant temperature shaker;

[0123] (2) Centrifuge the bacterial liquid at 12000g for 2 min to collect the bacterial cells, and aspirate the supernatant, and add 15 ml of RB solution (containing RNase A) to suspend the bacterial cells;

[0124] (3) Mix thoroughly, then add 15 ml of LB solution, and gently mix up and down for 5-6 times within 5 min to fully lyse the bacterial cells, forming a blue transparent solution;

[0125] (4) Add 21 ml of NB solution, and gently mix for 5-6 times until a compact yellow agglomeration is formed, and stand at room temperature for 2 min, and centrifuge at 12000g for 15 min;

[0126] (5) Carefully aspirate the supernatant and transfer it to a centrifugal column, and centrifuge at 8000g for 2 min, discard the effluent, and return the centrifugal column to the collection tube;

[0127] (6) Add 5 ml TB solution to the centrifugal column, stand at room temperature for 10 min, centrifuge at 8000 g for 2 min, discard the effluent;

[0128] (7) Add 5 ml WB solution to the centrifugal column, centrifuge at 8000 g for 2 min, discard the effluent; repeat once;

[0129] (8) Put the centrifugal column back into the collection tube, centrifuge at 8000 g for 5 min, completely remove the residual WB solution, and then place the centrifugal column in a fume hood and dry naturally for 10 min to completely volatilize the ethanol in the centrifugal column;

[0130] (9) Put the centrifugal column into a clean 50 ml centrifugal tube, add 2 ml of EB solution preheated at 70°C to the center of the membrane, stand at room temperature for 5 min, and then centrifuge at 8000 g for 2 min to elute the plasmid; repeat the elution once and store at -20°C for standby, each recombinant plasmid should be ≥1000 ng / μl.

[0131] 9. Double enzyme digestion verification of CSA-pKS001 / CPB-pKS001 / ETX-pKS001 recombinant plasmid

[0132] Each recombinant plasmid was digested with Hind III enzyme and Not I enzyme at 37°C water bath for 3 hours, and the enzyme digestion reaction system is shown in Table 3.

[0133] Table 3. Enzyme digestion reaction system

[0134]

[0135]

[0136] After enzyme digestion, samples were taken for agarose gel electrophoresis detection to check whether the inserted fragment size is correct, as shown in Figure 1 The results of double enzyme digestion verification of each recombinant plasmid are shown in the table, and the results show that the enzyme digestion is correct; among them, Figure 1 -A is the enzyme digestion result of recombinant plasmid CSA-pKS001, and the results show that 7100 bp and 1307 bp DNA bands appear at the same time, the 1307 bp DNA band contains a 1296 bp recombinant Clostridium perfringens alpha toxin protein coding gene sequence (as shown in SEQ ID NO: 1) and a total of 11 bp of residues of the Hind III enzyme digestion residue introduced at the N terminal of the recombinant Clostridium perfringens alpha toxin protein coding gene sequence and the Not I enzyme digestion residue introduced at the C terminal, the adenine (A) at the front end of the Hind III enzyme is removed, and the guanine (G) and cytosine (C) at the front end of the Not I enzyme are removed;

[0137] Among them, Figure 1-B is the enzyme digestion result of the recombinant plasmid CPB-pKS001, the results show that 7100 bp and 1019 bp DNA bands appear at the same time, the 1019 bp DNA band contains 1008 bp of the coding gene sequence of the recombinant Clostridium perfringens beta1 toxin protein (as shown in SEQ ID NO: 3) and 11 bp of the Hind III enzyme digestion residue introduced at the N terminal of the coding gene sequence of the recombinant Clostridium perfringens beta1 toxin protein and the Not I enzyme digestion residue introduced at the C terminal, the adenine (A) at the front end of the Hind III enzyme is removed, the guanine (G) and cytosine (C) at the front end of the Not I enzyme is removed during double enzyme digestion;

[0138] wherein as Figure 1 -C is the enzyme digestion result of the recombinant plasmid ETX-pKS001, the results show that 7100 bp and 1019 bp DNA bands appear at the same time, the 1019 bp DNA band contains 1008 bp of the coding gene sequence of the recombinant Clostridium perfringens epsilon toxin protein (as shown in SEQ ID NO: 5) and 11 bp of the Hind III enzyme digestion residue introduced at the N terminal of the coding gene sequence of the recombinant Clostridium perfringens epsilon toxin protein and the Not I enzyme digestion residue introduced at the C terminal, the adenine (A) at the front end of the Hind III enzyme is removed, the guanine (G) and cytosine (C) at the front end of the Not I enzyme is removed during double enzyme digestion.

[0139] Example 2 Transfection of CHO-K1 cells with CSA-pKS001 / CPB-pKS001 / ETX-pKS001 recombinant plasmid and monoclonal preservation

[0140] 1. Transfection of CHO-K1 cells with CSA-pKS001 / CPB-pKS001 / ETX-pKS001 recombinant plasmid Take out the electric conversion Buffer (Yidabio Biotechnology, H10305), CHO CD04 culture medium (QUACELL, A11004; +80 mM glutamine), D-PBS (Gibco, 14190-144) in advance and restore to room temperature.

[0141] (1) Counting: After taking out the CHO-K1 cells in the CO2 shaker and shaking, the cell suspension was taken into an EP tube, and the number of cells was counted by Count Star cell counter after trypan blue staining;

[0142] (2) Centrifugation: about 6x10 7 cells of cell suspension were taken into a 50 ml centrifuge tube, centrifuged at 1000 rpm for 5 min, and the cell precipitate was collected;

[0143] (3) D-PBS washing: discard the supernatant medium to obtain the cell precipitate, add 10 ml D-PBS to resuspend the cells, centrifuge at 1000 rpm for 5 min, collect the cell precipitate;

[0144] (4) Pre-plasmid: add a total content of 240 μg of recombinant plasmid into a sterile EP tube;

[0145] (5) Mix plasmid DNA, cells and electroporation liquid: after centrifugation in step 3), discard the D-PBS, add 300 μl of electroporation buffer, mix gently by blowing and transfer all to the EP tube in step 4), mix again by blowing, and then immediately electroshock;

[0146] (6) Electroporation: take 200 μl of recombinant plasmid and cell suspension and add it to the H1 electroporation cup along the wall (do not generate bubbles), then correctly insert the electroporation cup into the base of the electroshocking instrument to electroshock, and the electroshocking parameters are shown in Table 4;

[0147] Table 4. Electroshocking parameters

[0148]

[0149] (7) Culture: after electroshocking, immediately take the cell suspension in the electroshocking cup and blow it into a T75 bottle;

[0150] (8) Repeat: repeat steps 6) and 7) twice to complete the electroshocking;

[0151] (9) Culture: after the T75 bottle is covered and shaken evenly, place it in a 37°C, 5% CO2 incubator for 48 hours, remove it and transfer all the cell suspension to a clean 50 ml centrifuge tube, centrifuge at 1000 rpm for 5 min to collect the cell precipitate, then add 20 ml of CHO CD04 culture medium (+ 25 μM MSX), disperse by blowing and transfer to a T75 bottle for continued pressure culture.

[0152] 2. Preservation of CSA-pKS001-CHO / CPB-pKS001-CHO / ETX-pKS001-CHO engineering cells

[0153] (1) Centrifugation: remove the engineering cells after 1 generation of pressure culture in the incubator, transfer all to a sterile 50 ml centrifuge tube, centrifuge at 1000 rpm for 5 min, and collect the cell precipitate;

[0154] (2) Cell counting: add 10 ml of CHO CD04 culture medium (+ 25 μM MSX) to the cell precipitate in the tube, disperse by blowing, take 100 μl of cell suspension to an EP tube, stain with trypan blue, and count with a Count Star cell counter;

[0155] (3) Dilution: serially dilute each engineering cell by 10-fold to 1 cell / ml, and select 1 cell / ml, 10 cells / ml and 100 cells / ml for plating, respectively;

[0156] (4) Plating: plate each dilution of the engineering cells to a 96-well plate at 200 μl / well, with one plate for each dilution (also plate 4 wells of CHO-K1 negative cells as controls at 2000 cells / well), and then place in an incubator for 6-8 hours before observation and marking;

[0157] (5) Marking: carefully observe under an inverted microscope and mark the single cell wells in each plate;

[0158] (6) Culture: place the cell plates in a 37°C, 5% CO2 incubator for continued culture for 7-14 days, and observe the growth of the single clones in each plate daily;

[0159] (7) Subcloning and purification: select the single cell wells with good growth and repeat the above steps to screen for subclones with good expression for expansion;

[0160] (8) Expansion: gently blow apart the growing subclones and transfer them all to a 24-well cell plate, add 1 ml of CHO CD04 medium (+25M MSX), and place in a 37°C, 5% CO2 incubator for continued culture for 5-7 days;

[0161] (9) Expansion in petri dishes: gently blow apart the clones expanded in step 8) and transfer them all to a 10 cm petri dish, add 10 ml of CHO CD04 medium (+25M MSX), and place in a 37°C, 5% CO2 incubator for continued culture for 5-7 days;

[0162] (10) Cell shake flask culture: select 4-5 subclones with good growth from step 9) above, transfer them all to a clean 50 ml centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant and add 25-30 ml of CHO CD04 medium (+25M MSX), aspirate and blow to disperse, and then transfer to a 125 ml cell shake flask for suspension culture (37°C, 5% CO2, 120 rpm), take samples for counting with a Count Star cell counter every day after 3 days of culture, and select 2-3 stable proliferating clone cell lines for preservation from each;

[0163] (11) Preservation: when the cell density in the shake flask has proliferated to 5-7.5 x 10 6cells / ml, the original seed was frozen with cell freezing solution (30% fresh medium + 30% conditioned medium + 10% DMSO + 30% FBS, 4°C pre-cooled for standby) at a freezing density of 1-1.5 x 10 7 cells / ml, and after labeling (designated as CSA-pKS001-CHO strain, CPB-pKS001-CHO strain, and ETX-pKS001-CHO strain, respectively, labeled as P0 generation), the cells were placed in a freezing box and cooled overnight in a -80°C refrigerator, and then transferred to liquid nitrogen for long-term storage.

[0164] Example 3 Cell shake flask fermentation culture

[0165] (1) Inoculation: the base seed to be scaled up was proliferated to 5-8 x 10 6 cells / ml, 100-200 ml of cell suspension was taken after sampling and counting, and added to a 3L shake flask, and fresh CHO CD04 medium was added to 1L to make the inoculation density in the bottle 0.8-1 x 10 6 cells / ml, and then placed in a CO2 shaker for continued suspension culture (37°C, 5% CO2, 120 rpm);

[0166] (2) Counting: cell density and viability were monitored daily by sampling and counting, and samples were reserved;

[0167] (3) Fed-batch culture: when the cell density in the bottle reached 5 x 10 6 cells / ml, 5% of the culture volume of CHOFeed 02 feed medium was added, and the feed medium was added every other day;

[0168] (4) Sugar supplementation: when the cell density in the bottle reached 5 x 10 6 cells / ml, the glucose concentration in the medium was determined daily, and glucose concentrate was added in time to a final concentration of 5 g / L;

[0169] (5) Expression culture: when the cell density in the bottle increased to 1 x 10 7 cells / ml, the culture temperature was reduced to 32°C for expression culture, and SDS-PAGE gel electrophoresis was performed daily to determine the expression of the target protein.

[0170] The SDS-PAGE detection results are shown in Figure 2 , wherein Figure 2 A is the SDS-PAGE detection result of the supernatant of CSA-pKS001-CHO strain culture, the molecular weight of the recombinant Clostridium perfringens alpha toxin protein is about 48 KDa, and the target protein band expressed is indicated in the figure; Figure 2-B is the SDS-PAGE detection result of CPB-pKS001-CHO strain culture supernatant, the molecular weight of the recombinant Clostridium perfringens beta1 toxin protein is about 37KDa, and the identified protein band in the figure is the target protein expressed; Figure 2 -C is the SDS-PAGE detection result of ETX-pKS001-CHO strain culture supernatant, the molecular weight of the recombinant Clostridium perfringens epsilon toxin protein is about 35KDa, and the identified protein band in the figure is the target protein expressed; no protein band of corresponding size was found in the negative control.

[0171] The Western Blot detection result is shown in Figure 3 , wherein Figure 3 -A is the Western Blot detection result of CSA-pKS001-CHO strain culture supernatant, there is only one specific band with a size of about 48KDa at the identified position, which is the recombinant Clostridium sordellii alpha toxin protein; Figure 3 -B is the Western Blot detection result of CPB-pKS001-CHO strain culture supernatant, there is only one specific band with a size of about 37KDa at the identified position, which is the recombinant Clostridium perfringens beta1 toxin protein; Figure 3 -C is the Western Blot detection result of ETX-pKS001-CHO strain culture supernatant, there is only one specific band with a size of about 35KDa at the identified position, which is the recombinant Clostridium perfringens epsilon toxin protein; no protein band of corresponding size was found in the negative control, indicating that each target antigen protein was correctly expressed in the recombinant CHO-K1 cells.

[0172] (6) Harvest: After 7 days of expression culture, sample daily for Count Star cell counter counting. When the cell viability gradually decreases to 50% or less, harvest the cell culture, and use it as the harvest standard for the expression culture of the three strains of engineering cells.

[0173] Example 4 Purification of Target Protein

[0174] (1) Centrifugation: Harvest the cell culture, centrifuge at 4°C, 8000g for 20min to harvest the culture supernatant, filter it through a 0.45μm low protein adsorption filter membrane, and prepare it for use;

[0175] (2) Ion exchange chromatography medium selection: according to the isoelectric point pI of the recombinant Clostridium sordellii alpha toxin protein, which is 7.61, SP Sephrose FF ion exchange chromatography medium is selected; - 30S, Suzhou Na Micro) were purified respectively; - 30S, Suzhou Na Micro) were purified respectively;

[0176] (3) Column packing: 1L of ion exchange chromatography agarose was poured into the chromatography column respectively, and the column was packed at a compression coefficient of 1.05-1.10, first at a low flow rate and then at a high flow rate and constant pressure;

[0177] (4) Equilibrium: After the column was packed, 0.3M NaOH solution was used to completely sterilize the chromatography column at a flow rate of 40-50ml / min (about 2-3 times the column volume), and the pH value was stabilized at 13.5. Then the chromatography column was washed with 4-6 times the column volume of deionized water until the pH and UV values remained constant. Finally, the column was equilibrated with the corresponding pH, 0.01M PB buffer (pH corresponding to 6.5, 7.0, 7.5 and 8.0), at a flow rate of 20ml / min overnight, and the final pH and UV values remained constant;

[0178] (5) Sample loading: The pre-purified protein solution was diluted with the corresponding pH value PB buffer (equivalent volume dilution and equilibration for 2 hours), and then loaded into the chromatography column at a flow rate of 20-30ml / min, while monitoring the UV value of the sample flow to constant;

[0179] (6) Washing: After sample loading, 2-3 times the column volume of 0.02M Tris-HCl, 0.01M NaCl was loaded at a flow rate of 20-30ml / min to wash once, while monitoring the UV value of the sample flow to baseline equilibrium; Then use 0.02M Tris-HCl, 0.05M NaCl to wash the liquid at a flow rate of 20-30ml / min to wash to baseline equilibrium (about 3-4 times the column volume);

[0180] (7) Elution: The target proteins were eluted using PB solutions with corresponding pH values and different NaCl concentrations (7.0 and 0.3M, 7.5 and 0.5M, 7.5 and 0.5M for recombinant Clostridium perfringens alpha toxin protein, recombinant Clostridium perfringens beta1 toxin protein, and recombinant Clostridium perfringens epsilon toxin protein, respectively), at a flow rate of 10-20ml / min, while monitoring and collecting the ultraviolet absorption peak filtered protein;

[0181] (8) Content and concentration determination: After filtering the purified protein solution through a 0.22 μm low protein adsorption filter to remove bacteria, the total protein content of each purified protein was determined by BCA protein concentration determination kit, and the content and purity of each target protein were analyzed by SDS-PAGE gel electrophoresis and BandScan software.

[0182] (9) Storage: The content of each purified protein was not less than 800 μg / ml, and the purity was not less than 90%. After labeling, it was stored in a-80°C refrigerator for standby.

[0183] Example 5 Vaccine trial

[0184] (1) Antigen dilution: The recombinant Clostridium perfringens β1 toxin protein, ε toxin protein and recombinant Clostridium septicum α toxin protein were diluted to 120 μg / ml, 120 μg / ml and 60 μg / ml respectively with sterile PBS (pH = 7.2), and the components were mixed evenly (the final concentration of recombinant Clostridium septicum α toxin protein was 20 μg / ml, and the final concentrations of recombinant Clostridium perfringens β1 toxin protein and ε toxin protein were both 40 μg / ml);

[0185] (2) Emulsification: According to the use method of MONTANIDE TM ISA 206VG adjuvant, the emulsification conditions were set as follows: rotation speed 500 rpm, cooling water control temperature 28-30°C, shearing of oil phase adjuvant and water phase antigen suspension at a volume ratio of 1:1, stirring for 30-60 min, and then sampling for uniformity test;

[0186] (3) Property test: About 0.5 ml of vaccine was taken with a clean pipette and dropped on the surface of cold water, the first drop was cloud-like and quickly spread; 10 ml of vaccine was taken in a 15 ml graduated centrifuge tube and centrifuged at 3000 rpm for 15 min, no water phase was separated at the bottom of the tube; the test results are shown in Table 5 below, the trial vaccine is a milky white uniform suspension, the dosage form is cloud-like and does not separate;

[0187] (4) Viscosity test: According to Appendix 15 of Chinese Veterinary Pharmacopoeia (2020 edition, Part III), the test results are shown in Table 5 below, the viscosities are 20.1 mPa.s, 20.4 mPa.s and 20.2 mPa.s respectively;

[0188] (5) Sterility test: According to Appendix 28 of Chinese Veterinary Pharmacopoeia (2020 edition, Part III), the test results are shown in Table 5 below, and there is no sterile growth;

[0189] Table 5. Laboratory trial vaccine property and sterility test results

[0190]

[0191] (6) Preservation: The test vaccine that passed the test was stored in a refrigerator at 4°C for standby use.

[0192] Example 6 Safety Test

[0193] (1) Rabbit Test: Referring to the safety test method of "Sheep Fast Epidemic, Sudden Attack, Lamb Dysentery, and Intestinal Toxicemia Triple Four-Prevention Inactivated Vaccine" in the Chinese Veterinary Pharmacopoeia (2020 Edition, Volume 3), the test vaccine was injected into the neck muscles of 5 rabbits with a body weight of 1.5-2.0 kg, each with 3.0 ml (overdose, sheep immunization dose), and 5 control group rabbits were injected with the same volume of PBS. After injection, they were continuously observed for 7 days. The results of the test rabbits once overdosed are shown in Table 6 below.

[0194] Table 6. Results of test rabbits once overdosed

[0195]

[0196] Note: "Y" indicates rabbits injected with vaccine; "P" indicates rabbits injected with PBS.

[0197] The results showed that the test rabbits were all alive, with good mental state, normal food and water intake, and no swelling and necrosis at the injection site during the observation period. The body temperature of the rabbits in the injection group showed a transient increase, and the body temperature monitoring results are also shown in Table A below. The body temperature increased by no more than 1.3°C from the base temperature, gradually returned to normal on the 3rd day after injection, and no other systemic adverse reactions or abnormalities were observed. After the body temperature returned to normal, there was no difference in appetite, spirit and activity between the rabbits in the injection group and the PBS control group. This indicates that the test vaccine has good safety for rabbits. Figure 4

[0198] (2) Sheep Test: Referring to the safety test method of "Sheep Fast Epidemic, Sudden Attack, Lamb Dysentery, and Intestinal Toxicemia Triple Four-Prevention Inactivated Vaccine" in the Chinese Veterinary Pharmacopoeia (2020 Edition, Volume 3), the test vaccine was injected into the neck muscles of 5 one-month-old goat kids, each with 6.0 ml (overdose, 2 times the sheep immunization dose), and 5 control group kids were injected with the same volume of PBS. After injection, they were continuously observed for 7 days. The results of the test goat kids once overdosed are shown in Table 7 below.

[0199] Table 7. Results of test goat kids once overdosed

[0200]

[0201] Note: "R" indicates a goat kid injected with vaccine, with red ear number identification; "G" indicates a goat kid injected with PBS, with green ear number identification.

[0202] ​The results showed that all the tested lambs were healthy and alive during the observation period, and no swelling or necrosis was observed at the injection site. Occasionally, one lamb in the vaccine group showed slight lethargy and decreased appetite, and the body temperature was temporarily elevated (not higher than 40.8°C, all not more than 1.5°C higher than the basic body temperature). The body temperature gradually returned to normal on the 3rd day after vaccination, and the results of body temperature monitoring were also shown in Table 6. Figure 4 After the body temperature returned to normal, there was no visible difference in appetite, spirit and vitality between the test vaccine group and the PBS control group, indicating that the test vaccine was safe for lambs.

[0203] Example 7: Efficacy test

[0204] (1) Immunization: According to the "China Veterinary Drug Standards" (2020 edition, part three) "Sheep fast epidemic, sudden attack, lamb dysentery, enterotoxemia three-in-one four-prevention inactivated vaccine" efficacy test method, 20 rabbits with body weight of 1.5-2.0 kg and 20 young goats aged 8-12 months were selected (the mixed pre-immune serum was negative for the neutralizing antibody titers of the four toxins), and each animal was injected with vaccine in the neck muscle, 1.8 ml per rabbit and 3.0 ml per goat. Blood was collected 21 days after immunization to separate serum for neutralizing antibody titer detection and immune challenge test.

[0205] (2) Serum neutralization method: Collect the serum of rabbits and goats in the vaccine immunization group, mix 5 immune sera of the same group in equal amounts, take 0.4 ml of mixed serum, and mix it evenly with 0.8 ml of B-type Clostridium perfringens toxin (containing 8 mouse MLD), C-type Clostridium perfringens toxin (containing 16 mouse MLD), D-type Clostridium perfringens toxin (containing 16 mouse MLD), and Clostridium septicum toxin (containing 16 mouse MLD), and place it in a 37°C incubator for 40 min. Then, 2 mice of 18-20 g each were injected with 0.3 ml per mouse. At the same time, 2 mice of the same batch were used as controls, and each was injected with 1 MLD of the four toxins as controls. The mice in the neutralization B, C, and D groups were observed for 1 day, and the mice in the neutralization Clostridium septicum group were observed for 3 days. The number of protected mice was counted. The results of the neutralization test of the test vaccine immune serum are shown in Table 8.

[0206] Table 8. Results of neutralization test of test vaccine immune serum

[0207]

[0208] Note: "Y" indicates vaccinated rabbits; "B" indicates vaccinated goats, and blue ear number identification.

[0209] Results show that: four kinds of toxin control group mice were 2 / 2 lethal in the observation period, indicating that the toxicity of the four toxins was qualified; the serum of the vaccine immunized group of rabbits and the serum of the vaccine immunized group of goats were 2 (0.1 ml of immune serum neutralized 2 MLD toxin, and 2 / 2 healthy mice were observed) to B type Clostridium perfringens toxin, and 4 (0.1 ml of immune serum neutralized 4 MLD toxin, and 2 / 2 healthy mice were observed) to C and D type Clostridium perfringens toxin and Clostridium septicum toxin, indicating that the neutralizing antibody titer of the test vaccine immune serum was qualified, and was slightly higher than the inactivation vaccine efficacy test standard.

[0210] (3) Immune challenge method: 21 days after vaccination, four kinds of toxins qualified for rabbits and goats were used for toxin challenge test. Rabbit 1 MLD and goat 1 MLD of B, C, D type Clostridium perfringens toxin and Clostridium septicum toxin were used for ear / jugular vein injection challenge (2.0 ml of rabbit 1 MLD toxin was injected into each rabbit; 6.0 ml of goat 1 MLD toxin was injected into each goat), 5 per toxin; at the same time, 2 rabbits and 2 goats of the same batch were used as controls, and rabbit 1 MLD and goat 1 MLD of the four toxins were injected as controls, and the number of immune challenge protected animals was counted after challenge for 5 days. The immune challenge test results of the test vaccine are shown in Table 9.

[0211] Table 9. Immune challenge test results of the test vaccine

[0212]

[0213]

[0214] Note: "Y" indicates vaccinated rabbit; "W" indicates toxin control goat, white ear number identification; "B" indicates vaccinated goat, blue ear number identification.

[0215] Results show that the same toxin challenge control group of rabbits and goats were 0 / 2 healthy, indicating that the toxicity of the four toxins to rabbits and goats was qualified; the same toxin challenge vaccine immune group of rabbits and goats were 5 / 5 healthy, indicating that the challenge protection rate of the vaccine to the four toxins was 100%, and the immune protection efficacy was good.

[0216] In summary, the above examples are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art can easily understand and modify the technical solutions described in the foregoing examples, therefore, the present application is not limited to the specific details or the described examples without departing from the general concept defined by the claims and the equivalent scope.

Claims

1. An immune composition, characterized in that, Including recombinant Clostridium putrefactive α-toxin protein, recombinant Clostridium perfringens β1-toxin protein, and recombinant Clostridium perfringens ε-toxin protein; The amino acid sequence of the recombinant Clostridium putrefactive α-toxin protein is shown in SEQ ID NO:2; The amino acid sequence of the recombinant Clostridium perfringens β1 toxin protein is shown in SEQ ID NO:4; The amino acid sequence of the recombinant Clostridium perfringens ε toxin protein is shown in SEQ ID NO:6; The gene sequence encoding the recombinant Clostridium putrefactive α-toxin protein is shown in SEQ ID NO:1; the gene sequence encoding the recombinant Clostridium perfringens β1-toxin protein is shown in SEQ ID NO:3; and the gene sequence encoding the recombinant Clostridium perfringens ε-toxin protein is shown in SEQ ID NO:

5.

2. The immune composition according to claim 1, characterized in that, The mass ratio of recombinant Clostridium putrefactive α-toxin protein, recombinant Clostridium perfringens β1-toxin protein, and recombinant Clostridium perfringens ε-toxin protein in the immune composition is 1:2:

2.

3. The immune composition according to claim 1, characterized in that, It also includes pharmaceutically acceptable adjuvants, said adjuvant being at least one of SP 01 adjuvant, MONTANIDE™ ISA 201VG adjuvant, and MONTANIDE™ ISA 206VG adjuvant.

4. The immune composition according to claim 3, characterized in that, The volume ratio of the adjuvant to the immune composition is 1:

1.

5. A method for preparing the immune composition as described in claim 1, characterized in that, include: 1) Each target gene was cloned into a eukaryotic expression vector to obtain a recombinant vector; The target genes are the encoding genes for recombinant Clostridium putrefactive α-toxin protein, recombinant Clostridium perfringens β1-toxin protein, and recombinant Clostridium perfringens ε-toxin protein, respectively. 2) The recombinant vectors obtained in step 1) were transfected into CHO cells, and after culturing and screening, high-expression engineered cell lines were obtained; 3) The high-expression engineered cells obtained in step 2) are fermented and cultured to obtain the target proteins, namely, recombinant Clostridium putrefactive α-toxin protein, recombinant Clostridium perfringens β1-toxin protein and recombinant Clostridium perfringens ε-toxin protein. 4) The recombinant Clostridium putrefactive α-toxin protein, recombinant Clostridium perfringens β1-toxin protein and recombinant Clostridium perfringens ε-toxin protein obtained in step 3) are mixed to obtain the immune composition.

6. The preparation method according to claim 5, characterized in that, The eukaryotic expression vector is at least one of pcDNA3.1, pEE6.4, pEE12.4 and pKS001.

7. The preparation method according to claim 5, characterized in that, The CHO cells include at least one of the following: DG44, DXB11, CHO-K1, and CHO-S.

8. The use of the immune composition according to any one of claims 1 to 4 or the immune composition prepared by the method according to any one of claims 5 to 7 in the preparation of a medicament for the prevention of clostridial infection in sheep.

9. The use of the immune composition according to any one of claims 1 to 4 or the immune composition prepared by the method according to any one of claims 5 to 7 in the preparation of a trivalent and quadrivalent genetically engineered subunit vaccine for clostridial disease in sheep.

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