CN111808202B

CN111808202B - Clostridium perfringens gene engineering subunit vaccine, preparation method and application thereof

Info

Publication number: CN111808202B
Application number: CN202010953405.2A
Authority: CN
Inventors: 曹文龙; 孔迪; 滕小锘; 张大鹤
Original assignee: Suzhou Midi Biotechnology Co ltd; Suzhou Shinuo Biotechnology Co ltd
Current assignee: Suzhou Womei Biology Co ltd
Priority date: 2020-09-11
Filing date: 2020-09-11
Publication date: 2020-12-11
Anticipated expiration: 2040-09-11
Also published as: CN111808202A

Abstract

The invention discloses a clostridium perfringens gene engineering subunit vaccine, a preparation method and application thereof. The vaccine contains a protein composition and a pharmaceutically acceptable carrier, wherein the protein composition comprises three fusion proteins with sequences shown by SEQ ID NO. 2, SEQ ID NO. 6 and SEQ ID NO. 10 respectively. The vaccine provided by the invention has no toxicity, high safety and good immunogenicity, can generate stronger humoral immunity in an animal body, the immunized animal can resist strong toxicity and attack, and the vaccine can be prepared by large-scale serum-free suspension culture of a bioreactor, and has the advantages of easy quality control, stable batch-to-batch, low production cost and the like.

Description

Clostridium perfringens gene engineering subunit vaccine, preparation method and application thereof

Technical Field

The invention relates to a genetic engineering vaccine, in particular to a clostridium perfringens genetic engineering subunit vaccine, a preparation method and application thereof, belonging to the technical field of animal immunity drugs.

Background

Clostridium Perfringens (CP) is an important pathogen for zoonosis and can cause various diseases of human and livestock, such as cattle sniper, lamb dysentery and sheep enterotoxemia; flatulence and sudden death of pigs, necrotic enteritis of piglets; clostridial enteritis of horses, deer and rabbits. Not only threatens the health of human beings, but also causes huge economic loss to the animal husbandry. Because clostridium perfringens disease has the characteristics of acute morbidity, short course of disease and extremely high mortality, immunization is an effective method for preventing and controlling the disease. Clostridium perfringens has been demonstrated to exist in at least more than 15 exotoxins, of which alpha, beta, iota toxins are the four major pathogenic toxins, and clostridium perfringens can be classified into types A, B, C, D and E, depending on the type of toxin secreted. Many strains also produce enterotoxin (CPE), β 2 toxin, and Necrotic enteritis B-like toxin (NetB), among others. Clostridium perfringens alpha toxin (clostridium perfringens alpha toxin) is one of the most important toxins of clostridium perfringens, and has the characteristics of cytotoxicity, hemolytic activity, lethality, skin necrotizing property, platelet aggregation, increased vascular permeability and the like. Alpha toxin has very important significance for immunoprophylaxis of clostridium perfringens type A infection. Clostridium perfringens beta 1 toxin (clostridium perfringens beta 1 toxin) can only be isolated from clostridium perfringens types B and C, is lethal, forms tissue necrosis and is trypsin-stable, a neurotoxin. Clostridium perfringens beta 2 toxin (clostridium perfringens beta 2 toxin) is present in clostridium perfringens type C cultures and has similar biological activity to beta 1 toxin, and antibodies against beta 2 toxin recognize purified beta 2 toxin and react weakly with beta 1 toxin, however, antibodies against beta 1 toxin only react with beta 1 toxin but not with beta 2 toxin. Both the β 1 and β 2 toxins are cytotoxic and can trigger hemorrhagic necrosis of the intestinal mucosa. Clostridium perfringens toxin (clostridium perfringens epsilon toxin) is a necrotic and lethal toxin produced by clostridium perfringens types B and D, which causes the contraction of the aorta and other arteries of animals leading to elevated blood pressure, and also has the ability to bind to vascular epithelial receptors, which increases vascular permeability, ultimately leading to fatal edema. Iota toxin (iota-toxin) is secreted only by clostridium perfringens type E and is composed of two parts, Ia and Ib. Ia and Ib are nontoxic when existing independently and have cytotoxicity and lethality when existing simultaneously. NetB toxin can be separated from A-type and C-type clostridium perfringens, and the NetB toxin as a secreted toxin is very easy to act on a host immune system.

The current vaccine used for the immunization of clostridium perfringens disease is mainly a toxoid inactivated vaccine, and the production of the toxoid vaccine has the defects of high cost, long time consumption, pathogenic bacteria diffusion and the like because the culture of clostridium perfringens, the inactivation and the concentration of toxin are needed. However, genetic engineering vaccines such as CN110051834A and CN107308445A can only protect infection caused by one or more clostridia, and cannot provide comprehensive protection, and the recombinant protein is expressed in inclusion bodies, so the immunogenicity is poor. In addition, various microtoxins and bacterial metabolites in the culture supernatant often become allergens of immunized animals, and adverse reactions and even immune failure are easy to generate.

In view of the above, there is a need to develop a genetic engineering subunit vaccine that can simultaneously express multiple toxin proteins and has high safety and strong immunogenicity.

Disclosure of Invention

The invention mainly aims to provide a clostridium perfringens genetic engineering subunit vaccine, a preparation method and application thereof, so as to overcome the defects in the prior art.

In order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:

the embodiment of the invention provides a protein composition, which comprises: a first fusion protein having a sequence shown by SEQ ID NO. 2 or a sequence that is 95% or more identical to the full-length sequence of SEQ ID NO. 2, a second fusion protein having a sequence shown by SEQ ID NO. 6 or a sequence that is 95% or more identical to the full-length sequence of SEQ ID NO. 6, and a third fusion protein having a sequence shown by SEQ ID NO. 10 or a sequence that is 95% or more identical to the full-length sequence of SEQ ID NO. 10.

That is, the amino acid sequence of each fusion protein provided in the embodiments of the present invention may be the original sequence, an added or truncated sequence.

The embodiment of the invention also provides a genome composition, which comprises coding genes respectively used for coding the first fusion protein, the second fusion protein and the third fusion protein in the protein composition.

In some embodiments, the genomic composition comprises: a first nucleic acid molecule having a sequence shown by SEQ ID NO. 1 or a sequence that is 95% or more identical to the full-length sequence of SEQ ID NO. 1, a second nucleic acid molecule having a sequence shown by SEQ ID NO. 5 or a sequence that is 95% or more identical to the full-length sequence of SEQ ID NO. 5, and a third nucleic acid molecule having a sequence shown by SEQ ID NO. 9 or a sequence that is 95% or more identical to the full-length sequence of SEQ ID NO. 9.

The embodiment of the invention also provides a recombinant vector composition, which comprises:

a first recombinant vector comprising a gene encoding a first fusion protein in the protein composition;

a second recombinant vector comprising a gene encoding a second fusion protein in the protein composition; and

a third recombinant vector comprising a gene encoding a third fusion protein of the protein composition.

In some embodiments, the first, second, and third recombinant vectors include, but are not limited to, pFastBac1 or pVL1393, and pFastBac1 is preferably used.

Embodiments of the present invention also provide a host cell composition, including:

a first host cell comprising a gene encoding a first fusion protein in the protein composition;

a second host cell comprising a gene encoding a second fusion protein of the protein composition; and

a third host cell comprising a gene encoding a third fusion protein in the protein composition.

In some embodiments, the first, second, and third host cells are selected from insect cells, such as Sf9 cell lines, preferably Sf9 cell lines including but not limited to Sf9, High Five, or Sf21 cells, more preferably Sf9 cells.

The embodiment of the invention also provides an immune composition, which is characterized by comprising the following components: the protein composition described; and a pharmaceutically acceptable carrier.

In some embodiments, the pharmaceutically acceptable carrier includes, but is not limited to, any one or a combination of two or more of montainide ISA 206 VG, montainide ISA 201 VG, montainide ISA 15 VG, liquid paraffin, camphor oil, plant cell agglutinin, preferably, montainide ISA 201 VG.

The embodiment of the invention also provides a preparation method of the protein composition, which comprises the following steps:

providing genes encoding a first fusion protein, a second fusion protein, and a third fusion protein in the protein composition;

cloning the coding gene of the first fusion protein, the coding gene of the second fusion protein and the coding gene of the third fusion protein into shuttle vectors respectively to obtain a first recombinant shuttle vector, a second recombinant shuttle vector and a third recombinant shuttle vector containing corresponding target genes respectively;

respectively transforming the first recombinant shuttle vector, the second recombinant shuttle vector and the third recombinant shuttle vector into competent cells, and separating to obtain a first recombinant baculovirus genome plasmid, a second recombinant baculovirus genome plasmid and a third recombinant baculovirus genome plasmid which respectively contain corresponding target gene expression frames;

transfecting insect cells by using the first recombinant baculovirus genome plasmid, the second recombinant baculovirus genome plasmid and the third recombinant baculovirus genome plasmid respectively to obtain corresponding first recombinant baculovirus, second recombinant baculovirus and third recombinant baculovirus;

and respectively inoculating insect cells with the first recombinant baculovirus, the second recombinant baculovirus and the third recombinant baculovirus, culturing, and separating to obtain the first fusion protein, the second fusion protein and the third fusion protein.

In some embodiments, the shuttle vector includes, but is not limited to, pFastBac1 or pVL1393 and the like, preferably pFastBac 1.

In some embodiments, the host cell is selected from insect cells such as Sf9 cell line, for example, can include but is not limited to Sf9, High Five or Sf21 cells, and the like, preferably Sf9 cells.

The embodiment of the invention also provides a preparation method of the clostridium perfringens genetic engineering subunit vaccine, which comprises the following steps: preparing the first fusion protein, the second fusion protein and the third fusion protein by any one of the methods, and mixing the first fusion protein, the second fusion protein and the third fusion protein with a pharmaceutically acceptable carrier.

The embodiments also provide for the use of the protein composition or the immunological composition in the manufacture of a clostridium perfringens detection reagent, in the manufacture of a medicament for inducing an immune response against a clostridium perfringens antigen in a subject animal, or in the manufacture of a medicament for preventing infection of an animal with clostridium perfringens.

The embodiment of the invention also provides application of the protein composition or the immune composition in preparing clostridium perfringens genetic engineering subunit vaccines.

The embodiment of the invention provides a clostridium perfringens genetic engineering subunit vaccine which comprises any one of the immune compositions. Further, the vaccine may further comprise a pharmaceutically acceptable carrier.

The embodiment of the invention also provides application of a recombinant vector or a host cell containing the encoding gene of each fusion protein in the protein composition in producing a reagent for detecting the infection of animals by clostridium perfringens.

The embodiments also provide the use of a recombinant vector or host cell comprising the gene encoding each fusion protein in the protein composition in the manufacture of a medicament for inducing an immune response against a clostridium perfringens antigen in a subject animal.

The embodiment of the invention also provides the use of the recombinant vector or the host cell containing the coding gene of each fusion protein in the protein composition in the production of a medicament for preventing the animals from being infected by clostridium perfringens.

Embodiments also provide a method of inducing an immune response against a clostridium perfringens antigen, comprising administering the clostridium perfringens genetically engineered subunit vaccine to a subject animal.

Embodiments also provide a method of protecting a subject animal from infection by clostridium perfringens, the method comprising administering to the subject animal the clostridium perfringens genetically engineered subunit vaccine.

Embodiments also provide a vaccine suitable for generating an immune response against clostridium perfringens infection in a subject animal, the vaccine comprising: the protein compositions of the invention and adjuvant molecules.

An "adjuvant" as described in the present specification means any molecule added to the vaccine described in the present specification to enhance the immunogenicity of the antigen encoded by the encoding nucleic acid sequence described below. For example, the adjuvant can be IL-12, IL-15, IL-28, CTACK, TECK, Platelet Derived Growth Factor (PDGF), TNF α, TNF β, GM-CSF, Epidermal Growth Factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, IL-21, IL-31, IL-33, or a combination thereof; and in some embodiments, can be IL-12, IL-15, IL-28 or RANTES. Further, the adjuvant may preferably be related adjuvants produced by Suzhou Shino biotechnology, Inc. to improve the effect of the vaccine.

Compared with the prior art, the embodiment of the invention transforms the clostridium perfringens alpha toxin and beta 2 toxin and then performs fusion expression with the Ia toxin to form a first fusion protein (named as Plc-Ia-CPB 2), transforms the clostridium perfringens beta 1 toxin and performs fusion expression with NetB to form a second fusion protein (named as CPB 1-NetB), and transforms the clostridium perfringens toxin and Ib toxin to form a third fusion protein (named as ETX-Ib), and the fusion proteins can be prepared by a baculovirus insect cell expression system, suspension culture Sf9 cells and the like for expression and large-scale serum-free suspension culture, thereby greatly reducing the production cost of the vaccine, simultaneously, the antigenicity, the immunogenicity and the function of the obtained product are similar to those of natural proteins, the expression level is higher, the immunogenicity is strong, and the good immune effect can be provided only by a small amount, has no pathogenicity to animals, and is suitable for being widely applied as a recombinant genetic engineering subunit vaccine of clostridium perfringens.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a gel electrophoresis image of the PCR amplification product of the codon-optimized Plc-Ia-CPB2 gene of example 1, in which the band of interest appeared at the 1.4kbp position.

FIG. 2 is a gel electrophoresis chart of the colony PCR amplification product in example 1, wherein the band of interest appears at the 1.4kbp position.

FIG. 3 is a schematic diagram of the structure of the transfer vector pF-Plc-Ia-CPB2 in example 1.

FIG. 4 is a gel electrophoresis chart of the PCR-amplified product of CPB1-NetB gene after codon optimization in example 2, in which the band of interest appeared at the 0.8kbp position.

FIG. 5 is a gel electrophoresis chart of the colony PCR amplification product in example 2, in which the band of interest appears at the 0.8kbp position.

FIG. 6 is a schematic diagram of the structure of the transfer vector pF-CPB1-NetB in example 2.

FIG. 7 is a gel electrophoresis photograph of the PCR-amplified product of ETX-Ib gene after codon optimization in example 3, in which a desired band appears at the position of 1.0 kbp.

FIG. 8 is a gel electrophoresis chart of the PCR-amplified product of the colony in example 3, wherein the band of interest appears at the 1.0kbp position.

FIG. 9 is a schematic structural diagram of the transfer vector pF-ETX-Ib in example 3.

FIG. 10 is a SDS-PAGE detection profile of the cell culture obtained in example 5.

FIG. 11 is a graph showing the results of Western Blot detection of the products obtained in example 6 after SDS-PAGE electrophoresis.

FIG. 12 is a gray-scale scan of each purified fusion protein obtained in example 9.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

According to the embodiment of the invention, the clostridium perfringens alpha toxin, beta 2 toxin and Ia toxin are subjected to fusion expression, amino acids 266R and 294G of the clostridium perfringens alpha toxin are respectively mutated into P, H, amino acids 206D and 217S of the clostridium perfringens beta 2 toxin are respectively mutated into C, K, the toxicity of the two toxins is reduced, and finally the Plc-Ia-CPB2 fusion protein (defined as a first fusion protein) is formed, and meanwhile, Y at the 182 th position of the clostridium perfringens beta 1 toxin amino acid is removed and is subjected to fusion expression with NetB to form CPB1-NetB (defined as a second fusion protein), so that the clostridium perfringens alpha toxin, beta 2 toxin and Ia toxin are safe and nontoxic. Furthermore, the clostridium perfringens toxin and the clostridium perfringens Ib toxin are fused and expressed to form ETX-Ib protein (defined as a third fusion protein), and the selective fusion can ensure that the fused protein can be secreted and the expression quantity is increased, and simultaneously ensure that the fused protein is soluble, has higher activity and stronger immunogenicity. Furthermore, the three safe and nontoxic fusion proteins are used cooperatively, so that the invasion of clostridium perfringens can be comprehensively prevented.

Furthermore, the aforementioned Plc-Ia-CPB2, CPB1-NetB and ETX-Ib fusion proteins can be expressed by using Sf9 cells cultured in suspension based on a baculovirus insect cell expression system, and not only the expression level is high, but also the protein immunogenicity is good.

Further, the aforementioned Plc-Ia-CPB2, CPB1-NetB and ETX-Ib fusion proteins can be used for preparing a Clostridium perfringens genetically engineered subunit vaccine.

For example, in a specific embodiment of the present embodiments, a method for preparing a clostridium perfringens genetically engineered subunit vaccine can specifically comprise:

(1) preparing nucleic acid molecules for encoding the Plc-Ia-CPB2, CPB1-NetB and ETX-Ib fusion proteins, respectively;

(2) cloning the nucleic acid molecules of the fusion proteins prepared in the step (1) into shuttle vectors respectively to obtain corresponding recombinant shuttle vectors containing target genes;

(3) respectively transforming the recombinant shuttle vectors obtained in the step (2) into DH10Bac bacteria, selecting recombinant bacteria, extracting genome to transfect Sf9 cells (or other insect cells) to obtain recombinant baculovirus;

(4) incubating said Sf9 cells (or other insect cells as described previously) for recombinant expression to produce the Plc-Ia-CPB2, CPB1-NetB and ETX-Ib fusion proteins;

(5) and mixing the recombinant Plc-Ia-CPB2, the CPB1-NetB and the ETX-Ib fusion protein, and adding the mixture into an adjuvant to obtain the vaccine.

In the specific embodiment of the invention, Sf9 cells are used for expressing the fusion proteins Plc-Ia-CPB2, CPB1-NetB and ETX-Ib, the antigenicity, immunogenicity and function of the product are similar to those of natural proteins, the expression level is higher (100-200 mg/L), the immunogenicity is strong, no pathogenicity is caused to animals, and the vaccine can be prepared by using a bioreactor for large-scale serum-free suspension culture, so that the vaccine production cost is greatly reduced.

The clostridium perfringens genetic engineering subunit vaccine provided by the embodiment of the invention has no toxicity, high safety and good immunogenicity, can generate stronger humoral immunity in an animal body, and the immunized animal can resist strong toxicity attack, and also has a series of advantages of large-scale batch production, easy quality control, stable batch-to-batch, low production cost and the like.

When the clostridium perfringens gene engineering subunit vaccine provided by the embodiment of the invention is applied, only an effective amount of the clostridium perfringens gene engineering subunit vaccine needs to be inoculated to mammals such as human beings, cows, sheep, pigs, horses, deer, rabbits and the like. As used herein, the term "effective amount" refers to an amount sufficient to obtain, or at least partially obtain, a desired effect. For example, a disease-preventing effective amount refers to an amount sufficient to prevent, or delay the onset of disease; a therapeutically effective amount for a disease is an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. It is well within the ability of those skilled in the art to determine such effective amounts.

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The reagents and starting materials used in the following examples are commercially available, and the test methods in which specific conditions are not specified are generally carried out under conventional conditions or conditions recommended by the respective manufacturers. Further, unless otherwise indicated, the assays, detection methods, and preparations disclosed herein are performed using molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA techniques, and techniques conventional in the art. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, Sambrook et al: a LABORATORY MANUAL, Second edition, Cold Spring Harbor LABORATORY Press, 1989and Third edition, 2001; ausubel et al, Current PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; (iii) METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, chromatography Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.

Example 1 construction and identification of transfer vector pF-Plc-Ia-CPB2

Plc-Ia-CPB2 gene amplification and purification A codon-optimized Plc-Ia-CPB2 gene (SEQ ID NO: 1) was synthesized by Nanjing Kingsler Biotech Co., Ltd and cloned into a pUC17 vector to obtain a pUC-Plc-Ia-CPB2 plasmid vector. PCR amplification was performed using pUC-Plc-Ia-CPB2 plasmid as template and Plc-Ia-CPB2-F, Plc-Ia-CPB2-R as upstream and downstream primers (the gene sequences of Plc-Ia-CPB2-F, Plc-Ia-CPB2-R are shown in SEQ ID NO:3, 4), and the amplification system is shown in Table 1.

TABLE 1 Plc-Ia-CPB2 Gene amplification System

The reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 45 seconds, annealing at 54 ℃ for 45 seconds, extension at 72 ℃ for 1 minute, 35 cycles; extension at 72 ℃ for 10 min.

The PCR product was subjected to gel electrophoresis to verify the size of the target gene, and as shown in FIG. 1, a band of interest appeared at a position of 1.4kbp, and the target gene was successfully amplified and recovered and purified using a gel recovery and purification kit.

2. Enzyme digestion and purification the PCR amplification product of pFastBac1 plasmid and the Plc-Ia-CPB2 gene were digested simultaneously at 37 ℃ for 3 hours using BamHI and Hind III, and the specific digestion reaction systems are shown in tables 2 and 3.

And (3) performing gel electrophoresis on the enzyme digestion product, and purifying the enzyme digestion pFastBac1 plasmid and the alpha-iotaa (Ia) -beta 2 gene fragment by using a gel recovery and purification kit respectively.

TABLE 2 restriction system for Plc-Ia-CPB2 gene

TABLE 3 pFastBac1 plasmid digestion reaction System

3. Ligation the digested pFastBac1 plasmid and the digested product of the Plc-Ia-CPB2 gene were ligated overnight using T4 DNA ligase in a water bath at 16 ℃ in the system shown in Table 4.

TABLE 4 ligation System of the Plc-Ia-CPB2 Gene with the pFastBac1 plasmid

4. Mu.l of the ligation product was added to 100. mu.l of DH 5. alpha. competent cells, mixed well, heat-shocked at 42 ℃ for 90 seconds, ice-bathed for 2 minutes, added to 900. mu.l of LB medium without Amp, and incubated at 37 ℃ for 1 hour. 1.0 mL of the cell suspension was concentrated by centrifugation to 100. mu.l, and the concentrated solution was applied to LB solid medium containing Amp and cultured at 37 ℃ for 16 hours.

5. Colony PCR and sequencing identification single colonies on the selected plate are respectively inoculated into an LB liquid culture medium, cultured for 2 hours at 37 ℃, and colony PCR is carried out by taking a bacterial liquid as a template and taking Plc-Ia-CPB2-F and Plc-Ia-CPB2-R as primers. The size of the gene of interest was confirmed by subjecting the PCR product to gel electrophoresis, and as shown in FIG. 2, a sample showing a band of approximately 1.4kbp was positive. And (4) sending the bacterial liquid with positive colony PCR identification to a sequencing company for sequencing, and selecting the bacterial liquid with correct sequencing for storage. The schematic diagram of the constructed transfer vector containing the target gene pF-Plc-Ia-CPB2 is shown in FIG. 3.

Example 2 construction and characterization of transfer vector pF-CPB1-NetB

CPB1-NetB gene amplification and purification CPB1-NetB gene (SEQ ID NO: 5) after codon optimization is synthesized by Nanjing Kingsler Biotech Co., Ltd and cloned to pUC17 vector to obtain pUC-CPB1-NetB plasmid vector. PCR amplification was carried out using pUC-CPB1-NetB plasmid as template and CPB1-NetB-F, CPB1-NetB-R as upstream and downstream primers (the gene sequences of CPB1-NetB-F, CPB1-NetB-R are shown in SEQ ID NO:7, 8), and the amplification systems are shown in Table 5.

TABLE 5 CPB1-NetB Gene amplification System

The size of the target gene was verified by subjecting the PCR product to gel electrophoresis, and as shown in FIG. 4, the target gene was successfully amplified by the occurrence of a band of 0.8kbp, and was recovered and purified by a gel recovery and purification kit.

2. Enzyme digestion and purification the pFastBac1 plasmid and the PCR amplification product of the CPB1-NetB gene were digested simultaneously at 37 ℃ for 3 hours using BamHI and Hind III, and the specific digestion reaction systems are shown in tables 6 and 7.

And (3) performing gel electrophoresis on the enzyme digestion product, and purifying the enzyme digestion pFastBac1 plasmid and the CPB1-NetB gene fragment by using a gel recovery and purification kit respectively.

TABLE 6 enzyme digestion reaction system of CPB1-NetB gene

TABLE 7 pFastBac1 plasmid digestion reaction System

3. Ligation the digested pFastBac1 plasmid and the product of CPB1-NetB gene digestion were ligated overnight using T4 DNA ligase in a 16 ℃ water bath, the ligation system is shown in Table 8.

TABLE 8 ligation System of CPB1-NetB Gene and pFastBac1 plasmid

5. Colony PCR and sequencing identification single colonies on the selected plate are respectively inoculated into an LB liquid culture medium, cultured for 2 hours at 37 ℃, and colony PCR is carried out by taking a bacterial liquid as a template and CPB1-NetB-F and CPB1-NetB-R as primers. The size of the target gene was confirmed by subjecting the PCR product to gel electrophoresis, and as shown in FIG. 5, a sample showing a band of approximately 0.8kbp was found to be a positive sample. And (4) sending the bacterial liquid with positive colony PCR identification to a sequencing company for sequencing, and selecting the bacterial liquid with correct sequencing for storage. The schematic diagram of the constructed transfer vector pF-CPB1-NetB containing the target gene is shown in FIG. 6.

Example 3 construction and characterization of transfer vector pF-ETX-Ib

ETX-Ib gene amplification and purification ETX-Ib gene (SEQ ID NO: 9) obtained by synthesizing codon-optimized ETX-Ib gene in Nanjing Kingsry Biotechnology Co., Ltd is cloned to a pUC17 vector to obtain a pUC-ETX-Ib plasmid vector. PCR amplification was performed using pUC-ETX-Ib plasmid as template and ETX-Ib-F, ETX-Ib-R as upstream and downstream primers (the gene sequences of ETX-Ib-F, ETX-Ib-R are shown in SEQ ID NO:11, 12), and the amplification system is shown in Table 9.

TABLE 9 ETX-Ib Gene amplification System

The size of the target gene was verified by subjecting the PCR product to gel electrophoresis, and as shown in FIG. 7, the target gene was successfully amplified by the occurrence of a band of 1.0kbp, and was recovered and purified by a gel recovery and purification kit.

2. Enzyme digestion and purification the pFastBac1 plasmid and the PCR amplification product of the ETX-Ib gene were digested simultaneously for 3 hours at 37 ℃ with BamHI and Hind III, and the specific digestion reaction systems are shown in tables 10 and 11.

And (3) performing gel electrophoresis on the enzyme digestion product, and purifying the enzyme digestion pFastBac1 plasmid and the ETX-Ib gene fragment by using a gel recovery and purification kit respectively.

TABLE 10 restriction reaction system for ETX-Ib gene

TABLE 11 pFastBac1 plasmid digestion reaction System

3. Ligation the digested pFastBac1 plasmid and the product of ETX-Ib gene digestion were ligated overnight using T4 DNA ligase in a 16 ℃ water bath, the ligation system is shown in Table 12.

TABLE 12 ETX-Ib Gene and pFastBac1 plasmid ligation System

5. Colony PCR and sequencing identification single colonies on the selected plate are respectively inoculated into an LB liquid culture medium, cultured for 2 hours at 37 ℃, and colony PCR is carried out by taking a bacterial solution as a template and ETX-Ib-F and ETX-Ib-R as primers. The size of the target gene was confirmed by subjecting the PCR product to gel electrophoresis, and as shown in FIG. 8, a sample showing a band of approximately 1.0kbp was positive. And (4) sending the bacterial liquid with positive colony PCR identification to a sequencing company for sequencing, and selecting the bacterial liquid with correct sequencing for storage. The schematic diagram of the constructed transfer vector pF-ETX-Ib containing the target gene is shown in FIG. 9.

Example 4 construction of recombinant baculovirus genomes Bac-Plc-Ia-CPB2, Bac-CPB1-NetB and Bac-ETX-Ib

Mu.l of pF-Plc-Ia-CPB2 plasmid, pF-CPB1-NetB plasmid and pF-ETX-Ib plasmid in each of examples 1 to 3 above were taken and added to 100. mu.l of DH10Bac competent cells respectively to be mixed uniformly, ice-cooled for 30 minutes, water-bath heat shock at 42 ℃ for 90 seconds, ice-cooled for 2 minutes again, 900. mu.l of LB liquid medium without Amp was added, and cultured at 37 ℃ for 5 hours. After 100. mu.l of the diluted bacterial solution was diluted 81 times, 100. mu.l of the diluted bacterial solution was applied to LB solid medium containing gentamicin, kanamycin, tetracycline, X-gal and IPTG, and cultured at 37 ℃ for 48 hours.

2. Selecting a single colony, selecting a large white colony by using an inoculating needle, then streaking on an LB solid culture medium containing gentamicin, kanamycin, tetracycline, X-gal and IPTG, culturing for 48 hours at 37 ℃, selecting a single colony, inoculating an LB liquid culture medium containing gentamicin, kanamycin and tetracycline, culturing, preserving strains, and extracting plasmids. The recombinant plasmids Bacmid-Plc-Ia-CPB2, Bacmid-CPB1-NetB and Bacmid-ETX-Ib are respectively obtained.

Example 5 recombinant baculovirus transfection

Six well plates were seeded 0.8X 10 per well⁶The confluency of Sf9 cells is 50-70%. The following complexes were prepared for each well: diluting 4. mu.l of Cellffectin transfection reagent with 100. mu.l of transfection medium T1, and shaking briefly with vortex; mu.g of the recombinant Bacmid-Plc-Ia-CPB2 plasmid, recombinant Bacmid-CPB1-NetB plasmid and recombinant Bacmid-ETX-Ib plasmid of example 4 were diluted with 100. mu.l of transfection culture T1 medium, and the diluted transfection reagents and plasmids were mixed, gently and evenly blown, respectively, to prepare a transfection mixture. And adding the transfection compound after the cells adhere to the wall, incubating for 5 hours at 27 ℃, removing the supernatant, adding 2mLSF-SFM fresh culture medium, and culturing for 4-5 days at 27 ℃ to obtain the supernatant. Respectively obtaining recombinant baculovirus rBac-Plc-Ia-CPB2, rBac-CPB1-NetB and rBac-ETX-Ib, detecting virus titer of the harvested P1 generation recombinant baculovirus by using an MTT relative efficacy method, wherein the titer of the rBac-Plc-Ia-CPB 2P 1 virus is 7.5 multiplied by 10⁷pfu/mL, rBac-CPB1-NetB P1 virus titer is 3.7X 10⁸pfu/mL, rBac-ETX-Ib P1 virus titer 3.9 × 10⁷pfu/mL. Amplifying recombinant baculovirus rBac-Plc-Ia-CPB2, rBac-CPB1-NetB and rBac-ETX-Ib for later use as seed viruses.

Recombinant baculoviruses expressing the following (table 13) two control groups were also constructed as per the above example:

watch 13

Example 6 SDS-PAGE detection

Observing the cell cultures harvested in example 5, it can be seen that the cell cultures of control 1 and control 2 both show different degrees of apoptosis after 24 hours, and the cells show pathological morphology, probably because the non-mutated protein is toxic to insect cells and causes premature cell death.

The cell cultures of the groups harvested in example 5 were subjected to SDS-PAGE detection, while using Sf9 cells infected with empty baculovirus as a negative control. The specific operation is as follows: mu.l of the harvested cell culture was taken, 10. mu.l of 5 × loading buffer was added, the mixture was centrifuged in a boiling water bath for 5 minutes at 12000r/min for 1 minute, the supernatant was subjected to SDS-PAGE gel (12% strength gel) electrophoresis, and the gel was stained and decolored after electrophoresis to observe the band.

As shown in FIG. 10, the rBac-Plc-Ia-CPB2 cell culture showed a band of interest at a molecular weight of about 56kDa and the protein expression was higher than that of control 1; the rBac-CPB1-NetB cell culture has a target band at the molecular weight of about 30kDa, and the protein expression amount is higher than that of the control group 2; the rBac-ETX-Ib cell culture shows a target band at a molecular weight of about 37kDa, and the negative control has no band at the corresponding position.

Example 7 Western Blot assay

The SDS-PAGE products of example 6 were transferred to NC (nitrocellulose) membranes, blocked with 5% skim milk for 2 hours, rBac-Plc-Ia-CPB2, rBac-CPB1-NetB and rBac-ETX-Ib cell culture electrophoresed products were incubated with ovine anti-B, C, D Clostridium perfringens mixed toxin serum for 2 hours, rinsed, HRP-labeled rabbit anti-sheep polyclonal antibody secondary antibody was incubated for 2 hours, rinsed, and then added dropwise with enhanced chemiluminescent fluorescent substrate and photographed using a chemiluminescent imager. The results are shown in FIG. 11, where the recombinant baculovirus expression sample has a band of interest, and the negative control has no band of interest, indicating that the protein of interest was correctly expressed in Sf9 cells.

EXAMPLE 8 bioreactor serum-free suspension culture of insect cells

The Sf9 insect cells were aseptically cultured in 1000mL shake flasks for 3-4 days to a concentration of 3-5X 10⁶cell/mL, when the activity is more than 95%, inoculating the cells into a 5L bioreactor, wherein the inoculation concentration is 3-8 × 10⁵cell/mL. When the cell concentration reaches 3-55X 10⁶At cell/mL, cells were seeded into a 50L bioreactor until the cells grew to a concentration of 3-55X 10⁶cell/mL, inoculating into 500L bioreactor until cell concentration reaches 2-85 × 10⁶When cell/mL, rBac-Plc-Ia-CPB2 is inoculated, and the culture conditions of the reactor are that the pH value is 6.0-6.5, the temperature is 25-27 ℃, the dissolved oxygen is 30-80%, and the stirring speed is 100-180 rpm. In view of the optimum conditions for cell culture, it is preferable to set pH6.2, the temperature at the stage of cell culture at 27 ℃, the dissolved oxygen at 50%, and the stirring speed at 100-180 rpm. Culturing for 5-9 days after infection, adding one-thousandth final concentration BEI, acting at 37 deg.C for 48 hr, adding two-thousandth final concentration Na₂S₂O₃The inactivation is terminated. Cell culture supernatants were harvested by centrifugation or hollow fiber filtration and stored at 2-8 ℃ for the Plc-Ia-CPB2 protein stock. Meanwhile, protein stock solutions expressing CPB1-NetB, ETX-Ib and each control group were prepared in the same manner.

Example 9 protein purification

1. Purifying the harvested stock solution by cation exchange chromatography

The particle exchange chromatography was performed using strong cation particle chromatography packing POROS 50HS, which was sterilized with 0.5M NaOH before use. The vaccine stock was then equilibrated with microfiltration buffer at room temperature, and then loaded onto the column at a rate of 125 mL/min, followed by 8 column volumes eluted with rinse buffer a (0.05M MOPS (sodium salt), pH =7.0, 0.5M NaCl). Elution was then performed with a linear gradient from 0% buffer a to 100% buffer B (0.05M MOPS (sodium salt), pH =7.0, 1.5M NaCl), where a total of 10 column volumes were eluted by linear elution, and then the 10 column volumes were harvested on average. After linear elution, 2 column volumes were eluted with buffer B and collected separately. The collected sample was placed in a 2L sterile plastic bottle and placed at 4 ℃. The fractions collected under the last elution peak (A280) were then stored sterile filtered at 4 ℃.

2. Hydroxyapatite hydrophobic chromatography

Using a pre-packed Hydroxyapatite column (CHT;. Ceramic Hydroxyapatite Type II Media), first, 50 mM MOPS (sodium salt), pH =7.0, 1.25M NaCl was equilibrated, and then the above preliminary-purified sample was loaded at 90 cm/hour, and after loading, elution was carried out using 8 volumes of an equilibration solution until the UV value was zero. Then a gradient elution was performed using an eluent (0.2M phosphate, pH =7.0, 1.25M NaCl) with a concentration of eluent from 0% to 100%, the speed was still 90 cm/h and the elution volume was 4 column volumes. Purifying to obtain the target protein.

Quantifying the purified target protein by using BCA total protein, and then determining the purity of the target protein by combining gray scanning, wherein the purified protein is shown in figure 12, the concentration of the Plc-Ia-CPB2 protein is 186 mu g/mL, and the purity is 92%; the concentration of CPB1-NetB protein is 212 mug/mL, and the purity is 95%; the concentration of the ETX-Ib protein is 110 mu g/mL, and the purity is 93 percent.

Example 10 vaccine preparation

Introducing a biphasic oil adjuvant (such as adjuvant 201) into the oil phase tank, autoclaving at a temperature of at least 121 deg.C for 30 minutes, and cooling to room temperature. The recombinant protein stock solutions harvested in example 9 were mixed by dilution in equal proportions in the manner shown in Table 14, and the mixed recombinant fusion proteins were diluted to a final concentration of 100. mu.g/ml with PBS (pH 7.2, 0.01mol/L) and mixed well. Adding the water phase into an emulsifying tank, stirring at 80-100 r/min, slowly adding the oil phase according to the ratio of 1:1(V/V), and stirring for 20-30 min after the addition is finished. Sampling after emulsification, inspecting, subpackaging after being qualified, and storing at 4 ℃.

TABLE 14

Example 11 immunization experiment

Test one: safety test

40 healthy rabbits with the weight of 1.5-2.0 kg are taken and divided into 4 groups, each group of animals are injected with 8.0ml (four times of immune dose) of vaccines of an immune group, a control group A, a control group B and a control group C through muscle or subcutaneous injection respectively, the injection observation is carried out for 10 days, and the detailed results are shown in a table 15.

Watch 15

And (2) test II: efficacy test

Healthy rabbits which are negative to clostridium perfringens toxin antibodies and have the weight of 1.5-2.0 kg are selected and grouped according to the requirements of table 14, 4 rabbits in each group are injected with 2.0ml of vaccine subcutaneously or intramuscularly at the neck, and a negative control group is arranged, and normal saline with the same volume is injected in the same way. Blood is collected 14 days after inoculation, serum is separated, and the rabbits are subjected to secondary immunization in the same dose and the same way. Blood was collected 21 days after the second immunization, and serum was separated. The toxin antibody titer in the rabbit serum after the primary and secondary immunizations was determined by the following method: the method comprises the steps of equivalently mixing 4 immune rabbit sera of the same group and the same time, taking 0.4ml of the mixed sera, respectively mixing with 0.8ml of A-type clostridium perfringens toxin (containing 4 mouse MLDs), B-type clostridium perfringens toxin (containing 4 mouse MLDs), C-type clostridium perfringens toxin (containing 4 mouse MLDs), D-type clostridium perfringens toxin (containing 12 mouse MLDs) and E-type clostridium perfringens toxin (containing 4 mouse MLDs), reacting at 37 ℃ for 40min after mixing, respectively injecting 10 mice of 16-20 g and 0.3ml per mouse intravenously, detecting toxin neutralizing antibody titer and observing survival rate. The detailed results are shown in Table 16.

TABLE 16 results of efficacy testing

It is to be understood that the above-described embodiments are part of the present invention, and not all embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Sequence listing

<110> Suzhou Shino Biotechnology, Inc., Suzhou Midi Biotechnology, Inc

<120> clostridium perfringens gene engineering subunit vaccine, preparation method and application thereof

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Met Asn Tyr Lys Gln Ala Thr Phe Tyr Leu Gly Glu Ala Met His Tyr

1 5 10 15

Phe Gly Asp Ile Asp Thr Pro Tyr His Pro Ala Asn Val Thr Ala Val

20 25 30

Asp Ser Ala Gly His Val Lys Phe Glu Thr Phe Ala Glu Glu Arg Lys

35 40 45

Glu Gln Tyr Lys Ile Asn Thr Ala Gly Cys Lys Thr Asn Glu Asp Phe

50 55 60

Tyr Ala Asp Ile Leu Lys Asn Lys Asp Phe Asn Ala Trp Ser Lys Glu

65 70 75 80

Tyr Ala Arg Gly Phe Ala Lys Thr Gly Lys Ser Ile Tyr Tyr Ser His

85 90 95

Ala Ser Met Ser His Ser Trp Asp Asp Trp Asp Tyr Ala Ala Lys Val

100 105 110

Thr Leu Ala Asn Ser Gln Lys Gly Thr Ala Gly Tyr Ile Tyr Pro Phe

115 120 125

Leu His Asp Val Ser Glu Gly Asn Asp Pro Ser Val Gly Lys Asn Val

130 135 140

Lys Glu Leu Val Ala Tyr Ile Ser Thr Ser His Glu Lys Asp Ala Gly

145 150 155 160

Thr Asp Asp Tyr Met Tyr Phe Gly Ile Lys Thr Lys Asp Gly Lys Thr

165 170 175

Gln Glu Trp Glu Met Asp Asn Pro Gly Asn Asp Phe Met Thr Gly Ser

180 185 190

Lys Asp Thr Tyr Thr Phe Lys Leu Lys Asp Glu Asn Leu Lys Ile Asp

195 200 205

Asp Ile Gln Asn Met Trp Ile Arg Lys Ile His Leu Lys Leu Pro Lys

210 215 220

Asn Thr Gly Met Leu Pro Tyr Ile Asn Ser Asn Asp Val Lys Thr Leu

225 230 235 240

Ile Glu Gln Asp Tyr Ser Ile Lys Ile Asp Lys Ile Val Arg Ile Val

245 250 255

Ile Glu Gly Lys Gln Tyr Ile Lys Ala Glu Ala Ser Ile Val Asn Ser

260 265 270

Leu Asp Phe Lys Asp Asp Val Ser Lys Gly Asp Leu Trp Gly Lys Glu

275 280 285

Asn Tyr Ser Asp Trp Ser Asn Lys Leu Thr Pro Asn Glu Leu Ala Asp

290 295 300

Val Asn Glu Arg Val Asn Asn Val Glu Gln Tyr Arg Glu Met Leu Glu

305 310 315 320

Asp Phe Lys Tyr Asp Pro Asn Gln Gln Leu Lys Ser Phe Glu Ile Leu

325 330 335

Asn Ser Gln Lys Ser Asp Asn Lys Glu Ile Phe Asn Val Lys Thr Glu

340 345 350

Phe Leu Asn Gly Ala Ile Tyr Asp Met Glu Phe Thr Val Ser Ser Lys

355 360 365

Asp Gly Lys Leu Ile Val Ser Asp Met Glu Arg Thr Lys Val Glu Asn

370 375 380

Glu Gly Lys Tyr Ile Leu Thr Pro Ser Phe Arg Thr Gln Val Cys Thr

385 390 395 400

Trp Asp Asp Glu Leu Ala Gln Ala Ile Gly Gly Val Tyr Pro Gln Thr

405 410 415

Tyr Ser Asp Arg Phe Thr Tyr Tyr Ala Asp Asn Ile Leu Leu Asn Phe

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Arg Gln Tyr Ala Thr Ser Gly Ser Arg Asp Leu Lys Val Glu Tyr Ser

435 440 445

Val Val Cys His Trp Met Trp Lys Asp Asp Val Lys Ala Lys Gln Met

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Val Tyr Gly Gln Asn Pro Asp Ser Ala Arg Gln Ile Arg Leu Tyr Ile

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Glu Lys

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ggatccatga actataaaca ggcgaccttt tatctgggc 39

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gattttagca ccattcagac cgatcatagc accagcaaag cgagctggga taccaaattt 180

accgaaacca cccgcggcaa ctataacctg aaaagcaaca acccggtgta tggcaacgaa 240

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ccgaacggca ccgaagaaag cattattaaa gtgaaaatgg aacgcgaacg caactgctat 420

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ccggattttc gcaccattca gcgcaaagat gatgcgaacc tggcgagctg ggatattaaa 600

tttgtggaaa ccaaagatgg ctataacatt gatagctatc atgcgattta tggcaaccag 660

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ctgagcaccc tgattagcgg cggctttagc ccgaacatgg cgctggcgct gacctaa 777

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Met Val Ser Asn Thr Met Gly Tyr Lys Ile Gly Gly Ser Ile Glu Ile

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Glu Glu Asn Lys Pro Lys Ala Ser Ile Glu Ser Glu Tyr Ala Glu Ser

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Ser Thr Ile Glu Tyr Val Gln Pro Asp Phe Ser Thr Ile Gln Thr Asp

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His Ser Thr Ser Lys Ala Ser Trp Asp Thr Lys Phe Thr Glu Thr Thr

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Arg Gly Asn Tyr Asn Leu Lys Ser Asn Asn Pro Val Tyr Gly Asn Glu

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Met Phe Met Tyr Gly Arg Tyr Thr Asn Val Pro Ala Thr Glu Asn Ile

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Ile Pro Asp Tyr Gln Met Ser Lys Leu Ile Thr Gly Gly Leu Asn Pro

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Asn Met Ser Val Val Leu Thr Ala Pro Asn Gly Thr Glu Glu Ser Ile

115 120 125

Ile Lys Val Lys Met Glu Arg Glu Arg Asn Cys Tyr Lys Asp Val Ser

130 135 140

Asn Ser Ile Gly Tyr Ser Ile Gly Gly Asn Ile Ser Val Glu Gly Lys

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Thr Ala Gly Ala Gly Ile Asn Ala Ser Tyr Asn Val Gln Asn Thr Ile

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Ser Tyr Glu Gln Pro Asp Phe Arg Thr Ile Gln Arg Lys Asp Asp Ala

180 185 190

Asn Leu Ala Ser Trp Asp Ile Lys Phe Val Glu Thr Lys Asp Gly Tyr

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Asn Ile Asp Ser Tyr His Ala Ile Tyr Gly Asn Gln Leu Phe Met Lys

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Ser Arg Leu Tyr Asn Asn Gly Asp Lys Asn Phe Thr Asp Asp Arg Asp

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Leu Ser Thr Leu Ile Ser Gly Gly Phe Ser Pro Asn Met Ala Leu Ala

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Leu Thr

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agctggagcg attatattag ccagattgat agcattagcg cgagcctgat tctggatacc 480

ggcaacgaaa cctttgaacg ccgcgtggcg gcgaaagata gcagcaaccc ggaagataaa 540

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ggcctgctgt attttaacga aattccgatt gatgaaagct gcgtggaact gatttttgat 660

gataacaccg cgaacattat taaaaacagc ctgaaaaccc tggatgataa aaaaatttat 720

aacgtgaaac tggaacgcgg catgaacatt ctgattaaaa ccccgagcta ttttaccaac 780

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ggcctgcagg gcaccgcgaa cgaagtgaac ggcgaaacca aaattaccct gccgatgagc 900

aacctgaaac cgtataaacg ctatattttt agcggctata gcaaaagcag cagcgcgagc 960

aacagcctga acattaacat taaagcgtaa 990

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<211> 329

<212> PRT

<213> Artificial sequence (Artificial sequence)

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Lys Glu Ile Ser Asn Thr Val Ser Asn Glu Met Ser Lys Lys Ala Ser

1 5 10 15

Tyr Asp Asn Val Asp Thr Leu Ile Glu Lys Gly Arg Tyr Asn Thr Lys

20 25 30

Tyr Asn Tyr Leu Lys Arg Met Glu Lys Tyr Tyr Pro Asn Ala Met Ala

35 40 45

Tyr Phe Asp Lys Val Thr Ile Asn Pro Gln Gly Asn Asp Phe Tyr Ile

50 55 60

Asn Asn Pro Lys Val Glu Leu Asp Gly Glu Pro Ser Met Asn Tyr Leu

65 70 75 80

Glu Asp Val Tyr Val Gly Lys Ala Leu Leu Thr Asn Asp Thr Gln Gln

85 90 95

Glu Gln Lys Leu Lys Ser Gln Ser Phe Thr Cys Lys Asn Thr Asp Thr

100 105 110

Val Gln Ile Lys Leu Glu Thr Thr Gln Val Ser Gly Asn Phe Gly Thr

115 120 125

Lys Asn Asn Gln Gly Gln Ile Val Thr Glu Gly Asn Ser Trp Ser Asp

130 135 140

Tyr Ile Ser Gln Ile Asp Ser Ile Ser Ala Ser Leu Ile Leu Asp Thr

145 150 155 160

Gly Asn Glu Thr Phe Glu Arg Arg Val Ala Ala Lys Asp Ser Ser Asn

165 170 175

Pro Glu Asp Lys Thr Pro Glu Leu Thr Ile Gly Glu Ala Ile Glu Lys

180 185 190

Ala Phe Gly Ala Thr Lys Asn Gly Gly Leu Leu Tyr Phe Asn Glu Ile

195 200 205

Pro Ile Asp Glu Ser Cys Val Glu Leu Ile Phe Asp Asp Asn Thr Ala

210 215 220

Asn Ile Ile Lys Asn Ser Leu Lys Thr Leu Asp Asp Lys Lys Ile Tyr

225 230 235 240

Asn Val Lys Leu Glu Arg Gly Met Asn Ile Leu Ile Lys Thr Pro Ser

245 250 255

Tyr Phe Thr Asn Phe Asp Gly His Asn Thr Phe Pro Lys Ser Trp Ser

260 265 270

Asn Ile Asn Thr Gln Asn Lys Asp Gly Leu Gln Gly Thr Ala Asn Glu

275 280 285

Val Asn Gly Glu Thr Lys Ile Thr Leu Pro Met Ser Asn Leu Lys Pro

290 295 300

Tyr Lys Arg Tyr Ile Phe Ser Gly Tyr Ser Lys Ser Ser Ser Ala Ser

305 310 315 320

Asn Ser Leu Asn Ile Asn Ile Lys Ala

325

<210> 11

<211> 33

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 11

ggatccatgg gcgcgggcgg caaagtgagc tat 33

<210> 12

<211> 33

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 12

aagcttttac gctttaatgt taatgttcag gct 33

<210> 13

<211> 1449

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 13

atgaactata aacaggcgac cttttatctg ggcgaagcga tgcattattt tggcgatatt 60

gataccccgt atcatccggc gaacgtgacc gcggtggata gcgcgggcca tgtgaaattt 120

gaaacctttg cggaagaacg caaagaacag tataaaatta acaccgcggg ctgcaaaacc 180

aacgaagatt tttatgcgga tattctgaaa aacaaagatt ttaacgcgtg gagcaaagaa 240

tatgcgcgcg gctttgcgaa aaccggcaaa agcatttatt atagccatgc gagcatgagc 300

catagctggg atgattggga ttatgcggcg aaagtgaccc tggcgaacag ccagaaaggc 360

accgcgggct atatttatcg ctttctgcat gatgtgagcg aaggcaacga tccgagcgtg 420

ggcaaaaacg tgaaagaact ggtggcgtat attagcacca gcggcgaaaa agatgcgggc 480

accgatgatt atatgtattt tggcattaaa accaaagatg gcaaaaccca ggaatgggaa 540

atggataacc cgggcaacga ttttatgacc ggcagcaaag atacctatac ctttaaactg 600

aaagatgaaa acctgaaaat tgatgatatt cagaacatgt ggattcgcaa aattcatctg 660

aaactgccga aaaacaccgg catgctgccg tatattaaca gcaacgatgt gaaaaccctg 720

attgaacagg attatagcat taaaattgat aaaattgtgc gcattgtgat tgaaggcaaa 780

cagtatatta aagcggaagc gagcattgtg aacagcctgg attttaaaga tgatgtgagc 840

aaaggcgatc tgtggggcaa agaaaactat agcgattgga gcaacaaact gaccccgaac 900

gaactggcgg atgtgaacga acgcgtgaac aacgtggaac agtatcgcga aatgctggaa 960

gattttaaat atgatccgaa ccagcagctg aaaagctttg aaattctgaa cagccagaaa 1020

agcgataaca aagaaatttt taacgtgaaa accgaatttc tgaacggcgc gatttatgat 1080

atggaattta ccgtgagcag caaagatggc aaactgattg tgagcgatat ggaacgcacc 1140

aaagtggaaa acgaaggcaa atatattctg accccgagct ttcgcaccca ggtgtgcacc 1200

tgggatgatg aactggcgca ggcgattggc ggcgtgtatc cgcagaccta tagcgatcgc 1260

tttacctatt atgcggataa cattctgctg aactttcgcc agtatgcgac cagcggcagc 1320

cgcgatctga aagtggaata tagcgtggtg gatcattgga tgtggaaaga tgatgtgaaa 1380

gcgagccaga tggtgtatgg ccagaacccg gatagcgcgc gccagattcg cctgtatatt 1440

gaaaaataa 1449

<210> 14

<211> 777

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 14

atggtgagca acaccatggg ctataaaatt ggcggcagca ttgaaattga agaaaacaaa 60

ccgaaagcga gcattgaaag cgaatatgcg gaaagcagca ccattgaata tgtgcagccg 120

gattttagca ccattcagac cgatcatagc accagcaaag cgagctggga taccaaattt 180

accgaaacca cccgcggcaa ctataacctg aaaagcaaca acccggtgta tggcaacgaa 240

atgtttatgt atggccgcta taccaacgtg ccggcgaccg aaaacattat tccggattat 300

cagatgagca aactgattac cggcggcctg aacccgaaca tgagcgtggt gctgaccgcg 360

ccgaacggca ccgaagaaag cattattaaa gtgaaaatgg aacgcgaacg caactgctat 420

aaagatgtga gcaacagcat tggctatagc attggcggca acattagcgt ggaaggcaaa 480

accgcgggcg cgggcattaa cgcgagctat aacgtgcaga acaccattag ctatgaacag 540

ccgtatgatt ttcgcaccat tcagcgcaaa gatgatgcga acctggcgag ctgggatatt 600

aaatttgtgg aaaccaaaga tggctataac attgatagct atcatgcgat ttatggcaac 660

cagctgttta tgaaaagccg cctgtataac aacggcgata aaaactttac cgatgatcgc 720

gatctgagca ccctgattag cggcggcttt agcccgaaca tggcgctggc gctgacc 777

Claims

1. A protein composition comprising: a first fusion protein with the sequence shown as SEQ ID NO. 2, a second fusion protein with the sequence shown as SEQ ID NO. 6 and a third fusion protein with the sequence shown as SEQ ID NO. 10.

2. A gene composition comprising genes encoding the first fusion protein, the second fusion protein, and the third fusion protein of the protein composition of claim 1, respectively.

3. The genomic composition of claim 2, comprising: a first nucleic acid molecule with a sequence shown as SEQ ID NO. 1, a second nucleic acid molecule with a sequence shown as SEQ ID NO. 5 and a third nucleic acid molecule with a sequence shown as SEQ ID NO. 9.

4. A recombinant vector composition comprising:

a first recombinant vector comprising a gene encoding a first fusion protein of the protein composition of claim 1;

5. The recombinant vector composition of claim 4, wherein: the first, second and third recombinant vectors include pfastBac1 or pVL 1393.

6. A host cell composition comprising:

a first host cell comprising a gene encoding a first fusion protein of the protein composition of claim 1;

7. The host cell composition of claim 6, wherein: the first host cell, the second host cell and the third host cell are selected from Sf9 cell lines, and the Sf9 cell line comprises Sf9, High Five or Sf21 cells.

8. An immunological composition characterized by comprising: the protein composition of claim 1; and a pharmaceutically acceptable carrier.

9. The immunogenic composition of claim 8, wherein: the pharmaceutically acceptable carrier comprises one or more of MONTANIDE ISA 206 VG, MONTANIDE ISA 201 VG, MONTANIDE ISA 15 VG, liquid paraffin, camphor oil and plant cell agglutinin.

10. A method for preparing a protein composition, comprising:

providing genes encoding the first fusion protein, the second fusion protein, and the third fusion protein in the protein composition of claim 1;

11. The method of claim 10, wherein: the shuttle vector comprises pFastBac1 or pVL 1393; and/or, the insect cell is selected from the Sf9 cell line, the Sf9 cell line comprises Sf9, High Five or Sf21 cells.

12. A preparation method of a clostridium perfringens genetic engineering subunit vaccine is characterized by comprising the following steps: preparing a first fusion protein, a second fusion protein, a third fusion protein using the method of any one of claims 10-11, and admixing the first fusion protein, the second fusion protein, the third fusion protein with a pharmaceutically acceptable carrier.

13. Use of the protein composition of claim 1 or the immunogenic composition of claim 8 or 9 in the manufacture of a medicament for inducing an immune response against a clostridium perfringens antigen in a subject animal.

14. Use of the protein composition of claim 1 or the immunogenic composition of claim 8 or 9 in the manufacture of a medicament for preventing infection of an animal by clostridium perfringens.

15. Use of the protein composition according to claim 1 or the immunogenic composition according to claim 8 or 9 for the preparation of a clostridium perfringens genetically engineered subunit vaccine.