CN109111509B

CN109111509B - Mutant of alpha toxin of clostridium putrefactive bacteria, gene for expressing mutant, preparation method and vaccine of clostridium putrefactive bacteria

Info

Publication number: CN109111509B
Application number: CN201811096571.4A
Authority: CN
Inventors: 贺笋; 王钢; 唐慧芬; 刘强德; 李延涛
Original assignee: Tiankang Pharmaceutical Suzhou Co ltd
Current assignee: Tiankang Pharmaceutical Co ltd
Priority date: 2018-09-19
Filing date: 2018-09-19
Publication date: 2021-08-13
Anticipated expiration: 2038-09-19
Also published as: CN109111509A

Abstract

The invention provides a clostridium putrefaction alpha toxin mutant, a gene for expressing the same, a preparation method and a clostridium putrefaction vaccine, and relates to the technical field of biology. The mutant of the alpha toxin of the clostridium putrefaction comprises any one of (a1) - (a 3). Wherein (a1) is a protein obtained by mutating the 86 th amino acid residue of the alpha toxin of clostridium putrefaciens with the amino acid sequence shown as SEQ ID NO.1 from cysteine to leucine and the 189 th amino acid residue from serine to cysteine; (a2) a protein obtained by cleaving the signal peptide of (a 1); (a3) any one of the fusion proteins obtained by connecting labels to the N terminal and/or the C terminal of the protein shown in (a1) or (a2) has the advantages of low toxic and side effects and good immunogenicity. The clostridium putrefactive vaccine comprises a clostridium putrefactive alpha toxin mutant or a gene for coding the clostridium putrefactive alpha toxin mutant, and has good safety and good immunogenicity.

Description

Mutant of alpha toxin of clostridium putrefactive bacteria, gene for expressing mutant, preparation method and vaccine of clostridium putrefactive bacteria

Technical Field

The invention relates to the technical field of biology, in particular to a clostridium putrefactive alpha toxin mutant, a gene for expressing the mutant, a preparation method and a clostridium putrefactive vaccine.

Background

The Clostridium putrefaction (Clostridium septicum) is a gram-positive anaerobic bacillus, can cause diseases such as malignant edema disease, muscle necrosis, gas gangrene and necrotic enteritis of animals and people, and is also a pathogenic bacterium causing fast plague of sheep.

The bacterium can secrete a plurality of exotoxins such as alpha, beta, gamma and the like, wherein the alpha toxin is a main pathogenic virulence factor and an immunoprotective antigen. The alpha toxin of the clostridium putrefying is one of the perforative toxins of the aerolysin family, is a main lethal virulence factor of the clostridium putrefying, has the functions of hemolysis, lethality and necrosis, and has good immunogenicity, and the toxoid prepared by the alpha toxin can effectively resist the infection of the clostridium putrefying.

The spoilage clostridium is widely distributed in soil, excrement, dust, marsh and the digestive tract of animals, easily causes diseases such as malignant edema, muscle necrosis, gas gangrene and necrotic enteritis of animals such as sheep, horses, cows, pigs, dogs, cats, chickens and deer and human beings through wounds or the digestive tract by means of approaches such as feed, drinking water and surrounding environment polluted by spores, and the diseases are independent of the age, sex and variety of the animals. There are two common routes of infection, one is infection caused by spore contamination of clostridium putrefaciens due to trauma such as castration, tail breakage, childbirth, surgery or injection without strict disinfection; another type of infection through the digestive tract is usually endemic, and often occurs in seasons with sudden changes in climate in autumn, winter and early spring, and continuous rain and shade. The diseases caused by the clostridium putrefactive have the characteristics of rapid propagation, acute disease onset, short course of disease and high mortality in the sowing process. Among them, sheep are most susceptible to clostridium putrefaction, and the nutrition of diseased sheep is more than moderate, which is a great hazard to livestock and poultry breeding. Diseases caused by clostridium putrefactive bacteria, such as epidemic disease, generally have short disease course, and death often happens before the animals are treated. Therefore, vaccination is one of the most effective ways to prevent the disease.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a mutant of clostridium putrefaction alpha toxin, which has the advantages of low toxic and side effects and good immunogenicity.

The second purpose of the invention is to provide a gene for coding the mutant of the alpha toxin of the clostridium putrefaciens.

The third object of the present invention is to provide a method for producing the gene of the mutant of clostridium putrefaciens alpha toxin.

A fourth object of the present invention is to provide the above-mentioned mutant of α -toxin of clostridium putrefactive, a gene encoding the mutant of α -toxin of clostridium putrefactive, a method for producing the mutant of α -toxin of clostridium putrefactive, or an application of the protein produced by the method for producing the mutant of α -toxin of clostridium putrefactive.

A fifth object of the present invention is to provide a clostridium putrefactive vaccine comprising the above-mentioned mutant of clostridium putrefactive α -toxin or a gene encoding the above-mentioned mutant of clostridium putrefactive α -toxin.

In order to solve the technical problems, the invention adopts the following technical scheme:

the invention provides a mutant of clostridium putrefaciens alpha toxin, which comprises any one of the following (a1) - (a 3):

(a1) protein obtained by mutating the 86 th amino acid residue of clostridium putrefaciens alpha toxin with the amino acid sequence shown as SEQ ID NO.1 from cysteine to leucine and the 189 th amino acid residue from serine to cysteine;

(a2) a protein obtained by cleaving the signal peptide of (a 1);

(a3) and (b) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein represented by (a1) or (a 2).

Preferably, the mutant of clostridium putrefaciens alpha toxin has an amino acid sequence shown as SEQ ID No. 3.

The invention also provides a gene for coding the clostridium putrefaction alpha toxin mutant.

Preferably, the nucleotide sequence is shown as SEQ ID NO. 4.

The invention also provides a preparation method of the clostridium putrefactive alpha toxin mutant, which comprises the following steps: expressing the gene encoding the mutant of the alpha toxin of the clostridium putrefactive in a host cell.

Preferably, the preparation method comprises: expressing a gene encoding the mutant clostridium putrefactive alpha toxin using a mammalian expression system;

preferably, the gene of the mutant of clostridium putrefactive alpha toxin is expressed using a CHO cell expression system; the CHO cell is preferably a CHO-S cell line.

Preferably, the gene encoding the alpha toxin mutant of the clostridium putrefactive is cloned to an expression vector, then is introduced into a host cell, and then the host cell expressing the alpha toxin mutant of the clostridium putrefactive is screened and cultured, and the protein expressed by the host cell is purified to obtain the alpha toxin mutant of the clostridium putrefactive;

preferably, the expression vector comprises pcDNA3, preferably pcDNA^TM

The invention also provides the mutant of the alpha toxin of the clostridium putrefactive bacterium, a gene for coding the mutant of the alpha toxin of the clostridium putrefactive bacterium, a preparation method of the mutant of the alpha toxin of the clostridium putrefactive bacterium or application of the mutant of the alpha toxin of the clostridium putrefactive bacterium prepared by the preparation method in the following (x1) - (x 5);

(x1) preparing a clostridium putrefaction vaccine;

(x2) preparing antibodies to clostridium putrefaciens alpha toxin;

(x3) preparing reagents and/or kits for detecting clostridium putrefactive alpha toxin;

(x4) preparing a reagent and/or a kit for detecting antibodies to clostridium putrefaciens alpha toxin;

(x5) preparing a clostridium putrefaciens alpha toxin diagnostic antigen.

The invention also provides a clostridium putrefactive vaccine, which comprises the clostridium putrefactive alpha toxin mutant or a gene encoding the clostridium putrefactive alpha toxin mutant.

Preferably, the clostridium putrefactive vaccine comprises a mutant of clostridium putrefactive alpha toxin; the clostridium putrefaction alpha toxin mutant has an amino acid sequence shown as SEQ ID NO. 3; the mutant of the alpha toxin of the clostridium putrefactive is obtained by expression of a mammalian expression system;

preferably, the concentration of the mutant of clostridium putrefactive alpha toxin in the clostridium putrefactive vaccine is 30-50 μ g/mL; preferably 35-45 mug/mL; more preferably 40. mu.g/mL;

preferably, the vaccine further comprises adjuvants including one or more of vaccine adjuvants, stabilizers and antibiotics;

preferably, the vaccine adjuvant comprises aluminum hydroxide gel, Freund's complete adjuvant, Freund's incomplete adjuvant, white oil adjuvant, MF59 adjuvant or Montanide ISA series adjuvant; preferably Montanide ISA series adjuvants are used; more preferably, ISA15A adjuvant is used.

Compared with the prior art, the invention has the following beneficial effects:

the mutant of the alpha toxin of the clostridium putrefactive is mainly protein obtained by mutating the 86 th amino acid residue of the alpha toxin of the clostridium putrefactive with the amino acid sequence shown as SEQ ID NO.1 from cysteine to leucine and the 189 th amino acid residue from serine to cysteine; or further removing the signal peptide; or a fusion protein obtained after connecting a label at the N end and/or the C end of the amino acid sequence of the fusion protein, so as to facilitate the subsequent separation, purification and identification of the protein. The mutant of the alpha toxin of the clostridium putrefactive has no toxic or side effect, greatly reduces the biological safety risk, and effectively keeps the immunogenicity of the alpha toxin.

The present invention also provides a gene encoding the mutant of alpha toxin of clostridium putrefactive bacteria and a method for producing the mutant of alpha toxin of clostridium putrefactive bacteria, wherein the gene of the fusion protein is expressed in a host.

The alpha toxin mutant of the clostridium putrefactive bacterium, the gene for coding the alpha toxin mutant of the clostridium putrefactive bacterium, the preparation method of the alpha toxin mutant of the clostridium putrefactive bacterium, or the protein prepared by the preparation method of the alpha toxin mutant of the clostridium putrefactive bacterium, which are provided by the invention, have wide application, and can be applied to the preparation of vaccines of various diseases caused by the clostridium putrefactive bacterium; preparing antibodies to clostridium putrefactive alpha toxin; preparing a reagent and/or a kit for detecting clostridium putrefactive alpha toxin; preparing a reagent and/or a kit for detecting the clostridium putrefactive alpha toxin antibody and preparing a clostridium putrefactive alpha toxin diagnostic antigen.

The clostridium putrefactive vaccine provided by the invention comprises the clostridium putrefactive alpha toxin mutant or the gene for coding the clostridium putrefactive alpha toxin mutant. The alpha toxin mutant attenuated to animals is obtained by codon optimization and contains 2 amino acid site mutations, and has good safety and good immunogenicity.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 shows pcDNA^TM

A plasmid map of (a);

FIG. 2 shows the result of gel electrophoresis of recombinant proteins of mutant α toxin of Clostridium putrefactive in example 1;

FIG. 3 shows the result of western blot detection of recombinant proteins of the mutant α toxin of Clostridium putrefactive in example 1 of the present invention.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present 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. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

(a1) mutating the 86 th amino acid residue of the alpha toxin of the clostridium putrefaciens with the amino acid sequence shown as SEQ ID NO.1 from cysteine to leucine; and, the 189 th amino acid residue is mutated from serine to cysteine to obtain protein;

(a2) a protein obtained by cleaving the signal peptide of (a 1);

The total length of the gene of the detected alpha toxin mutant of the clostridium putrefaction is 1323bp, and the gene has a sequence shown as SEQ ID NO.2, codes 440 amino acids and has an amino acid sequence shown as SEQ ID NO. 1. The protein is about 48kDa in size and comprises a 31 amino acid signal peptide sequence. Studies of their functional sites show that:

the 398 th arginine on the KKRRGKR398SVD fragment is essential for the in vitro activation of the alpha toxin, this site is the eukaryotic furin (furin) recognition site containing the residue RGKR, and the pro-toxin is proteolytically cleaved to form an activated form of 41-44kDa size and a small propeptide. The WDWnW fragment at 333NGYSEWDWKWV343 is the binding site for the alpha toxin to the receptor Glycosylphosphatidylol (GPI) -anchored protein, where n denotes that the site is any amino acid. The amphiphilic transmembrane structure 203KIGVKTSFKVGLEAIADSKVETSFEFNAE231 is the main functional region for alpha toxin to perforate the cell membrane.

The invention discovers that the following two sites are mutated simultaneously: the 86 th cysteine is mutated into leucine, and the 189 th serine is mutated into cysteine, so that the toxicity of alpha toxin can be effectively reduced, the non-toxic mutant of the alpha toxin of the clostridium putrefaction is obtained, the biological safety risk is greatly reduced, and meanwhile, the immunogenicity of the alpha toxin is effectively maintained.

Meanwhile, the mutant of the alpha toxin of the clostridium putrefactive can also be protein with a signal peptide removed. The signal peptide is a fragment of the protein, which directs the ribosome and locates in a channel on the endoplasmic reticulum, allows the ribosome to attach to the endoplasmic reticulum and to pass the growing protein chain through the channel, whereupon the signal peptide is cleaved off, the synthesized protein is released into the lumen of the endoplasmic reticulum, and the protein is finally transported to the outside of the cell. It can be seen that immunogenic epitopes are less likely present on the signal peptide, and that cleavage of the signal peptide can also further promote soluble expression of the protein.

Further, the mutant of clostridium putrefaciens alpha toxin provided by the invention can also be a fusion protein obtained after connecting a tag to the N end and/or the C end of the amino acid sequence of the mutant, so as to facilitate the subsequent separation, purification and identification of the protein, wherein the tag can be, for example, but not limited to, a His tag, a Flag tag, a GST tag, an MBP tag, a NusA tag and a SUMO tag.

In some preferred embodiments, the clostridium putrefactive alpha toxin mutant has an amino acid sequence as shown in SEQ ID No. 3. The amino acid sequence is obtained by mutating the 86 th amino acid residue of the amino acid sequence shown in SEQ ID NO.1 from cysteine to leucine; and the 189 th amino acid residue is mutated from serine to cysteine. Then, 31 amino acid residues at the N end of the amino acid sequence shown in SEQ ID NO.1 are removed, namely, the signal peptide part of the clostridium putrefactive alpha toxin mutant is removed. Finally, a His tag is added to the C-terminal of SEQ ID NO.1, 6 histidines are selected in the embodiment, and in some other embodiments, 6-10 histidines commonly used in molecular biology technology can be selected, which is not limited by the invention. The non-toxic mutant of the clostridium putrefaction alpha toxin is obtained by optimizing the amino acid sequence shown in SEQ ID NO.1, and has the sequence shown in SEQ ID NO. as follows.

The invention also provides a gene for coding the clostridium putrefaction alpha toxin mutant. The gene encoding the mutant of the alpha toxin of the clostridium putrefying comprises a nucleotide sequence for expressing the mutant of the alpha toxin of the clostridium putrefying, and can also comprise a nucleotide sequence for regulating protein expression and/or assisting protein separation and purification, such as but not limited to a promoter sequence, an enhancer sequence and/or a sequence for expressing a protein tag. In some preferred embodiments, the gene of the mutant α toxin of clostridium putrefaciens has a nucleotide sequence as shown in SEQ ID No. 4.

The method for producing the mutant may be carried out by expressing the gene encoding the mutant of clostridium putrefaciens alpha toxin in a host, and may be, for example, but not limited to, an escherichia coli expression system, a yeast expression system, an insect expression system, a plant expression system, or a mammalian expression system.

However, because of the prokaryotic system, the expression product is usually in the form of insoluble inclusion body, and the expression of soluble protein is rarely reported. Since the expression product in inclusion bodies is biologically inactive, denaturation and renaturation treatments are required. The denaturation and renaturation of protein are extremely complex processes, the renaturation conditions of different proteins are different, and the renaturation rate is difficult to improve, which is a main restriction factor limiting the application of the protein. Meanwhile, the prokaryotic expression system has the problems of low expression quantity, high purification cost and endotoxin. Thus in a preferred embodiment, a mammalian expression system is used for the soluble expression of mutant clostridium putrefaciens alpha toxin. Mammalian expression systems also have the advantage that the expressed proteins are more similar in structure and biological properties to the native proteins after translational processing. Examples of host cells for use in the mammalian expression system include, but are not limited to, Chinese Hamster Ovary (CHO) cells, mouse kidney (BHK) cells, monkey kidney (COS) cells, mouse NSO thymoma cells, and mouse myeloma SP2/0 cells.

In some preferred embodiments, the mutant clostridium putrefactive alpha toxin is preferably expressed using CHO cells.

CHO (Chinese Hamster Ovary, CHO) cells were isolated from the ovaries of an adult female Chinese Hamster in 1957 by the university of colorado, usa, Theodore t. The safe host engineering cell approved by FDA is a better expression system for expressing target protein, especially complex modified macromolecular protein.

The CHO-S and CHO-K1 cell lines currently in common use are derived from CHO blasts. Both cell lines belong to fibroblasts, are non-secretory cells and rarely secrete CHO endogenous proteins per se. The CHO cell expression system has the following advantages:

(1) CHO belongs to fibroblast, secretes little endogenous protein of the CHO, and is beneficial to the separation of exogenous protein.

(2) Has accurate post-translational folding and modifying functions, and the expressed protein is most similar to natural protein molecules in the aspects of molecular structure, physical and chemical properties and biological functions.

(3) The material has the adherent growth characteristic and has higher shear force and osmotic pressure resistance; suspension culture, large-scale suspension culture in serum-free medium, and higher cell density in bioreactor can also be performed.

(4) Has the extracellular secretion function of the product, and is convenient for separating and purifying downstream products; has the high-efficiency amplification and expression capacity of the recombinant gene.

In some preferred embodiments, the preparation method comprises: cloning the gene encoding the alpha toxin mutant of the clostridium putrefactive to an expression vector, then introducing the gene into a host cell, screening and culturing the host cell expressing the alpha toxin mutant of the clostridium putrefactive, and purifying the protein expressed by the host cell to obtain the alpha toxin mutant of the clostridium putrefactive. Wherein the expression vector preferably uses pcDNA3, more preferably pcDNA^TM

pcDNA^TM

The vector is a bicistronic cloning vector, and the vector is designed to be used in most mammalian cells, so that transgenic recombinant proteins with extremely high expression levels can be obtained. PcdDNA^TM

The vector is compatible with TOPO isomerase and can be prepared by>The cloning efficiency of 85% contained the PCR product of the gene of interest. The gene for coding the clostridium putrefaction alpha toxin is inserted into a TOPO cloning site, and a CMV promoter is arranged at the upstream of the gene for promoting the transcription of a target gene; a TKpA terminator is arranged at the downstream; CMV forward binding primer binding sites and TKpA reverse binding primer binding sites are arranged on two sides of the target gene and are used for plasmid sequencing to ensure that the target gene sequence is correct; the vector contains two resistance marker genes, one is amp, namely ampicillin resistance gene, and is used for screening escherichia coli positive transformants; the other is neo, i.e. neomycin resistance gene, used to select cell lines stably transfected with CHO cells.

The present invention also provides applications of the above-described clostridium putrefactive alpha toxin mutant, the above-described gene encoding the above-described clostridium putrefactive alpha toxin mutant, the above-described method for producing the above-described clostridium putrefactive alpha toxin mutant, or a protein produced by the above-described method for producing the clostridium putrefactive alpha toxin mutant, to the following (x1) to (x 5): (x1) preparing a clostridium putrefaction vaccine; (x2) preparing antibodies to clostridium putrefaciens alpha toxin; (x3) preparing reagents and/or kits for detecting clostridium putrefactive alpha toxin; (x4) preparing a reagent and/or a kit for detecting antibodies to clostridium putrefaciens alpha toxin; (x5) preparing a clostridium putrefaciens alpha toxin diagnostic antigen.

The alpha toxin mutant of the clostridium putrefactive has no toxic or side effect and better immunogenicity, so that the alpha toxin mutant of the clostridium putrefactive can be used for preparing a vaccine of the clostridium putrefactive, and can be used for preventing diseases of animals such as sheep, horses, cattle, pigs, dogs, cats, chickens, deer and the like caused by the clostridium putrefactive. The antibodies prepared by using the mutant of the alpha toxin of the clostridium putrefactive bacteria and the alpha toxin of the clostridium putrefactive bacteria can be applied to the preparation of various detection reagents and kits, for example, an ELISA kit containing the antibodies of the mutant of the alpha toxin of the clostridium putrefactive bacteria is used for detecting the clostridium putrefactive bacteria.

The invention also provides a clostridium putrefactive vaccine, which comprises the clostridium putrefactive alpha toxin mutant or the gene for coding the clostridium putrefactive alpha toxin mutant. The clostridium putrefactive vaccine provided by the invention can be prepared into a clostridium putrefactive alpha toxin subunit vaccine by taking the clostridium putrefactive alpha toxin mutant as a main immunogen; the clostridium putrefaction alpha toxin mutant can also be used as a component in an immune composition to act together with other immunogen substances to play a role in synergy; also disclosed is a DNA vaccine comprising the gene encoding a mutant of Clostridium putrefaciens alpha toxin.

The clostridium putrefaction vaccine prepared by the invention is subjected to codon optimization and contains 2 amino acid site mutations, so that the alpha toxin mutant attenuated for animals is obtained, and the vaccine presents very good safety and good immunogenicity in rabbit animals.

In some alternative embodiments, the clostridium putrefactive vaccine comprises a clostridium putrefactive alpha toxin mutant having an amino acid sequence as set forth in SEQ ID No.3, and the clostridium putrefactive alpha toxin mutant is expressed from a CHO cell expression system.

The vaccine is a recombinant subunit vaccine of the alpha toxin of the clostridium putrefaction, and the alpha toxin mutant attenuated to an animal body is obtained by codon optimization and mutation containing 2 amino acid sites, and a mammalian expression system of the vaccine can efficiently secrete and express recombinant alpha toxin protein and is very easy to purify; exhibits very good safety and good immunogenicity in rabbit animals. Therefore, the recombinant protein is an ideal candidate vaccine for updating the clostridium putrefaction toxin vaccine, and optimizes the existing problems of an escherichia coli expression system.

In some preferred embodiments, the concentration of the mutant alpha toxin of clostridium putrefactive in the clostridium putrefactive vaccine is 30-50 μ g/mL; preferably 35-45 mug/mL; more preferably 40. mu.g/mL. Preferably, the vaccine further comprises adjuvants including one or more of vaccine adjuvants, stabilizers and antibiotics. The vaccine adjuvant can be, for example, but not limited to, aluminum hydroxide gel, Freund's complete adjuvant, Freund's incomplete adjuvant, white oil adjuvant, MF59 adjuvant or Montanide ISA series adjuvant; preferably Montanide ISA series adjuvants are used; more preferably, ISA15A adjuvant is used.

The advantageous effects of the present invention will be further described with reference to preferred embodiments.

Example 1

The nucleotide sequence shown in SEQ ID NO.4 (artificially synthesized) is inserted into pcDNA^TM

The TOPO cloning site on the carrier has CMV promoter in the upstream and TKpA terminator in the downstream to obtain pcDNA3.3-alpha toxin plasmid for transfection, and the pcDNA3.3-alpha toxin plasmid is linearized with ApaLI enzyme and the linearized recombinant carrier is electrically shocked and transformed into mammal cell CHO-S to obtain mammal cell recombinant cell strain CHO-S a-toxin. The recombinant cell line was inoculated into CHO-S-SFM II medium. Then, a cell strain expressing the mutant of the alpha toxin of the clostridium putrefaciens is selected, and the temperature is 37 ℃ and the CO content is 5 percent₂The culture medium is cultured in suspension for 10 days, the supernatant is collected by centrifugation, and SDS-PAGE and WEST-BLOT detection are carried out simultaneously, the detection result is shown in figure 2 and figure 3, wherein, lane 2 and lane 3 in figure 2 and figure 3 are recombinant proteins of the alpha toxin mutant of the clostridium putrefactive bacteria, and the CHO-S cell strain can successfully express the alpha toxin mutant of the clostridium putrefactive bacteria.

CHO cell recovery and passage: 1. the water bath kettle is opened for 37 ℃ in advance; the medium was preheated at 37 ℃ in advance. 2. The frozen CHO cells were removed from the liquid nitrogen tank. 3. The frozen cells were immediately placed in a 37 ℃ water bath, thawed rapidly by gentle shaking (about 1min), and removed. 4. The outer wall of the tube was sterilized with 75% ethanol, placed in a biosafety cabinet, transferred to a15 mL centrifuge tube containing 10mL of medium, centrifuged at 800rpm for 5 min. 5. Removing supernatant from centrifuged cells, taking a little fresh culture medium to resuspend the cells, transferring the cells to a culture flask, and adding freshGently shaking the culture medium to disperse cells uniformly, counting cells and detecting activity, and controlling density at 3-4 × 10⁵cells/mL, viability>95 percent. 6. Placing in an incubator at 110rpm, 37 deg.C, 5% CO₂And (5) culturing. 7. The cells are cultured for 2-3 days, and the density reaches 2.0 multiplied by 10⁶cells need to be passaged around cells/mL. 8. Removing part of the culture from the flask and discarding it, adding fresh medium to the flask, diluting the remaining cell culture (the remaining cell culture is determined by cell density, culture volume, density after dilution, etc. 9, placing in an incubator at 110rpm, 37 deg.C, 5% CO₂And continuing culturing. 10. The cell density and the cell activity are detected every day when the cell density reaches 2 multiplied by 10⁶cells were passaged around cell/mL.

Transfection of Alpha-toxin plasmid into CHO cells: 1. CHO cells were cultured in suspension to 300mL at a seeding density of 1X 10 1 day before transfection⁶cells/mL, placed in an incubator at 110rpm, 37 ℃ and 5% CO₂And (5) culturing. 2. The cell density of the transfected angel is controlled to be 1-1.5X 10⁶About cells/mL. 3. DNA-transfection reagent mixture: DNA and transfection reagents were added to the transfection buffer and mixed well and incubated at 37 ℃. 4. The DNA-transfection reagent mixture was added to the cells to be transfected and placed in an incubator at 110rpm, 37 ℃ and 5% CO₂And (5) culturing. 5. Collecting: about 4-6 days after transfection, the cell culture is removed, centrifuged, and the supernatant or cells are collected. 6. The supernatant was affinity purified by a nickel column, and then the obtained protein was quantified.

Example 2

The embodiment provides a clostridium putrefaction vaccine, which comprises the protein provided in the embodiment 1, wherein the protein content is 30 mug/mL, and the protein and the commercial adjuvant ISA15A are uniformly mixed according to the volume ratio of 1:1 to prepare the vaccine.

Example 3

The embodiment provides a clostridium putrefaction vaccine, which comprises the protein provided in the embodiment 1, wherein the protein content is 40 mug/mL, and the protein and the commercial adjuvant ISA15A are uniformly mixed according to the volume ratio of 1:1 to prepare the vaccine.

Example 4

The embodiment provides a clostridium putrefaction vaccine, which comprises the protein provided in the embodiment 1, wherein the protein content is 50 mug/mL, and the protein and the commercial adjuvant ISA15A are uniformly mixed according to the volume ratio of 1:1 to prepare the vaccine.

Example 5

The embodiment provides a clostridium putrefaction vaccine, which comprises the protein provided in the embodiment 1, wherein the protein content is 30 mug/mL, and the protein and the commercial adjuvant MF59 are uniformly mixed according to the volume ratio of 1:1 to prepare the vaccine.

Example 6

The embodiment provides a clostridium putrefaction vaccine, which comprises the protein provided in the embodiment 1, wherein the protein content is 40 mug/mL, and the protein and the commercial adjuvant MF59 are uniformly mixed according to the volume ratio of 1:1 to prepare the vaccine.

Example 7

The embodiment provides a clostridium putrefaction vaccine, which comprises the protein provided in the embodiment 1, wherein the protein content is 50 mug/mL, and the protein and the commercial adjuvant MF59 are uniformly mixed according to the volume ratio of 1:1 to prepare the vaccine.

Example 8

The embodiment provides a clostridium putrefaction vaccine, which comprises the protein provided in the embodiment 1, wherein the protein content is 30 mug/mL, and the protein and the commercial adjuvant ISA206 are uniformly mixed according to the volume ratio of 1:1 to prepare the vaccine.

Example 9

The embodiment provides a clostridium putrefaction vaccine, which comprises the protein provided in the embodiment 1, wherein the protein content is 40 mug/mL, and the protein and the commercial adjuvant ISA206 are uniformly mixed according to the volume ratio of 1:1 to prepare the vaccine.

Example 10

The embodiment provides a clostridium putrefaction vaccine, which comprises the protein provided in the embodiment 1, wherein the protein content is 50 mug/mL, and the protein and the commercial adjuvant ISA206 are uniformly mixed according to the volume ratio of 1:1 to prepare the vaccine.

Effect example 1

Safety test 45 healthy rabbits (1.5-2.0 kg) were used, and divided into 9 groups of 5 rabbits each, and 2.0ml of the vaccine provided in examples 2-10 was injected into the rabbits each by muscle or subcutaneous injection, and the rabbits were all alive after 10 days of observation, and no necrosis was observed in the muscle or skin at the injection site.

Effect example 2

The vaccines provided in examples 2-9 were tested for efficacy, and each set of experiments was as follows:

4 healthy rabbits (1.5-2.0 kg) were injected with 1.0ml of vaccine subcutaneously or intramuscularly in each neck. Blood is collected 14-21 days after inoculation, and serum is separated. The serum of 4 immunized rabbits is mixed in equal amount, 0.4ml of mixed serum is mixed with the putrefactive clostridial toxin (containing 4 mouse MLD), the mixture is put at 37 ℃ for 40min, and then 2 mice of 16-20g are injected intravenously, wherein each mouse is 0.3 ml. At the same time, 2 mice from the same batch were each injected with 1MLD of the same toxin as the toxin serum mixture. The observation was carried out for 1 day, and the results were judged. The control mice all died, and the serum neutralization titer reached 1 for the clostridium putrefaction toxin (0.1ml of immune animal serum neutralized 1MLD toxin), namely the control mice were judged to be qualified. And (3) testing the efficacy: in the vaccines provided in the examples, the serum neutralization titer reached 1 for clostridial putrefactive toxin, and the vaccines were effective. The efficacy results indicated that the adjuvant was IS15A most effective. The results are shown in Table 1.

Table 1 efficacy results for the vaccines provided in the examples

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Sequence listing

<110> Tiankang biological products Ltd

<120> mutant of clostridium putrefactive alpha toxin, gene expressing same, preparation method and clostridium putrefactive vaccine

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<170> SIPOSequenceListing 1.0

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<211> 440

<212> PRT

<213> Clostridium putrefactive (Clostridium septicum)

<400> 1

Met Ser Lys Lys Ser Phe Ala Lys Lys Val Ile Cys Thr Ser Met Ile

1 5 10 15

Ala Ile Gln Cys Ala Ala Val Val Pro His Val Gln Ala Tyr Ala Leu

20 25 30

Thr Asn Leu Glu Glu Gly Gly Tyr Ala Asn His Asn Asn Ala Ser Ser

35 40 45

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

50 55 60

Ile Gln Asp Pro Glu Phe Ile Arg Asn Trp Ala Asn Val Ala His Ser

65 70 75 80

Leu Gly Phe Gly Trp Cys Gly Gly Thr Ala Asn Pro Asn Val Gly Gln

85 90 95

Gly Phe Glu Phe Lys Arg Glu Val Gly Ala Gly Gly Lys Val Ser Tyr

100 105 110

Leu Leu Ser Ala Arg Tyr Asn Pro Asn Asp Pro Tyr Ala Ser Gly Tyr

115 120 125

Arg Ala Lys Asp Arg Leu Ser Met Lys Ile Ser Asn Val Arg Phe Val

130 135 140

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

145 150 155 160

Ala Pro Leu Asn Ser Ala Ser Phe Asp Leu Ile Asn Glu Ser Lys Thr

165 170 175

Glu Ser Lys Leu Ser Lys Thr Phe Asn Tyr Thr Thr Ser Lys Thr Val

180 185 190

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

195 200 205

Ser Phe Lys Val Gly Leu Glu Ala Ile Ala Asp Ser Lys Val Glu Thr

210 215 220

Ser Phe Glu Phe Asn Ala Glu Gln Gly Trp Ser Asn Thr Asn Ser Thr

225 230 235 240

Thr Glu Thr Lys Gln Glu Ser Thr Thr Tyr Thr Ala Thr Val Ser Pro

245 250 255

Gln Thr Lys Lys Arg Leu Phe Leu Asp Val Leu Gly Ser Gln Ile Asp

260 265 270

Ile Pro Tyr Glu Gly Lys Ile Tyr Met Glu Tyr Asp Ile Glu Leu Met

275 280 285

Gly Phe Leu Arg Tyr Thr Gly Asn Ala Arg Glu Asp His Thr Glu Asp

290 295 300

Arg Pro Thr Val Lys Leu Lys Phe Gly Lys Asn Gly Met Ser Ala Glu

305 310 315 320

Glu His Leu Lys Asp Leu Tyr Ser His Lys Asn Ile Asn Gly Tyr Ser

325 330 335

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

340 345 350

Asn Ser Tyr Asp Ala Leu Thr Ser Arg Lys Leu Gly Gly Ile Ile Lys

355 360 365

Gly Ser Phe Thr Asn Ile Asn Gly Thr Lys Ile Val Ile Arg Glu Gly

370 375 380

Lys Glu Ile Pro Leu Pro Asp Lys Lys Arg Arg Gly Lys Arg Ser Val

385 390 395 400

Asp Ser Leu Asp Ala Arg Leu Gln Asn Glu Gly Ile Arg Ile Glu Asn

405 410 415

Ile Glu Thr Gln Asp Val Pro Gly Phe Arg Leu Asn Ser Ile Thr Tyr

420 425 430

Asn Asp Lys Lys Ile Asp Ile Asn

435 440

<210> 2

<211> 1323

<212> DNA

<213> Clostridium putrefactive (Clostridium septicum)

<400> 2

atgtcaaaaa aatcttttgc taaaaaagta atttgtacat ctatgattgc aattcagtgt 60

gcggcagtag taccacatgt acaagcttat gcacttacaa atcttgaaga ggggggatat 120

gcaaatcata ataatgcttc ttcaattaaa atatttggat atgaagacaa tgaagattta 180

aaagctaaaa ttattcaaga tccagagttt ataagaaatt gggcaaatgt agctcattca 240

ttaggatttg gatggtgcgg tggaacggct aatccaaacg ttggacaagg ttttgaattt 300

aaaagagaag ttggggcagg tggaaaagta tcttatttat tatctgctag atacaatcca 360

aatgatcctt atgcaagtgg gtatcgtgca aaagatagac tttctatgaa aatatcaaat 420

gttagatttg ttattgataa tgattctata aaattaggta cacctaaagt gaaaaaatta 480

gcacctttaa actctgctag ttttgattta ataaatgaaa gtaaaactga gtctaaatta 540

tcaaaaacat ttaattatac aacttctaaa acagtttcta aaacagataa ctttaaattt 600

ggagaaaaaa taggagtaaa aacatcattt aaagtaggtc ttgaagctat agctgacagt 660

aaagttgaga caagctttga atttaatgca gaacaaggtt ggtcaaatac aaatagtact 720

actgaaacta aacaagaaag tactacatat actgcaacag tttctccaca aactaaaaag 780

agattattcc tagatgtgtt aggatcacaa attgatattc cttatgaagg aaaaatatat 840

atggaatacg acatagaatt aatgggattt ttaagatata caggaaatgc tcgtgaagat 900

catactgaag atagaccaac agttaaactt aaatttggta aaaacggtat gagtgctgag 960

gaacatctta aagatttata tagtcataag aatattaatg gatattcaga atgggattgg 1020

aaatgggtag atgagaaatt tggttattta tttaaaaatt catacgatgc tcttactagt 1080

agaaaattag gaggaataat aaaaggctca tttactaaca ttaatggaac aaaaatagta 1140

attagagaag gtaaagaaat tccacttcct gataagaaga gaagaggaaa acgttcagta 1200

gattctttag atgctagatt acaaaatgaa ggtattagaa tagaaaatat tgaaacacaa 1260

gatgttccag gatttagact aaatagcata acatacaatg ataaaaaaat tgatattaat 1320

taa 1323

<210> 3

<211> 415

<212> PRT

<213> Artificial sequence ()

<400> 3

Leu Thr Asn Leu Glu Glu Gly Gly Tyr Ala Asn His Asn Asn Ala Ser

1 5 10 15

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

20 25 30

Ile Ile Gln Asp Pro Glu Phe Ile Arg Asn Trp Ala Asn Val Ala His

35 40 45

Ser Leu Gly Phe Gly Trp Leu Gly Gly Thr Ala Asn Pro Asn Val Gly

50 55 60

Gln Gly Phe Glu Phe Lys Arg Glu Val Gly Ala Gly Gly Lys Val Ser

65 70 75 80

Tyr Leu Leu Ser Ala Arg Tyr Asn Pro Asn Asp Pro Tyr Ala Ser Gly

85 90 95

Tyr Arg Ala Lys Asp Arg Leu Ser Met Lys Ile Ser Asn Val Arg Phe

100 105 110

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

115 120 125

Leu Ala Pro Leu Asn Ser Ala Ser Phe Asp Leu Ile Asn Glu Ser Lys

130 135 140

Thr Glu Ser Lys Leu Ser Lys Thr Phe Asn Tyr Thr Thr Cys Lys Thr

145 150 155 160

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

165 170 175

Thr Ser Phe Lys Val Gly Leu Glu Ala Ile Ala Asp Ser Lys Val Glu

180 185 190

Thr Ser Phe Glu Phe Asn Ala Glu Gln Gly Trp Ser Asn Thr Asn Ser

195 200 205

Thr Thr Glu Thr Lys Gln Glu Ser Thr Thr Tyr Thr Ala Thr Val Ser

210 215 220

Pro Gln Thr Lys Lys Arg Leu Phe Leu Asp Val Leu Gly Ser Gln Ile

225 230 235 240

Asp Ile Pro Tyr Glu Gly Lys Ile Tyr Met Glu Tyr Asp Ile Glu Leu

245 250 255

Met Gly Phe Leu Arg Tyr Thr Gly Asn Ala Arg Glu Asp His Thr Glu

260 265 270

Asp Arg Pro Thr Val Lys Leu Lys Phe Gly Lys Asn Gly Met Ser Ala

275 280 285

Glu Glu His Leu Lys Asp Leu Tyr Ser His Lys Asn Ile Asn Gly Tyr

290 295 300

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

305 310 315 320

Lys Asn Ser Tyr Asp Ala Leu Thr Ser Arg Lys Leu Gly Gly Ile Ile

325 330 335

Lys Gly Ser Phe Thr Asn Ile Asn Gly Thr Lys Ile Val Ile Arg Glu

340 345 350

Gly Lys Glu Ile Pro Leu Pro Asp Lys Lys Arg Arg Gly Lys Arg Ser

355 360 365

Val Asp Ser Leu Asp Ala Arg Leu Gln Asn Glu Gly Ile Arg Ile Glu

370 375 380

Asn Ile Glu Thr Gln Asp Val Pro Gly Phe Arg Leu Asn Ser Ile Thr

385 390 395 400

Tyr Asn Asp Lys Lys Ile Asp Ile Asn His His His His His His

405 410 415

<210> 4

<211> 1245

<212> DNA

<213> Artificial sequence ()

<400> 4

cttacaaatc ttgaagaggg gggatatgca aatcataata atgcttcttc aattaaaata 60

tttggatatg aagacaatga agatttaaaa gctaaaatta ttcaagatcc agagtttata 120

agaaattggg caaatgtagc tcattcatta ggatttggat ggttaggtgg aacggctaat 180

ccaaacgttg gacaaggttt tgaatttaaa agagaagttg gggcaggtgg aaaagtatct 240

tatttattat ctgctagata caatccaaat gatccttatg caagtgggta tcgtgcaaaa 300

gatagacttt ctatgaaaat atcaaatgtt agatttgtta ttgataatga ttctataaaa 360

ttaggtacac ctaaagtgaa aaaattagca cctttaaact ctgctagttt tgatttaata 420

aatgaaagta aaactgagtc taaattatca aaaacattta attatacaac ttgtaaaaca 480

gtttctaaaa cagataactt taaatttgga gaaaaaatag gagtaaaaac atcatttaaa 540

gtaggtcttg aagctatagc tgacagtaaa gttgagacaa gctttgaatt taatgcagaa 600

caaggttggt caaatacaaa tagtactact gaaactaaac aagaaagtac tacatatact 660

gcaacagttt ctccacaaac taaaaagaga ttattcctag atgtgttagg atcacaaatt 720

gatattcctt atgaaggaaa aatatatatg gaatacgaca tagaattaat gggattttta 780

agatatacag gaaatgctcg tgaagatcat actgaagata gaccaacagt taaacttaaa 840

tttggtaaaa acggtatgag tgctgaggaa catcttaaag atttatatag tcataagaat 900

attaatggat attcagaatg ggattggaaa tgggtagatg agaaatttgg ttatttattt 960

aaaaattcat acgatgctct tactagtaga aaattaggag gaataataaa aggctcattt 1020

actaacatta atggaacaaa aatagtaatt agagaaggta aagaaattcc acttcctgat 1080

aagaagagaa gaggaaaacg ttcagtagat tctttagatg ctagattaca aaatgaaggt 1140

attagaatag aaaatattga aacacaagat gttccaggat ttagactaaa tagcataaca 1200

tacaatgata aaaaaattga tattaatcat catcatcatc atcat 1245

Claims

1. A mutant of Clostridium putrefaction alpha toxin, which is any one of the following (a1) - (a 2):

(a1) protein obtained by mutating the 86 th amino acid residue of the alpha toxin of the clostridium putrefaciens of the amino acid sequence shown as SEQ ID NO.1 from cysteine to leucine and the 189 th amino acid residue from serine to cysteine;

(a2) and (d) a protein obtained by cleaving the signal peptide of (a 1).

2. The mutant of the alpha toxin of the clostridium putrefactive is characterized in that the amino acid sequence of the mutant of the alpha toxin of the clostridium putrefactive is shown as SEQ ID NO. 3.

3. A gene encoding a mutant of clostridium putrefaciens alpha toxin according to claim 1 or 2.

4. The gene encoding a mutant α toxin of clostridium putrefactive bacteria of claim 3, wherein the nucleotide sequence of said gene is shown in SEQ ID No. 4.

5. A method of producing a mutant of clostridium putrefactive α toxin according to claim 1 or 2, comprising: expressing a gene encoding the mutant clostridium putrefactive alpha toxin in a host cell.

6. The method of claim 5, comprising: expressing a gene encoding the mutant clostridium putrefactive alpha toxin using a mammalian expression system.

7. The method of claim 6, wherein the gene of the mutant alpha toxin of Clostridium putrefactive is expressed using a CHO cell expression system.

8. The method according to claim 7, wherein the CHO cell is a CHO-S cell line.

9. The method according to any one of claims 5 to 8, wherein the gene encoding the mutant α toxin of clostridium putrefactive is cloned into an expression vector, and then introduced into a host cell, and the host cell expressing the mutant α toxin of clostridium putrefactive is selected and cultured, and the protein expressed by the host cell is purified to obtain the mutant α toxin of clostridium putrefactive.

10. The method of claim 9, wherein the expression vector is pcDNA3.

11. According to the claimsThe method according to claim 9, wherein the expression vector is pcDNA^TM3.3-TOPO^R。

12. Use of a clostridium putrefactive alpha toxin mutant according to claim 1 or 2, a gene according to claim 3 or 4, a method for producing a clostridium putrefactive alpha toxin mutant according to any one of claims 5 to 11, or a clostridium putrefactive alpha toxin mutant produced by the production method, for any one of the following (x1) to (x 4):

(x1) preparing a clostridium putrefaction vaccine;

(x2) preparing antibodies to clostridium putrefaciens alpha toxin;

(x4) preparing a reagent and/or a kit for detecting antibodies to clostridium putrefaciens alpha toxin.

13. A Clostridium putrefactive vaccine comprising the mutant of alpha toxin of Clostridium putrefactive of claim 1 or 2 or the gene of claim 3 or 4.

14. The clostridium putrefactive vaccine of claim 13, wherein the clostridium putrefactive vaccine comprises a clostridium putrefactive alpha toxin mutant; the amino acid sequence of the clostridium putrefaction alpha toxin mutant is shown in SEQ ID NO. 3; the mutant of the alpha toxin of the clostridium putrefactive is obtained by expression of a mammalian expression system.

15. The clostridium putrefactive vaccine of claim 14, wherein the concentration of the clostridium putrefactive alpha toxin mutant in the clostridium putrefactive vaccine is 30-50 μ g/mL.

16. The clostridium putrefactive vaccine of claim 15, wherein the concentration of the clostridium putrefactive alpha toxin mutant in the clostridium putrefactive vaccine is 35-45 μ g/mL.

17. The clostridium putrefactive vaccine of claim 16, wherein the concentration of clostridium putrefactive alpha toxin mutant in the clostridium putrefactive vaccine is 40 μ g/mL.

18. The clostridium putrefactive vaccine according to claim 13, further comprising adjuvants including one or more of vaccine adjuvants, stabilizers, and antibiotics.

19. The clostridium putrefaction vaccine of claim 18, wherein the vaccine adjuvant is aluminum hydroxide gel, freund's complete adjuvant, freund's incomplete adjuvant, white oil adjuvant, MF59 adjuvant or Montanide ISA series adjuvant.

20. A clostridium putrefaction vaccine according to claim 19, wherein said vaccine adjuvant uses Montanide ISA series adjuvants.

21. The clostridium putrefaction vaccine of claim 20, wherein the vaccine adjuvant uses an ISA15A adjuvant.