CN107812183A - A kind of clostridium septicum alpha toxin recombinant subunit vaccine and its production method - Google Patents
A kind of clostridium septicum alpha toxin recombinant subunit vaccine and its production method Download PDFInfo
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Abstract
The present invention relates to a kind of clostridium septicum alpha toxin recombinant subunit vaccine and its production method.54th cysteine mutation of wild type clostridium septicum alpha toxin maturation toxin is leucine by the present invention, 264th asparagine mutation is alanine, 269th hyte Histidine mutations are alanine, 310th tryptophan sports alanine, obtain for the nontoxic alpha toxin mutant of animal body, and it is histidine-tagged plus 6 in the C-terminal of mutain, as antigen for vaccine.Also disclose recombinant expression carrier and recombinant host cell containing the clostridium septicum alpha toxin non-toxic mutant encoding gene.Clostridium septicum alpha toxin recombinant subunit vaccine its effect prepared using the present invention is far above existing vaccine, and greatly reduces the bio-safety risk in vaccine production process.In addition, by the high advantage of vaccine semi-finished product protein concentration, without increasing the dosage of connection seedling, you can prepare connection seedling.
Description
Technical field
The present invention relates to a kind of clostridium septicum alpha toxin recombinant subunit vaccine and its production method.Belong to biology system for animals
Product field.
Background technology
Clostridium septicum (Clostridium septium) is a kind of Gram-positive anaerobic bacillus(cillus anaerobicus), can cause animal and people
Disease, the sheep such as malignant edema disease, muscular death, gas gangrene and necrotic enteritis draw via the infection of digestive canal bacterium
The sheep braxy (Braxy) risen is a kind of common, multiple, non-contact, acute and lethal infectious diseases.The bacterium can secrete α, β, γ and
A variety of exotoxins such as δ, wherein alpha toxin are its main pathogenic virulence factor and immune protective antigen, and the toxin has molten
Blood, lethal and downright bad three kinds of bioactivity, can make gastrointestinal mucosal (especially abomasum mucous membrane) be inflamed and necrosis, pass through simultaneously
Blood circulation enters internal, can stimulate central nervous system, causes animal acute shock and rapid dead.
Clostridium septicum is distributed widely in soil, excrement, dust, marsh and the alimentary canal of animal, easily by dirty by gemma
The approach such as feed, drinking-water and the surrounding environment of dye, cause the animals such as sheep, horse, ox, pig, dog, cat, chicken, deer through wound or alimentary canal
With the disease such as malignant edema disease, muscular death, emphysematous gangrene and the necrotic enteritis of people, age of the morbidity with animal, sex
And kind is unrelated.Its routes of infection typically has two kinds, and one kind is due to wound such as castration, docking, childbirth, surgical operation, injection
Etc. no strict sterilization, clostridium septicum gemma is caused to pollute and cause infection, such case is mostly to distribute；Another is through digestion
Road infects, now more popular in place, is mainly in autumn, winter and early spring weather cataclysm, season cloudy and drizzly for days on end.Experimental animal is small
Mouse, cavy and rabbit are susceptible.In addition, dove can also infect, fast, morbidity is anxious, the course of disease is short, dead with propagating for morbidity communication process
Die the characteristics of rate is high.Sheep is the most susceptible to sheep braxy, and the nutrition of the sheep that falls ill is more more than medium, the harm to livestock and poultry breeding industry
It is larger.Therefore, the research to the bacterium has highly important aetology and public hygienics meaning.
For example fast epidemic disease of the disease as caused by clostridium septicum, the general course of disease is extremely very brief, often has little time treatment animal and occurs
It is dead.Therefore, immunity inoculation is one of prevention maximally efficient approach of the disease.At present, immunoprophylaxis of the China to sheep braxy, is adopted more
With inactivated vaccine, there are sheep braxy and black disease Combined vaccine or sheep braxy, sudden subcutaneous ulcer, enterotoxemia triple vaccine.Immunization method is, no matter greatly
Lamb, equal 5.0m L/ only subcutaneously or intramuscularly injecting immunes.Class is made through formaldehyde detoxification with clostridium septicum α elements after purification in foreign countries
Toxin, then it is mixed with that toxoid vaccine is made using aluminate or phosphate gel as adjuvant, after animal is immunized, body is to different shaped
Clostridium septicum and its gemma have resistance.
The commercialized vaccine used at present is mainly inactivated vaccine, although in terms of animal clostridium septicum associated diseases are prevented
Certain effect is achieved, but these vaccines still expose some defects in use, such as vaccine immunity easily causes
Animal local inflammation and toxic reaction etc.；It is related to ectotoxic inactivation in preparation process, toxin be present and leaks or inactivate not
The bio-safety hidden danger such as thorough；Exempt from addition, the metabolite of the various small toxin and bacterium in culture supernatant often turns into
The sensibiligen of epidemic disease animal, inoculation animal are also easy to produce adverse reaction, cause immune effect to decline even immuning failure.Therefore, develop
The clostridium septicum toxin gene engineered vaccine that security is good, Effective Antigens content is high, immunogenicity is strong, it is following development side
To.
The standby clostridium septicum alpha toxin recombinant subunit vaccine of patent system of the present invention, is to be optimized using codon, contained
The restructuring clostridium septicum alpha toxin protein production of 4 amino acid mutations, i.e., retain the integrality of natural toxin albumen to greatest extent
And space conformation, so as to keep its immunogenicity, it turn avoid the bio-safety hidden danger that single amino acids mutation is brought.Meanwhile
The vaccine also has the advantages that preparation technology is simple, immunizing dose is low, immune efficacy is good, is the existing clostridium septicum toxin epidemic disease in China
The ideal candidates vaccine of seedling upgrading.
The content of the invention
Present invention aims at clostridium septicum alpha toxin recombinant subunit vaccine is prepared, for preventing by clostridium septicum sense
Disease caused by dye.
Technical solution of the present invention
1. a kind of clostridium septicum alpha toxin recombinant subunit vaccine, it is characterised in that the vaccine contains uncommon using large intestine angstrom
The restructuring clostridium septicum alpha toxin albumen of Salmonella expression；Seedling is the big of recombination expression clostridium perfringens alpha toxin albumen with strain
Intestines Escherichia is named as ETEC (Escherichia coli) BL21/mCSA strains, and the bacterial strain is in 2017
Deliver Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica Chinese microorganism strain within 21 days 09 month
The common micro-organisms center preservation of preservation administration committee, deposit number are：CGMCC No.14650.
2. clostridium septicum alpha toxin recombinant subunit vaccine of the present invention, it is characterised in that the restructuring clostridium septicum α poison
Fibroin, compared with wild type clostridium septicum alpha toxin maturation toxin, contain 4 amino acid mutations, respectively the 54th half Guang ammonia
Acid mutation is leucine, and the 264th asparagine mutation is alanine, and the 269th hyte Histidine mutations are alanine, the 310th
Tryptophan sports alanine.It is meanwhile histidine-tagged plus 6 in C-terminal.
3. clostridium septicum alpha toxin recombinant subunit vaccine of the present invention, it is characterised in that the restructuring clostridium septicum α poison
Fibroin, the gene order codon optimization of its recombinant expression carrier, it is easier to high expression is realized in Escherichia coli.
4. clostridium septicum alpha toxin recombinant subunit vaccine of the present invention, it is characterised in that the restructuring clostridium septicum α poison
Fibroin is non-toxic mutant, greatly reduces the bio-safety risk in vaccine production process.
5. clostridium septicum alpha toxin recombinant subunit vaccine of the present invention, it is characterised in that the restructuring clostridium septicum α poison
Fibroin, its C-terminal contain 6 histidines (6His) label, are easy to the purifying of albumen.
6. the preparation method of clostridium septicum alpha toxin recombinant subunit vaccine of the present invention, it is characterised in that with the table
Up to clostridium septicum alpha toxin recombinant protein ETEC BL21/mCSA strains as production of vaccine bacterial strain, fermented culture,
After induced expression, bacterial cell disruption, inclusion body isolate and purify, two-phase oil emulsion adjuvant is added to be mixed.
The specific embodiment of the invention
1. the preparation of clostridium septicum alpha toxin recombinant subunit vaccine
(1) strain：Seedling with strain be recombinantly express clostridium septicum alpha toxin albumen ETEC BL21/mCSA
Strain, the strain expression clostridium septicum alpha toxin albumen contain 4 amino acid mutations (the 54th cysteine mutation is leucine,
264th asparagine mutation is alanine, and the 269th hyte Histidine mutations are alanine, and the 310th tryptophan sports the third ammonia
Acid), and to the addition of 6 histidine-tagged for the C-terminal of recombinant protein.The bacterial strain delivered Chaoyang District, Beijing City on 09 21st, 2017
The institute 3 of North Star West Road 1 China Committee for Culture Collection of Microorganisms of Institute of Microorganism, Academia Sinica common micro-organisms
Center preservation, deposit number are：CGMCC No.14650；Identified, taken care of and supplied by China Veterinery Drug Inspection Office.
(2) first order seed breeding and identification：Freeze-drying lactobacillus is melted with a small amount of LB fluid nutrient mediums, streak inoculation is in containing card
The LB solid plates of that mycin, put 37 DEG C and cultivate 12~16 hours, choose standard compliant single bacterium colony, inoculation contains kanamycins
LB fluid nutrient mediums, put 37 DEG C cultivate 8~12 hours, dispensed after being mixed with 50% glycerine equal proportion, it is qualified through purely examining
Afterwards, as seedling first order seed.
(3) secondary seed breeding and identification：First order seed is taken, the LB Liquid Cultures containing kanamycins are inoculated with by 1% amount
Base, puts 37 DEG C of shaken cultivations 8~12 hours, as secondary seed.
(4) prepared by antigen for vaccine：Qualified secondary seed is taken, by 2% inoculation of culture medium total amount containing kanamycins
LB fluid nutrient mediums, fermentation tank is interior to be cultivated.Set culture parameters be：37 DEG C of cultivation temperature, pH value 7.0, dissolved oxygen 40%.Work as culture
Thing OD600Be worth for 10~15 when, cool the temperature to 28 DEG C, add final concentration of 0.3mmol/L IPTG Fiber differentiations 4h.
(5) bacterium is broken：Thalline is collected by centrifugation, adds the 10ml lysates (0.02mol/L of pH value 7.2 by every gram of thalline weight in wet base
Tris buffer solutions, 0.3mol/L NaCl) ratio thalline is resuspended, with cryogenic high pressure homogenizer with 800bar pressure under the conditions of 4 DEG C
Power crushes thalline 3 times.Lysate centrifuges 30min through 4 DEG C of 10000r/min, abandoning supernatant, collects the inclusion body of precipitation.
(6) purify：By every gram of inclusion body 5ml lysates (6mol/L guanidine hydrochlorides, 20mmol/L Tris-HCl, pH value
8.0) after ratio is dissolved 2 hours with 200r/min vibrations at room temperature, 4 DEG C of 10000r/min centrifuge 30min, collect supernatant
(7) protein content detects：Protein content is detected with BCA methods.0.4mg/ml should be not less than.Through SDS-PAGE detections simultaneously
Gray scale scanning is carried out to band, purity of protein should be not less than 60%.
(8) steriling test：By existing《Chinese veterinary pharmacopoeia》(the Chinese veterinary pharmacopoeia committee, Republic of China Veterinary Pharmacopoeia,
Two 〇 year First Five-Year Plan versions three, Chinese agriculture publishing house, 2011, hereinafter referred to as《Chinese veterinary pharmacopoeia》) annex progress.Answer asepsis growth.
(7) vaccine formulation：Two-phase oil adjuvant (such as 201 adjuvants) is imported in oil phase tank, with least 121 DEG C of autoclavings 30
Minute, it is standby to be cooled to room temperature.According to determining the protein quantity result, it is qualified to be examined with PBS (0.01mol/L of pH value 7.2)
Mixed after purifying protein suitably dilution.Aqueous phase is added in emulsion tank, stirred with 80~100r/min, while by 1:1 (V/V's)
Ratio, oil phase is slowly added to, 20~30min is stirred after adding.Sampling is tested after emulsification, is dispensed after qualified.
2. the inspection of clostridium septicum alpha toxin recombinant subunit vaccine
(1) character
Appearance milky white emulsion.
Formulation is in water-in-oil-in water (W/O/W).A cleaning suction pipe is taken, a small amount of vaccine is drawn and drips in cleaning cold water surface,
Should be in that cloud spreads.
Stability is drawn vaccine 10ml and added in centrifuge tube, centrifuges 15min with 3000r/min, should not be demulsified, and ttom of pipe
The aqueous phase of precipitation should be not more than 0.5ml.
Viscosity is pressed《Chinese veterinary pharmacopoeia》Annex is measured, and should meet regulation.
(2) loading quantity inspection is pressed《Chinese veterinary pharmacopoeia》Annex is checked, should meet regulation.
(3) steriling test is pressed《Chinese veterinary pharmacopoeia》Annex is tested, and answers asepsis growth.
(4) safety verification 1.5~2.0kg of body weight healthy rabbits 4, each muscle or hypodermic injection vaccine 4.0ml, observation
10 days.Work should be all good for.
(5) efficacy test 1.5~2.0kg of body weight healthy rabbits 4, each neck subcutaneously or intramuscularly vaccinates 2.0ml.
14~21 days after inoculation, blood sampling, serum is separated.By the serum mixed in equal amounts of 4 immunizing rabbits, take pooled serum 0.4ml with
0.8ml clostridium septicums toxin (contains 4 mouse MLD), mixes rearmounted 37 cooperation 40min, is then injected intravenously 16~20g mouse
2,0.3ml/ is only.It is simultaneously each to criticize mouse 2 with same, 1MLD is injected respectively to be opposed with toxin serum mixture identical toxin
According to.Observation 1 day, result of determination.
It is all dead to compare mouse, serum neutralization titer reaches 1 to clostridium septicum toxin, and (0.1ml immune serums neutralize
1MLD toxin), that is, it is qualified to be judged to.
Brief description of the drawings
Fig. 1：The SDS-PAGE qualification results of mCSA gene engineering recombinant bacteriums, in figure：M:Protein marker；Lane
1:After pET-30a (+) induction；Lane 2:Lysate after pET30a-mCSA inductions；Lane 3:Sunk after pET30a-mCSA inductions
Form sediment；Lane 4:Precipitated after pET30a-mCSA inductions.
Fig. 2：In Western blot (using anti-His antibody) qualification result figure of mCSA gene engineering recombinant bacteriums：M:
Protein marker；Lane 1:After pET-30a (+) induction；Lane 2:Lysate after pET30a-mCSA inductions；Lane 3:
Precipitated after pET30a-mCSA inductions；Lane 4:Precipitated after pET30a-mCSA inductions.
The present invention relates to biomaterial resource information
Microorganism of the present invention is：(it is respectively that the 54th cysteine mutation is bright ammonia containing 4 amino acid mutations
Acid, the 264th asparagine mutation are alanine, and the 269th hyte Histidine mutations are alanine, and the 310th tryptophan sports
Alanine), and the C-terminal of recombinant protein with the addition of the 6 histidine-tagged large intestines angstrom for recombinantly expressing clostridium septicum alpha toxin albumen and wish
Salmonella BL21/mCSA strains.The bacterial strain has delivered Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 China on 09 21st, 2017
The preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms center of institute of microbiology of the academy of sciences, deposit number are：
CGMCC No.14650。
The positive effect of the present invention
The present invention relates to a kind of clostridium septicum alpha toxin recombinant subunit vaccine and its production method.The present invention is by wild type
54th cysteine mutation of clostridium septicum alpha toxin maturation toxin is leucine, and the 264th asparagine mutation is the third ammonia
Acid, the 269th hyte Histidine mutations are alanine, and the 310th tryptophan sports alanine, obtained nontoxic for animal body
Alpha toxin mutant, meanwhile, it is histidine-tagged plus 6 in the C-terminal of mutain, as antigen for vaccine.It is of the invention further public
Recombinant expression carrier and recombinant host cell containing the clostridium septicum alpha toxin non-toxic mutant encoding gene are opened.This hair
The clostridium perfringens alpha toxin non-toxic mutant of bright recombination expression totally nontoxic in Mice Body, and presented in Rabbit Model
Good immunogenicity and immune protective.The clostridium septicum alpha toxin non-toxic mutant or its encoding gene of the present invention can answer
For preparing preventing corruption perfringens alpha toxin subunit vaccine.Using clostridium septicum alpha toxin recombinant subunit epidemic disease proposed by the present invention
Seedling, there is obvious advantage compared with the clostridium septicum alpha toxin inactivated vaccine of the current commercialization in China, not only greatly reduce vaccine life
Bio-safety risk during production, and prepared vaccine potency is far above existing vaccine.In addition, rely on vaccine semi-finished product
The high advantage of protein concentration, when preparing connection seedling jointly with other antigens, without increasing the dosage of connection seedling, you can prepare connection
Seedling, greatly facilitate the exploitation of connection seedling.
Embodiment
Following examples are more preferable explanation technical scheme, but technical solution of the present invention are not construed as limiting.
Embodiment 1
--- structure, expression and the identification of the amino acid mutants of clostridium septicum alpha toxin 4
1. gene chemical synthesis
The application after codon optimization, devises 4 ammonia according to the sequence of clostridium septicum alpha toxin native protein gene
Base acid mutation, it is respectively leucine by the 54th cysteine mutation of wild type clostridium septicum alpha toxin maturation toxin, the 264th
Position asparagine mutation is alanine, and the 269th hyte Histidine mutations are alanine, and the 310th tryptophan sports alanine, from
And obtain for the nontoxic clostridium septicum alpha toxin mutant of animal body.Meanwhile the C-terminal of mutain adds 6 histidine marks
Label.With the method for chemical synthesis, this section of gene order is synthesized, altogether comprising 1254 nucleotides.Wherein, 1-1233 positions are corruption
Perfringens alpha toxin maturation toxin sequence (containing 4 amino acid mutations) is lost, 1234-1254 positions are 6 His-Tag sequences.Specifically
Nucleotide sequence as shown in SEQ ID No.1, shown in amino acid sequence as SEQ ID No.2.
2. the structure of nonfusion expression vector
Using artificial synthesized clostridium septicum alpha toxin full-length gene as template, using primer pair CSA-F/CSA-R (sequences 3/
Sequence 4) enter performing PCR amplification.
Wherein sense primer CSA-F sequences are：
5 '-CGCGGATCCATGACTAATCTGGAG-3 ' 24 (sequence 3), its 5 ' end introduce restriction enzyme BamH I
Point and protectiveness base；
Anti-sense primer CSA-R sequences are：
5 '-CCGCTCGAGTTAGTGGTGATGATG-3 ' 24 (sequence 4), its 5 ' end introduce restriction enzyme Xho I
Point and protectiveness base.
PCR system is：10 × EX Taq Buffer 5,4 μ l of μ l μ l, dNTPs, each 2 μ l, EX Taq enzyme of upstream and downstream primer
0.5 μ l, the μ l of full-length gene DNA profiling 2, supplement ddH2O is to 50 μ l systems.PCR reaction conditions are：94 DEG C of pre-degeneration 10min；94
DEG C denaturation 30s, 53 DEG C annealing 30s, 72 DEG C extension 1min30s, totally 33 circulation；Last 72 DEG C of rings extend 10min.
Obtained target DNA band is expanded, it is double digested using I/Xho of BamH I after recovered, and by same enzyme
The pET30a carriers connection of digestion is cut, obtains inserting the positive colony pET30-mCSA of clostridium septicum alpha toxin full-length gene.Will be even
The plasmid conversion DH5 α competent cells connected, in the LB fluid nutrient mediums containing kanamycins, 37 DEG C are shaken picking monoclonal
Overnight incubation is swung, extraction plasmid is standby.
3. recombinantly express the structure of the engineering strain of clostridium septicum alpha toxin full-length gene
Obtained plasmid will be extracted, converts e. coli bl21 (DE3) competent cell, picking monoclonal extremely contains and blocks that
In the LB fluid nutrient mediums of mycin, 37 DEG C of shaken cultivations are stayed overnight, and after PCR identifications contain target DNA fragment, are named as large intestine angstrom
Uncommon Salmonella (E.coli) BL21/mCSA strains, and 50% isometric glycerine LB is added, -70 DEG C freeze, and the bacterial strain is in 2017
Deliver Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica Chinese microorganism strain within 21 days 09 month
The common micro-organisms center preservation of preservation administration committee, deposit number are：(bacterial strain is also known as CGMCC No.14650 in the present invention
For：The amino acid mutants of clostridium septicum alpha toxin 4).
4. the expression and identification of destination protein
Genetic engineering bacterium ETEC (E.coli) BL21/ of clostridium septicum alpha toxin full-length gene will be recombinantly expressed
MCSA strains are inoculated in the LB fluid nutrient mediums that 100ml contains kanamycins, after 37 DEG C of shaken cultivation 4h, cool the temperature to 28
DEG C, add final concentration of 0.3mmol/L IPTG solution Fiber differentiations 4h.Thalline is collected by centrifugation after the completion of bacterium solution culture, by every
Gram thalline adds the ratio of 10ml lysates [0.02mol/L Tris buffer solutions (pH value 7.2), 0.3mol/L NaCl] that bacterium is resuspended
Body, ultrasonication thalline 30min, broken condition are in ice-water bath：Work 9s, interval 9s, ultrasonic power 400W.Will be broken
Bacterium solution afterwards centrifuges 30min in 4 DEG C, with 12000r/min, abandoning supernatant, collects the inclusion body of precipitation.By every gram of inclusion body
Shaken at room temperature with 200r/min with the ratio of 5ml lysates (6mol/L guanidine hydrochlorides, 20mmol/L Tris-HCl, pH value 8.0)
After swinging dissolving 2 hours, 4 DEG C of 10000r/min centrifuge 30min, collect supernatant.Take 30 μ l supernatants add 10 μ l × SDS-
PAGE sample-loading buffers, 70 DEG C of effect 10min, carry out 12%SDS-PAGE electrophoresis, as a result see Fig. 1.It will be seen from figure 1 that mesh
Albumen be largely present in the precipitation of cellular lysate liquid, expressed in inclusion body, expression quantity is good, and expression quantity is up to 20mg/L.
The most suitable induced expression condition of destination protein is 37 DEG C, induced expression 4h.
5. the Western blot identifications of destination protein
Using the destination protein expression product under different inductive conditions in above-mentioned steps, using anti-His antibody, to purpose egg
It is white to carry out Western blot identifications, as a result see Fig. 2.As can be seen from Figure 2,37 DEG C, 4h induction cell cracking precipitation in, purpose egg
White expression highest, account for the 60% of the total expression quantity of destination protein；37 DEG C, 4h induction cell cracking supernatant in, destination protein
Expression it is also not low, account for the 40% of the total expression quantity of destination protein.Because cell is cracked in supernatant in the purpose of solubility expression
Albumen, space structure are closest with wild-type toxin.Comprehensive SDS-PAGE and Western blot qualification results, further really
The most suitable induced expression condition for determining destination protein is 37 DEG C, induced expression 4h.
Embodiment 2
--- virulence test of the amino acid mutants of clostridium septicum alpha toxin 4 to mouse
By determining virulence of the amino acid mutants of clostridium septicum alpha toxin 4 to mouse, to verify the mutant in vivo
Actual attenuating effects.The amino acid mutation of clostridium septicum alpha toxin 4 of the amino acid mutants of clostridium septicum alpha toxin 4 activation is recombinated into egg
White mCSA, and wild type clostridium septicum alpha toxin, respectively with different dosage, 16~18g mouse are inoculated with through tail vein, every dose
Amount injection 5,0.2ml/ is only.As a result when dosage of inoculation is 0.1mg, all mouse are strong living and have no adverse reaction, and wild
Type control group can cause mouse 5/5 dead when being inoculated with 10ng.The result shows that clostridium septicum alpha toxin mutant is in mouse
It is nontoxic in vivo, is confirmed as toxin non-toxic mutant.
Virulence of the recombinant protein mCSA of table 1 to mouse
Embodiment 3
--- the Study On Immunogenicity of the amino acid mutants of clostridium septicum alpha toxin 4
(1) bacterium solution culture：The ETEC BL21/mCSA of clostridium septicum alpha toxin mutant protein will be recombinantly expressed
Strain culture bacterium solution, the LB fluid nutrient mediums containing kanamycins are inoculated with by the 2% of culture medium total amount, are cultivated in fermentation tank.Training is set
Foster parameter is：37 DEG C of cultivation temperature, pH value 7.0, dissolved oxygen 40%.As culture OD600Be worth for 10~15 when, cool the temperature to 28
DEG C, add final concentration of 0.3mmol/L IPTG Fiber differentiations 4h.
(2) bacterium is broken：Thalline is collected by centrifugation, adds the 10ml lysates (0.02mol/L of pH value 7.2 by every gram of thalline weight in wet base
Tris buffer solutions, 0.3mol/L NaCl) ratio thalline is resuspended, with cryogenic high pressure homogenizer with 800bar pressure under conditions of 4
Power crushes thalline 3 times.Lysate is through 4.Lysate centrifuges 30min through 4 DEG C of 10000r/min, abandoning supernatant, collects precipitation
Inclusion body.
(3) purify：By every gram of inclusion body 5ml lysates (6mol/L guanidine hydrochlorides, 20mmol/L Tris-HCl, pH value
8.0) after ratio is dissolved 2 hours with 200r/min vibrations at room temperature, 4 DEG C of 10000r/min centrifuge 30min, collect supernatant
(4) protein content detects：Protein content is detected with BCA methods.As a result protein content is 8.3mg/ml..Through SDS-
PAGE is detected and is carried out gray scale scanning, purity of protein 74% to band.
(5) steriling test：By existing《Chinese veterinary pharmacopoeia》Annex is carried out.As a result equal asepsis growth.
(6) vaccine formulation：Two-phase oil adjuvant (such as 201 adjuvants) is imported in oil phase tank, with least 121 DEG C of autoclavings 30
Minute, it is standby to be cooled to room temperature.According to determining the protein quantity result, it is qualified to be examined with PBS (0.01mol/L of pH value 7.2)
Purifying protein mixes after being diluted to final concentration of 100 μ g/ml.Aqueous phase is added in emulsion tank, stirred with 80~100r/min, together
When press 1:1 (V/V) ratio, is slowly added to oil phase, and 20~30min is stirred after adding.Sampling is tested after emulsification, after qualified
Packing.
(7) Study On Immunogenicity：According to《Chinese veterinary pharmacopoeia》Defined method is carried out.Specifically test method is：Use body weight
1.5~2.0kg healthy rabbits 4, each neck subcutaneously or intramuscularly vaccinates 2.0ml.14 days after inoculation, blood sampling, serum is separated.
Meanwhile secondary immunity is carried out to rabbit with same dose, identical approach.Two exempt from latter 21 days, blood sampling, separation serum.Using following
Method, respectively to primary immune response and secondary immunity after, the toxin antibody potency in rabbit anteserum is measured：
By the serum mixed in equal amounts of 4 immunizing rabbits, pooled serum 0.4ml is taken (to contain 4 with 0.8ml clostridium septicums alpha toxin
Mouse MLD), rearmounted 37 cooperation 40min is mixed, is then injected intravenously 16~20g mouse 2,0.3ml/ is only.It is simultaneously each to use together
Mouse 2 is criticized, 1MLD is injected respectively and is compared with toxin serum mixture identical toxin.Observation 1 day, result of determination.It is if right
All dead according to mouse, serum neutralization titer reaches 1 (in 0.1ml immune serums and 1MLD toxin) to clostridium septicum alpha toxin,
It is qualified to be judged to.
After measured, after primary immune response, (i.e. 0.1ml rabbit anteserums can neutralize 6MLD for 6 for malicious antibody titer in rabbit anteserum
Clostridium septicum alpha toxin)；After secondary immunity, (i.e. 0.1ml rabbit anteserums can neutralize for 15 for malicious antibody titer in rabbit anteserum
15MLD clostridium septicum alpha toxin).
《Chinese veterinary pharmacopoeia》Provide, the malicious antibody titer in rabbit anteserum reaches 1 to clostridium septicum toxin, you can is judged to epidemic disease
Clostridium septicum toxin moiety is qualified in seedling.Therefore, the vaccine of the application production, in antigenic content as little as 50 μ g/ml situation
Under, either to rabbit primary immune response, or secondary immunity, exceed existing《Chinese veterinary pharmacopoeia》Regulation, it was demonstrated that the epidemic disease
Seedling has good immunogenicity.
In view of the existing commercial clostridial toxin vaccine in China must use formalin-inactivated detoxification, bio-safety hidden danger be present,
It has impact on the security that vaccine uses in field；Existing commercialized vaccine is in process of production, also unstable in the presence of production poison simultaneously,
The problem of causing vaccine potency unstable.Therefore, the vaccine of the application production is the existing clostridium septicum inactivated vaccine upgrading in China
The ideal candidates vaccine of the replacement.
Sequence table
<110>China Veterinery Drug Inspection Office
<120>A kind of clostridium septicum alpha toxin recombinant subunit vaccine and its production method
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 1494
<212> DNA
<213>It is artificial synthesized
<223>Description to artificial sequence：Clostridium septicum alpha toxin full-length gene
<400> 1
atggagttta gcgcgaaatt cgttgctgca tggaccctga aggcagcagc catctctatc 60
accgagatca aaggtgtgat cgtacaccgt atcgaaacca ttctgttcgc tccaagcatc 120
attaacaact attacatgca gcagtaccag aacagcatgg acggtccagg tctggagtcc 180
accaacattc tgcaacgtat gcgtgatctg agcctgcaaa gcgctaacgg tggcggttct 240
actaatctgg aggaaggtgg ttacgcgaat cataacaacg catctagcat taagatcttc 300
ggctacgaag ataacgagga tctgaaagcc aagatcattc aggacccaga gttcattcgc 360
aactgggcaa acgtggcaca cagcctgggt ttcggttggc tgggtggcac cgcgaaccca 420
aacgtgggcc agggtttcga gttcaaacgc gaggttggtg caggtggcaa ggtgtcctac 480
ctgctgtctg ctcgctataa cccaaacgac ccgtacgcca gcggttatcg tgctaaagat 540
cgcctgtcca tgaagatttc taacgtgcgt ttcgttatcg acaacgattc tatcaaactg 600
ggcactccga aggttaagaa actggctccg ctgaacagcg ccagcttcga tctgattaac 660
gagagcaaga ccgagtctaa actgtccaag acctttaact acactacctc taagaccgtg 720
tccaagactg acaacttcaa attcggcgag aagatcggcg taaagacctc tttcaaggta 780
ggtctggaag ctatcgctga cagcaaagtg gagacttcct ttgagttcaa cgcggagcag 840
ggttggtcca ataccaactc tactaccgaa accaaacagg agtccactac ttacactgcg 900
actgtttctc cgcaaactaa gaaacgtctg tttctggacg tactgggcag ccagattgac 960
attccatacg aaggtaagat ctacatggaa tacgacatcg aactgatggg ctttctgcgc 1020
tatactggtg cggcacgtga agatgcaact gaggaccgtc caaccgtgaa actgaaattc 1080
ggcaagaacg gtatgtctgc tgaagagcat ctgaaagacc tgtattctca caagaacatc 1140
aacggctata gcgaatggga ttggaaagct gttgatgaga agtttggcta cctgtttaag 1200
aactcctatg atgctctgac cagccgcaag ctgggtggca ttatcaaagg ttccttcacc 1260
aacatcaacg gtactaagat cgttatccgt gaaggcaaag aaattccgct gccggacaag 1320
aaacgtcgtg gcaaacgctc tgtagattct ctggacgcac gtctgcaaaa tgaaggtatc 1380
cgtattgaga acattgaaac ccaggacgtg ccaggtttcc gcctgaactc tatcacctac 1440
aacgataaga aactgattct gatcaacaac atccatcacc atcatcacca ctaa 1494
<210> 2
<211> 497
<212> PRT
<213>It is artificial synthesized
<223>Description to artificial sequence：The amino acid sequence of clostridium septicum alpha toxin
<400> 2
Met Glu Phe Ser Ala Lys Phe Val Ala Ala Trp Thr Leu Lys Ala Ala
5 10 15
Ala Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu
20 25 30
Thr Ile Leu Phe Ala Pro Ser Ile Ile Asn Asn Tyr Tyr Met Gln Gln
35 40 45
Tyr Gln Asn Ser Met Asp Gly Pro Gly Leu Glu Ser Thr Asn Ile Leu
50 55 60
Gln Arg Met Arg Asp Leu Ser Leu Gln Ser Ala Asn Gly Gly Gly Ser
65 70 75 80
Thr Asn Leu Glu Glu Gly Gly Tyr Ala Asn His Asn Asn Ala Ser Ser
85 90 95
Ile Lys Ile Phe Gly Tyr Glu Asp Asn Glu Asp Leu Lys Ala Lys Ile
100 105 110
Ile Gln Asp Pro Glu Phe Ile Arg Asn Trp Ala Asn Val Ala His Ser
115 120 125
Leu Gly Phe Gly Trp Leu Gly Gly Thr Ala Asn Pro Asn Val Gly Gln
130 135 140
Gly Phe Glu Phe Lys Arg Glu Val Gly Ala Gly Gly Lys Val Ser Tyr
145 150 155 160
Leu Leu Ser Ala Arg Tyr Asn Pro Asn Asp Pro Tyr Ala Ser Gly Tyr
165 170 175
Arg Ala Lys Asp Arg Leu Ser Met Lys Ile Ser Asn Val Arg Phe Val
180 185 190
Ile Asp Asn Asp Ser Ile Lys Leu Gly Thr Pro Lys Val Lys Lys Leu
195 200 205
Ala Pro Leu Asn Ser Ala Ser Phe Asp Leu Ile Asn Glu Ser Lys Thr
210 215 220
Glu Ser Lys Leu Ser Lys Thr Phe Asn Tyr Thr Thr Ser Lys Thr Val
225 230 235 240
Ser Lys Thr Asp Asn Phe Lys Phe Gly Glu Lys Ile Gly Val Lys Thr
245 250 255
Ser Phe Lys Val Gly Leu Glu Ala Ile Ala Asp Ser Lys Val Glu Thr
260 265 270
Ser Phe Glu Phe Asn Ala Glu Gln Gly Trp Ser Asn Thr Asn Ser Thr
275 280 285
Thr Glu Thr Lys Gln Glu Ser Thr Thr Tyr Thr Ala Thr Val Ser Pro
290 295 300
Gln Thr Lys Lys Arg Leu Phe Leu Asp Val Leu Gly Ser Gln Ile Asp
305 310 315 320
Ile Pro Tyr Glu Gly Lys Ile Tyr Met Glu Tyr Asp Ile Glu Leu Met
325 330 335
Gly Phe Leu Arg Tyr Thr Gly Ala Ala Arg Glu Asp Ala Thr Glu Asp
340 345 350
Arg Pro Thr Val Lys Leu Lys Phe Gly Lys Asn Gly Met Ser Ala Glu
355 360 365
Glu His Leu Lys Asp Leu Tyr Ser His Lys Asn Ile Asn Gly Tyr Ser
370 375 380
Glu Trp Asp Trp Lys Ala Val Asp Glu Lys Phe Gly Tyr Leu Phe Lys
385 390 395 400
Asn Ser Tyr Asp Ala Leu Thr Ser Arg Lys Leu Gly Gly Ile Ile Lys
405 410 415
Gly Ser Phe Thr Asn Ile Asn Gly Thr Lys Ile Val Ile Arg Glu Gly
420 425 430
Lys Glu Ile Pro Leu Pro Asp Lys Lys Arg Arg Gly Lys Arg Ser Val
435 440 445
Asp Ser Leu Asp Ala Arg Leu Gln Asn Glu Gly Ile Arg Ile Glu Asn
450 455 460
Ile Glu Thr Gln Asp Val Pro Gly Phe Arg Leu Asn Ser Ile Thr Tyr
465 470 475 480
Asn Asp Lys Lys Leu Ile Leu Ile Asn Asn Ile His His His His His
485 490 495
His
497
<210> 3
<211> 30
<212> DNA
<213>It is artificial synthesized
<223>Description to artificial sequence：Expand the sense primer CSA-F of Gene of Clostridium septicum
<400> 3
CGCGGATCCA TGGAGTTTAG CGCGAAATTC 30
<210> 4
<211> 31
<212> DNA
<213>It is artificial synthesized
<223>Description to artificial sequence：Expand the sense primer CSA-FR of Gene of Clostridium septicum.
<400> 4
CCGCTCGAGT TAGTGGTGAT GATGGTGATG G 31
4
Claims (6)
1. a kind of clostridium septicum alpha toxin recombinant subunit vaccine, it is characterised in that the vaccine contains using Bacillus coli expression
Restructuring clostridium septicum alpha toxin albumen；Seedling with strain be recombinantly express clostridium perfringens alpha toxin albumen E
Bacterium is named as ETEC (Escherichia coli) BL21/mCSA strains, and the bacterial strain is on 09 21st, 2017
Deliver the Chinese microorganism strain preservation management of Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica
Committee's common micro-organisms center preservation, deposit number are：CGMCC No.14650.
2. clostridium septicum alpha toxin recombinant subunit vaccine as claimed in claim 1, it is characterised in that the restructuring clostridium septicum α
Toxin protein, compared with wild type clostridium septicum alpha toxin maturation toxin, contain 4 amino acid mutations, respectively the 54th half Guang
Histidine mutations are leucine, and the 264th asparagine mutation is alanine, and the 269th hyte Histidine mutations are alanine, the 310th
Position tryptophan sports alanine.It is meanwhile histidine-tagged plus 6 in the C-terminal of mutain.
3. clostridium septicum alpha toxin recombinant subunit vaccine as claimed in claim 1, it is characterised in that the restructuring clostridium septicum α
Toxin protein, the gene order codon optimization of its recombinant expression carrier, it is easier to high expression is realized in Escherichia coli.
4. clostridium septicum alpha toxin recombinant subunit vaccine as claimed in claim 1, it is characterised in that the restructuring clostridium septicum α
Toxin protein is non-toxic mutant, greatly reduces the bio-safety risk in vaccine production process.
5. clostridium septicum alpha toxin recombinant subunit vaccine as claimed in claim 1, it is characterised in that the restructuring clostridium septicum α
Toxin protein, its C-terminal contain 6 histidines (6His) label, are easy to the purifying of albumen.
6. the preparation method of clostridium septicum alpha toxin recombinant subunit vaccine as claimed in claim 1, it is characterised in that with the table
Up to clostridium septicum alpha toxin recombinant protein ETEC BL21/mCSA strains as production of vaccine bacterial strain, fermented culture,
After induced expression, bacterial cell disruption, inclusion body isolate and purify, two-phase oil adjuvant is added to be mixed.
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|---|---|---|---|
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108904791A (en) * | 2018-07-20 | 2018-11-30 | 中国兽医药品监察所 | A kind of clostridium perfringens alpha toxin recombinant subunit vaccine and its production method |
| CN109111509A (en) * | 2018-09-19 | 2019-01-01 | 天康生物股份有限公司 | Clostridium septicum alpha toxin mutant, the gene for expressing it, preparation method and clostridium septicum vaccine |
| CN109395072A (en) * | 2018-10-30 | 2019-03-01 | 中国兽医药品监察所 | A kind of Gene of Clostridium septicum engineered vaccine and its production method |
| CN110157655A (en) * | 2019-05-22 | 2019-08-23 | 中国兽医药品监察所 | A kind of nontoxicity clostridium chauvei genetic engineering subunit vaccine strain and its application |
| CN111944838A (en) * | 2020-09-02 | 2020-11-17 | 天康生物股份有限公司 | Application of gram-positive bacterium expression system in expression of clostridium putrefactive toxin, preparation method of clostridium putrefactive alpha toxin and vaccine |
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2017
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108904791A (en) * | 2018-07-20 | 2018-11-30 | 中国兽医药品监察所 | A kind of clostridium perfringens alpha toxin recombinant subunit vaccine and its production method |
| CN109111509A (en) * | 2018-09-19 | 2019-01-01 | 天康生物股份有限公司 | Clostridium septicum alpha toxin mutant, the gene for expressing it, preparation method and clostridium septicum vaccine |
| CN109111509B (en) * | 2018-09-19 | 2021-08-13 | 天康制药(苏州)有限公司 | Mutant of alpha toxin of clostridium putrefactive bacteria, gene for expressing mutant, preparation method and vaccine of clostridium putrefactive bacteria |
| CN109395072A (en) * | 2018-10-30 | 2019-03-01 | 中国兽医药品监察所 | A kind of Gene of Clostridium septicum engineered vaccine and its production method |
| CN109395072B (en) * | 2018-10-30 | 2021-11-30 | 中国兽医药品监察所 | Genetically engineered vaccine of clostridium putrefactive alpha toxin and production method thereof |
| CN110157655A (en) * | 2019-05-22 | 2019-08-23 | 中国兽医药品监察所 | A kind of nontoxicity clostridium chauvei genetic engineering subunit vaccine strain and its application |
| CN110157655B (en) * | 2019-05-22 | 2021-06-01 | 中国兽医药品监察所 | Strain for non-toxic clostridium emphysema genetic engineering subunit vaccine and application thereof |
| CN111944838A (en) * | 2020-09-02 | 2020-11-17 | 天康生物股份有限公司 | Application of gram-positive bacterium expression system in expression of clostridium putrefactive toxin, preparation method of clostridium putrefactive alpha toxin and vaccine |
| CN111944838B (en) * | 2020-09-02 | 2022-08-16 | 天康制药(苏州)有限公司 | Application of gram-positive bacterium expression system in expression of clostridium putrefactive toxin, preparation method of clostridium putrefactive alpha toxin and vaccine |
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