CN109111509A - Clostridium septicum alpha toxin mutant, the gene for expressing it, preparation method and clostridium septicum vaccine - Google Patents
Clostridium septicum alpha toxin mutant, the gene for expressing it, preparation method and clostridium septicum vaccine Download PDFInfo
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Abstract
The present invention provides a kind of clostridium septicum alpha toxin mutant, the gene for expressing it, preparation method and clostridium septicum vaccines, are related to field of biotechnology.The clostridium septicum alpha toxin mutant includes any one in (a1)-(a3).Wherein, it (a1) by cysteine mutation is leucine and the 189th amino acids residue by the protein that mutant serine is that cysteine obtains will to have the 86th amino acids residue of clostridium septicum alpha toxin of the amino acid sequence as shown in SEQ ID NO.1；(a2) protein obtained for the signal peptide of excision (a1)；(a3) any one in the fusion protein obtained for the N-terminal of the protein shown in (a1) or (a2) and/or C-terminal connection label, has toxic side effect low, the good advantage of immunogenicity.The clostridium septicum vaccine includes clostridium septicum alpha toxin mutant or the gene for encoding it, has good safety and good immunogenicity.
Description
Technical field
The present invention relates to field of biotechnology, more particularly, to clostridium septicum alpha toxin mutant, the gene for expressing it, system
Preparation Method and clostridium septicum vaccine.
Background technique
Clostridium septicum (Clostridium septium) is a kind of Gram-positive anaerobic bacillus(cillus anaerobicus), can cause animal and people
The diseases such as malignant edema disease, muscular death, emphysematous gangrene and necrotic enteritis, and cause the pathogen of sheep braxy.
The bacterium can secrete a variety of exotoxins such as α, β and γ, and wherein alpha toxin is its principal causative virulence factor and immune guarantor
Shield property antigen.Clostridium septicum alpha toxin be gas lysin family perforation toxin one kind, be clostridium septicum main lethal virulence because
Son has the function of haemolysis, lethal and downright bad, and furthermore the toxin also has good immunogenicity, toxoid prepared therefrom
It is effective against the infection of clostridium septicum.
Clostridium septicum is distributed widely in soil, excrement, dust, marsh and the alimentary canal of animal, easily by by gemma dirt
The approach such as feed, drinking-water and the ambient enviroment of dye cause sheep, horse, ox, pig, dog, cat, chicken and deer etc. dynamic through wound or alimentary canal
The diseases such as the malignant edema of object and people disease, muscular death, emphysematous gangrene and necrotic enteritis, the age of morbidity and animal,
Gender and kind are unrelated.There are two types of its routes of infection is general, one is due to wound for example castration, docking, childbirth, surgical operation or
There is no strict sterilization during injection, clostridium septicum gemma is caused to pollute and cause to infect；Another is infected through alimentary canal,
It is mostly popular in place at this time, it is mainly in autumn, winter and early spring weather cataclysm, season cloudy and drizzly for days on end.Disease caused by clostridium septicum
Have the characteristics that broadcast process and have and propagates that fast, morbidity is anxious, the course of disease is short, the death rate is high.Wherein, sheep is the easiest to clostridium septicum
Sense, the nutrition for the sheep that falls ill are larger to the harm of livestock and poultry breeding industry mostly more than medium.The disease as caused by clostridium septicum is for example fast
Epidemic disease, the general course of disease is extremely very brief, often has little time treatment animal and death occurs.Therefore, immunity inoculation is to prevent the disease the most
One of effective way.
In view of this, the present invention is specifically proposed.
Summary of the invention
The first purpose of this invention is to provide a kind of clostridium septicum alpha toxin mutant have toxic side effect low, is immunized
The good advantage of originality.
Second object of the present invention is to provide a kind of gene for encoding above-mentioned clostridium septicum alpha toxin mutant.
Third object of the present invention is to provide a kind of preparation side of the gene of above-mentioned clostridium septicum alpha toxin mutant
Method.
Fourth object of the present invention is to provide a kind of above-mentioned clostridium septicum alpha toxin mutant, the above-mentioned corrupt shuttle of coding
The gene of bacterium alpha toxin mutant, the preparation method of above-mentioned clostridium septicum alpha toxin mutant are dashed forward by above-mentioned clostridium septicum alpha toxin
The application for the albumen that variant preparation method is prepared.
Of the invention the 5th is designed to provide a kind of clostridium septicum vaccine, which includes above-mentioned clostridium septicum α poison
The gene of plain mutant or the above-mentioned clostridium septicum alpha toxin mutant of coding.
In order to solve the above technical problems, spy of the present invention adopts the following technical scheme that
The present invention provides a kind of clostridium septicum alpha toxin mutant, including any one in following (a1)-(a3):
(a1) by the 86th amino acids residue of clostridium septicum alpha toxin with the amino acid sequence as shown in SEQ ID NO.1
It is leucine and the 189th amino acids residue by the protein that mutant serine is that cysteine obtains by cysteine mutation；
(a2) protein that the signal peptide of (a1) obtains is cut off；
(a3) fusion protein that the N-terminal of the protein shown in (a1) or (a2) and/or C-terminal connection label obtain.
Preferably, the clostridium septicum alpha toxin mutant has the amino acid sequence as shown in SEQ ID NO.3.
The present invention also provides a kind of genes for encoding above-mentioned clostridium septicum alpha toxin mutant.
Preferably, there is the nucleotide sequence as shown in SEQ ID NO.4.
The present invention also provides a kind of preparation methods of above-mentioned clostridium septicum alpha toxin mutant, comprising: will encode the corruption
The gene of perfringens alpha toxin mutant is lost in host cell expression.
Preferably, the preparation method includes: to encode the clostridium septicum alpha toxin using mammalian expression systems expression
The gene of mutant；
Preferably, using the gene of clostridium septicum alpha toxin mutant described in expressing cho cell system expression；Chinese hamster ovary celI is excellent
Select CHO-S cell line.
Preferably, the gene cloning of the coding clostridium septicum alpha toxin mutant is then introduced into expression vector
Host cell, then the host cell for expressing the clostridium septicum alpha toxin mutant is screened and cultivates, by the egg of host cell expression
It is white it is purified after obtain the clostridium septicum alpha toxin mutant；
Preferably, the expression vector includes pcDNA3, preferably pcDNATM
The present invention also provides a kind of above-mentioned clostridium septicum alpha toxin mutant, the above-mentioned clostridium septicum alpha toxin mutant of coding
Gene, above-mentioned clostridium septicum alpha toxin mutant preparation method or the clostridium septicum alpha toxin that is prepared of above-mentioned preparation method
Application of the mutant in following (x1)-(x5)；
(x1) clostridium septicum vaccine is prepared；
(x2) clostridium septicum alpha toxin antibody is prepared；
(x3) reagent and/or kit of preparation detection clostridium septicum alpha toxin；
(x4) reagent and/or kit of preparation detection clostridium septicum alpha toxin antibody；
(x5) clostridium septicum alpha toxin diagnostic antigen is prepared.
The present invention also provides a kind of clostridium septicum vaccine, which includes above-mentioned clostridium septicum alpha toxin mutant or volume
The gene of the above-mentioned clostridium septicum alpha toxin mutant of code.
Preferably, the clostridium septicum vaccine includes clostridium septicum alpha toxin mutant；The clostridium septicum alpha toxin mutation
Body has the amino acid sequence as shown in SEQ ID NO.3；The clostridium septicum alpha toxin mutant is by mammalian expression systems
Expression obtains；
Preferably, the concentration of clostridium septicum alpha toxin mutant is 30-50 μ g/mL in the clostridium septicum vaccine；Preferably
35-45μg/mL；More preferably 40 μ g/mL；
Preferably, the vaccine further includes auxiliary material, and the auxiliary material includes one of vaccine adjuvant, stabilizer and antibiotic
Or it is a variety of；
Preferably, the vaccine adjuvant includes aluminium hydroxide gel, Freund's complete adjuvant, incomplete Freund's adjuvant, white oil assistant
Agent, MF59 adjuvant or Montanide ISA series of adjuvants；It is preferable to use Montanide ISA series of adjuvants；More preferably use
ISA15A adjuvant.
Compared with prior art, the invention has the following beneficial effects:
Clostridium septicum alpha toxin mutant provided by the invention will predominantly have the amino acid sequence as shown in SEQ ID NO.1
The 86th amino acids residue of clostridium septicum alpha toxin of column by cysteine mutation be leucine and the 189th amino acids residue by
Mutant serine is the protein that cysteine obtains；Or further remove the protein after its signal peptide；Or in its amino acid
The fusion protein obtained after N-terminal and/or C-terminal the connection label of sequence, in order to the separation, purifying and identification of following protein.It should
Clostridium septicum alpha toxin mutant has no toxic side effect, and bio-safety risk greatly reduces, while effectively keeping alpha toxin
Immunogenicity.
The present invention also provides the genes that can encode above-mentioned clostridium septicum alpha toxin mutant and above-mentioned clostridium septicum α poison
The preparation method of plain mutant, by expressing the gene of above-mentioned fusion protein in host.
Above-mentioned clostridium septicum alpha toxin mutant provided by the invention, the above-mentioned clostridium septicum alpha toxin mutant of above-mentioned coding
Gene, the preparation method of above-mentioned clostridium septicum alpha toxin mutant are prepared by above-mentioned clostridium septicum alpha toxin mutation preparation
Obtained albumen can be applied to the vaccine for preparing a variety of diseases as caused by clostridium septicum using very extensive；Prepare corrupt shuttle
Bacterium alpha toxin antibody；The reagent and/or kit of preparation detection clostridium septicum alpha toxin；Preparation detection clostridium septicum alpha toxin antibody
Reagent and/or kit and preparation clostridium septicum alpha toxin diagnostic antigen.
Clostridium septicum vaccine provided by the invention includes above-mentioned clostridium septicum alpha toxin mutant or above-mentioned coding corruption shuttle
The gene of bacterium alpha toxin mutant.It is codon optimization, containing 2 amino acid sites mutation, obtains to animal body attenuated
Alpha toxin mutant, have good safety and good immunogenicity.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is pcDNATM Plasmid map；
Fig. 2 is the gel electrophoresis result for the recombinant protein that the present invention implements 1 clostridium septicum alpha toxin mutant provided；
Fig. 3 is the western blot inspection for the recombinant protein that the present invention implements 1 clostridium septicum alpha toxin mutant provided
Survey result.
Specific embodiment
Technical solution of the present invention is clearly and completely described below in conjunction with attached drawing, it is clear that described implementation
Example is a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, ordinary skill
Personnel's every other embodiment obtained without making creative work, shall fall within the protection scope of the present invention.
The person that is not specified actual conditions in embodiment, carries out according to conventional conditions or manufacturer's recommended conditions.Agents useful for same or instrument are not
Production firm person is indicated, is the conventional products that can be obtained by commercially available purchase.
The present invention provides a kind of clostridium septicum alpha toxin mutant, including any one in following (a1)-(a3):
(a1) by the 86th amino acids residue of clostridium septicum alpha toxin with the amino acid sequence as shown in SEQ ID NO.1
It is leucine by cysteine mutation；With the albumen that the 189th amino acids residue is obtained by mutant serine for cysteine
Matter；
(a2) protein that the signal peptide of (a1) obtains is cut off；
(a3) fusion protein that the N-terminal of the protein shown in (a1) or (a2) and/or C-terminal connection label obtain.
In the full length gene 1323bp of the existing clostridium septicum alpha toxin mutant having been detected by, there is such as SEQ ID
Sequence shown in NO.2 encodes 440 amino acid, has the amino acid sequence as shown in SEQ ID NO.1.Albumen size about 48kDa,
Signal peptide sequence comprising 31 amino acid.To its functional site research shows that:
The 398th arginine in KKRRGKR398SVD segment, the Activated in Vitro for alpha toxin are required, this positions
Point is eukaryon furin (furin) recognition site containing residue RGKR, precursor toxin shape after protease hydrolytic acts on
At the activated form of 41-44kDa size and a small molecule propetide.WDWnW segment on 333NGYSEWDWKWV343 is α poison
Element and receptor glycosyl phosphatidyl alcohol (GPI) anchorin binding site, wherein n indicates that the site is arbitrary amino acid.It is double
Close transmembrane structure 203KIGVKTSFKVGLEAIADSKVETSFEFNAE231 is the major function that alpha toxin is perforated on cell membrane
Area.
It is a discovery of the invention that following two sites of simultaneous mutation: being leucine by the 86th cysteine mutation, and by the
189 mutant serines are cysteine, can effectively reduce the toxicity of alpha toxin, obtain clostridium septicum alpha toxin non-toxic mutant
Bio-safety risk, while the effective immunogenicity for keeping alpha toxin greatly reduces in body.
Meanwhile clostridium septicum alpha toxin mutant provided by the invention may be the protein removed after its signal peptide.Letter
Number peptide is a segment of protein, and signal peptide guidance ribosomes is simultaneously positioned in endoplasmic reticulum channel, makes ribosomes
It is attached in endoplasmic reticulum, and the protein chain constantly extended is penetrated through into channel, subsequent signal peptide is cut, has synthesized
At protein be discharged into endoplasmic, albumen is finally transported to extracellular.As it can be seen that being deposited on signal peptide with lesser possibility
In the epitope with immunogenicity, and cuts off signal peptide and can also further promote the solubility expression of albumen.
Further, clostridium septicum alpha toxin mutant provided by the invention may be the N-terminal in its amino acid sequence
And/or the fusion protein obtained after C-terminal connection label, in order to the separation, purifying and identification of following protein, the label is for example
It can be but be not limited to His label, Flag label, GST label, MBP label, NusA label, SUMO label.
In some preferred embodiments, the clostridium septicum alpha toxin mutant has as shown in SEQ ID NO.3
Amino acid sequence.The amino acid sequence is by the 86th amino acids residue of amino acid sequence shown in SEQ ID NO.1 by half
Cystine sports leucine；With the 189th amino acids residue by mutant serine be cysteine.Then remove SEQ ID
31 amino acid residues of amino acid sequence N-terminal shown in NO.1 remove the signal peptide moiety of clostridium septicum alpha toxin mutant.Most
His label is added in the C-terminal of SEQ ID NO.1 again afterwards, present embodiment selection has the His label of 6 histidines, some
In other embodiments, the His label of common 6-10 histidine in Protocols in Molecular Biology, the present invention couple also can choose
This is with no restrictions.By the above-mentioned optimization to amino acid sequence shown in SEQ ID NO.1, it is nontoxic prominent to obtain clostridium septicum alpha toxin
Variant has the sequence as shown in SEQ ID NO..
The present invention also provides the genes for encoding above-mentioned clostridium septicum alpha toxin mutant.Coding of the present invention is above-mentioned
The gene of clostridium septicum alpha toxin mutant includes the nucleotide sequence for expressing above-mentioned clostridium septicum alpha toxin mutant, can also be wrapped
The nucleotide sequence isolated and purified for regulatory protein expression and/or auxilin is included, such as can be but be not limited to start
Subsequence, enhancer sequence and/or the sequence for expressing protein tag.In some preferred embodiments, the corruption
The gene of perfringens alpha toxin mutant has the nucleotide sequence as shown in SEQ ID NO.4.
The present invention also provides a kind of preparation methods of above-mentioned clostridium septicum alpha toxin mutant, comprising: will encode the corruption
The gene of perfringens alpha toxin mutant is lost in host cell expression.
The preparation method is by expressing the gene of the above-mentioned coding clostridium septicum alpha toxin mutant in host
Can, such as can be but be not limited in escherichia expression system, yeast expression system, insect expression system, plant expression
System or mammalian expression systems.
But due to using prokaryotic system, expression product is usually with the presence of insoluble inclusion bodies, soluble protein
The report of expression is considerably less.Because the expression product in inclusion body does not have biological activity, thus is denaturalized and is answered
Property processing.The denaturation of albumen and renaturation are an extremely complex processes, and the denaturing conditions of different albumen are different, and renaturation yield is often very
Hardly possible improves, this is the main restricting factor for limiting its application.Prokaryotic expression system simultaneously, expression quantity is low, purifies at high cost, endogenous toxic material
Plain Questions topic.Therefore in one preferred embodiment, using mammalian expression systems to clostridium septicum alpha toxin mutant into
The soluble expression of row.After mammalian expression systems also have the translated processing of the albumen of expression, structure and biological characteristics
The advantages of property is closer to native protein.Host cell used in the mammalian expression systems for example can be but unlimited
In for Chinese hamster ovary (CHO) cell, small hamster kidney (BHK) cell, MK cells (COS) cell, mouse NSO thymoma it is thin
Born of the same parents and mouse myeloma SP2/0 cell etc..
In some preferred embodiments, it is preferable to use clostridium septicum alpha toxin mutant described in expressing cho cell.
CHO (Chinese Hamster Ovary, CHO) cell is the Theodore T.Puck of Univ Colorado-Boulder USA
Doctor separated out of an Adult female Chinese hamster ovary and obtains in nineteen fifty-seven.FDA certification safe host's engineering cell, be
Express express target protein, the preferable expression system of especially complicated modificationization high molecular weight protein.
CHO-S and CHO-K1 cell line commonly used at present is derived by CHO mother cell.Both cell lines
Fibroblast is belonged to, is a kind of nonsecreting type cell, few secretion CHO intrinsic protein itself.Expressing cho cell system tool
It has the following advantages:
(1) CHO belongs to fibroblast, seldom secretes the endogenous protein of itself, conducive to the separation of foreign protein.
(2) there is folding and rhetorical function after accurately translating, the albumen of expression is in molecular structure, physicochemical property and biology
Function aspect is learned closest to native protein molecule.
(3) there is adherent growth characteristic, tolerance shearing force and osmotic pressure ability with higher；It can also carry out suspension training
It supports, suspend culture on a large scale in serum free medium, higher cell density in bioreactor.
(4) have the function of product exocytosis, isolated and purified convenient for downstream product；Efficient amplification with recombination and
Ability to express.
In some preferred embodiments, the preparation method includes: that the coding clostridium septicum alpha toxin is dashed forward
The gene cloning of variant is then introduced into host cell to expression vector, then screens and cultivate the expression clostridium septicum alpha toxin and dash forward
The host cell of variant, by the albumen of host cell expression it is purified after obtain the clostridium septicum alpha toxin mutant.Wherein table
Up to carrier, it is preferable to use pcDNA3, more preferably pcDNATM
pcDNATM Carrier is bicistronic mRNA cloning vector, and it is dynamic that carrier design can be used for most of lactations
In object cell, it is made to obtain the transgenosis recombinant protein of high expression.pcDNATM Carrier and TOPO isomery
Enzyme is compatible, can include the PCR product of target gene with > 85% cloning efficiency.The gene of coding clostridium septicum alpha toxin exists
There are CMV promoter in the insertion of TOPO cloning site, upstream, can promote target gene transcription；There is TKpA terminator in downstream；Purpose base
Because there are CMV forward direction combination primer binding site and TKpA reverse primer binding site in two sides, for plasmid order-checking to ensure purpose
Gene order is correct；It is amp, that is, ampicillin resistance gene that the carrier, which contains there are two resistant maker gene, one, for screening
Escherichia coli positive transformants；Another is neo, that is, neomycin resistance gene, for screening the cell strain of stable transfection Chinese hamster ovary celI.
The present invention also provides a kind of above-mentioned clostridium septicum alpha toxin mutant, above-mentioned clostridium septicum alpha toxin mutant, on
State the preparation method of the gene, above-mentioned clostridium septicum alpha toxin mutant that encode above-mentioned clostridium septicum alpha toxin mutant or by above-mentioned
Application of the albumen that clostridium septicum alpha toxin mutation preparation is prepared in following (x1)-(x5): (x1) preparation corruption
Clostridial vaccine；(x2) clostridium septicum alpha toxin antibody is prepared；(x3) reagent and/or reagent of preparation detection clostridium septicum alpha toxin
Box；(x4) reagent and/or kit of preparation detection clostridium septicum alpha toxin antibody；(x5) diagnosis of preparation clostridium septicum alpha toxin is anti-
It is former.
Since clostridium septicum alpha toxin mutant provided by the invention has no toxic side effect, while there is preferable immunogenicity,
Therefore it can be used for preventing the animals such as sheep, horse, ox, pig, dog, cat, chicken and deer by corrupt shuttle to prepare clostridium septicum vaccine
Microbial disease.The antibody and the clostridium septicum alpha toxin prepared using the clostridium septicum alpha toxin mutant can be applied to
Prepare a variety of detection reagents and kit, for example, using the antibody containing clostridium septicum alpha toxin mutant ELISA kit with
Detect clostridium septicum.
The present invention also provides a kind of clostridium septicum vaccines, include above-mentioned clostridium septicum alpha toxin mutant or above-mentioned coding
The gene of clostridium septicum alpha toxin mutant.Clostridium septicum vaccine provided by the invention can be mutated with above-mentioned clostridium septicum alpha toxin
Clostridium septicum alpha toxin subunit vaccine is made as main immunogene in body；Above-mentioned clostridium septicum alpha toxin can also be mutated
Body as one of immune composition component, with other immunogenic substances collective effects, play synergistic effect；Also
Can be include above-mentioned coding clostridium septicum alpha toxin mutant gene DNA vaccination.
Clostridium septicum vaccine prepared by the present invention is codon optimization, containing 2 amino acid sites mutation, is obtained
To the alpha toxin mutant of animal body attenuated, extraordinary safety and good immunogenicity are presented in rabbit animal body.
In some alternative embodiments, the clostridium septicum alpha toxin mutant that the clostridium septicum vaccine includes, has
The amino acid sequence as shown in SEQ ID NO.3, and the clostridium septicum alpha toxin mutant is by expressing cho cell system table
It reaches.
The vaccine is clostridium septicum alpha toxin recombinant subunit vaccine, is codon optimization, contains 2 amino acid positions
Point mutation, obtains a Toxin mutants to animal body attenuated, and mammalian expression systems being capable of efficient secretory expression recombination
A toxin protein is highly susceptible to purifying；Extraordinary safety and good immunogenicity are presented in rabbit animal body.Therefore
Be clostridium septicum toxin vaccine upgrading ideal candidates vaccine, optimize escherichia expression system presently, there are ask
Topic.
In some preferred embodiments, the concentration of clostridium septicum alpha toxin mutant is in the clostridium septicum vaccine
30-50μg/mL；Preferably 35-45 μ g/mL；More preferably 40 μ g/mL.Preferably, the vaccine further includes auxiliary material, described auxiliary
Material includes one of vaccine adjuvant, stabilizer and antibiotic or a variety of.The vaccine adjuvant for example can be but be not limited to
Aluminium hydroxide gel, Freund's complete adjuvant, incomplete Freund's adjuvant, white-oil adjuvant, MF59 adjuvant or Montanide ISA series assistant
Agent；It is preferable to use Montanide ISA series of adjuvants；More preferably use ISA 15A adjuvant.
Beneficial effects of the present invention are further illustrated below with reference to preferred embodiment.
Embodiment 1
By (artificial synthesized) the insertion pcDNA of nucleotide sequence shown in SEQ ID NO.4TM On carrier
There are CMV promoter in TOPO cloning site, upstream, and there is TKpA terminator in downstream, obtain transfection pcDNA3.3- alpha toxin plasmid,
PcDNA3.3- alpha toxin plasmid is linearized with ApaLI enzyme, and the recombinant vector after linearisation is electroporated thin to mammal
In born of the same parents CHO-S, mammalian cell recombinant cell strain CHO-S a-toxin is obtained.Above-mentioned recombinant cell strain is inoculated into CHO-
In S-SFM II culture medium.Then filter out expression clostridium septicum alpha toxin mutant cell strain, 37 DEG C, 5%CO2Condition
Lower suspension is cultivated 10 days, and supernatant is collected by centrifugation, while carrying out SDS-PAGE and WEST-BLOT detection, testing result such as Fig. 2 and figure
Shown in 3, wherein the swimming lane 2 in Fig. 2 and Fig. 3 and swimming lane 3 are the recombinant protein of clostridium septicum alpha toxin mutant, and having on figure can be with
Find out CHO-S cell strain successful expression clostridium septicum alpha toxin mutant.
Chinese hamster ovary celI recovery and passage: 1, in advance water-bath is opened 37 DEG C；Culture medium is preheated at 37 DEG C in advance.2, from liquid
The Chinese hamster ovary celI frozen is taken out in nitrogen tank.3, freeze-stored cell is put into immediately in 37 DEG C of water-baths, gentle agitation makes its fast melt
(about 1min) takes out.4, it with 75% ethanol disinfection pipe outer wall, is put into Biohazard Safety Equipment, is transferred to the culture medium containing 10mL
In 15mL centrifuge tube, it is centrifuged 800rpm, 5min.5, the cell after being centrifuged removes supernatant, takes a little fresh culture that cell is resuspended,
It is then transferred in culture bottle, fresh culture is added and gently shakes, so that cell is uniformly dispersed, takes cell count and viability examination,
Make density domination in 3-4 × 105Cells/mL, vigor > 95%.6, it is placed in 110rpm in incubator, 37 DEG C, 5%CO2Culture.7,
Cell culture 2-3 days, density reached 2.0 × 106Cell need to be passed on when cells/mL or so.8, it is taken out from culture bottle
Part culture discards, then fresh culture is added into culture bottle, and to the dilution of remaining cell culture, (cell left is trained
Object is supported depending on density etc. after cell density, volume of culture, dilution.9, it is put into 110rpm in incubator, 37 DEG C, 5%CO2,
Continue to cultivate.It 10, daily need to be to cell density and viability examination, when cell density reaches 2 × 106When cells/mL or so,
Cell is passed on.
Alpha-toxin plasmid transfection Chinese hamster ovary celI: 1, it transfects first 1 day and Chinese hamster ovary celI is suspended into culture to 300mL, be inoculated with close
Degree is 1 × 106Cells/mL, is placed in 110rpm in incubator, and 37 DEG C, 5%CO2Culture.2, transfection is when the control of angel's cell density
In 1-1.5 × 106Cells/mL or so.3, DNA and transfection reagent DNA- transfection reagent mixtures: is added into transfection buffer
It mixes, 37 DEG C of incubations.4, DNA- transfection reagent mixtures are added in cell to be transfected, are put into 110rpm in incubator, 37 DEG C,
5%CO2Culture.5, it collects: about 4-6 days after transfection, taking out cell culture, supernatant or cell are collected in centrifugation.6, supernatant passes through
Nickel column affinity purification, then by the protein quantification of acquisition.
Embodiment 2
A kind of clostridium septicum vaccine is present embodiments provided, includes the albumen that embodiment 1 provides in the vaccine, wherein albumen
Content is 30 μ g/mL, and is uniformly mixed according to antigen with the volume ratio of commodity adjuvant ISA 15A for 1:1, and vaccine is made.
Embodiment 3
A kind of clostridium septicum vaccine is present embodiments provided, includes the albumen that embodiment 1 provides in the vaccine, wherein albumen
Content is 40 μ g/mL, and is uniformly mixed according to antigen with the volume ratio of commodity adjuvant ISA 15A for 1:1, and vaccine is made.
Embodiment 4
A kind of clostridium septicum vaccine is present embodiments provided, includes the albumen that embodiment 1 provides in the vaccine, wherein albumen
Content is 50 μ g/mL, and is uniformly mixed according to antigen with the volume ratio of commodity adjuvant ISA 15A for 1:1, and vaccine is made.
Embodiment 5
A kind of clostridium septicum vaccine is present embodiments provided, includes the albumen that embodiment 1 provides in the vaccine, wherein albumen
Content is 30 μ g/mL, and is uniformly mixed according to antigen with the volume ratio of commodity adjuvant MF59 for 1:1, and vaccine is made.
Embodiment 6
A kind of clostridium septicum vaccine is present embodiments provided, includes the albumen that embodiment 1 provides in the vaccine, wherein albumen
Content is 40 μ g/mL, and is uniformly mixed according to antigen with the volume ratio of commodity adjuvant MF59 for 1:1, and vaccine is made.
Embodiment 7
A kind of clostridium septicum vaccine is present embodiments provided, includes the albumen that embodiment 1 provides in the vaccine, wherein albumen
Content is 50 μ g/mL, and is uniformly mixed according to antigen with the volume ratio of commodity adjuvant MF59 for 1:1, and vaccine is made.
Embodiment 8
A kind of clostridium septicum vaccine is present embodiments provided, includes the albumen that embodiment 1 provides in the vaccine, wherein albumen
Content is 30 μ g/mL, and is uniformly mixed according to antigen with the volume ratio of commodity adjuvant ISA206 for 1:1, and vaccine is made.
Embodiment 9
A kind of clostridium septicum vaccine is present embodiments provided, includes the albumen that embodiment 1 provides in the vaccine, wherein albumen
Content is 40 μ g/mL, and is uniformly mixed according to antigen with the volume ratio of commodity adjuvant ISA206 for 1:1, and vaccine is made.
Embodiment 10
A kind of clostridium septicum vaccine is present embodiments provided, includes the albumen that embodiment 1 provides in the vaccine, wherein albumen
Content is 50 μ g/mL, and is uniformly mixed according to antigen with the volume ratio of commodity adjuvant ISA206 for 1:1, and vaccine is made.
Effect example 1
Safety verification weight 1.5-2.0kg healthy rabbits 45, are divided into 9 groups, and every group 5, the muscle of each group rabbit
Or the vaccine 2.0ml that subcutaneous injection embodiment 2- embodiment 10 provides, observe 10, it is all strong to live, and injection site muscle or
Skin is without necrosis.
Effect example 2
The effect for the vaccine for examining embodiment 2- embodiment 9 to provide, every group of experiment are as follows:
With weight 1.5-2.0kg healthy rabbits 4, each neck subcutaneously or intramuscularly vaccinates 1.0ml.14-21 after inoculation
Day, blood sampling separates serum.By the serum mixed in equal amounts of 4 immunizing rabbits, pooled serum 0.4ml is taken (to contain with clostridium septicum toxin
4 mouse MLD), 37 DEG C of effect 40min of postposition are mixed, are then injected intravenously 16-20g mouse 2,0.3ml/ is only.It is each simultaneously to use
With criticizing mouse 2,1MLD toxin identical with toxin serum mixture is injected respectively and is compared.Observation 1 day determines result.It is right
All dead according to mouse, serum neutralization titer reaches 1 (in 0.1ml immune serum and 1MLD toxin) to clostridium septicum toxin,
It is judged to qualification.Efficacy test: in the vaccine that each embodiment provides, serum neutralization titer reaches 1 to clostridium septicum toxin, vaccine
Effectively.Efficacy results show that adjuvant is that IS15A effect is best.The results are shown in Table 1.
The efficacy results for the vaccine that each embodiment of table 1 provides
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: its according to
So be possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features into
Row equivalent replacement；And these are modified or replaceed, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution
The range of scheme.
Sequence table
<110>Tian Kang Biological Co., Ltd.
<120>clostridium septicum alpha toxin mutant, the gene for expressing it, preparation method and clostridium septicum vaccine
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 440
<212> PRT
<213>clostridium septicum (Clostridium septium)
<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 septicum (Clostridium septium)
<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 (10)
1. a kind of clostridium septicum alpha toxin mutant, which is characterized in that including any one in following (a1)-(a3):
(a1) by the 86th amino acids residue of clostridium septicum alpha toxin with the amino acid sequence as shown in SEQ ID NO.1 by half
Cystine sports the protein that leucine and the 189th amino acids residue are obtained by mutant serine for cysteine；
(a2) protein that the signal peptide of (a1) obtains is cut off；
(a3) fusion protein that the N-terminal of the protein shown in (a1) or (a2) and/or C-terminal connection label obtain.
2. clostridium septicum alpha toxin mutant according to claim 1, which is characterized in that the clostridium septicum alpha toxin mutation
Body has the amino acid sequence as shown in SEQ ID NO.3.
3. encoding the gene of clostridium septicum alpha toxin mutant as claimed in claim 1 or 2.
4. the gene of clostridium septicum alpha toxin mutant according to claim 3, which is characterized in that have such as SEQ ID
Nucleotide sequence shown in NO.4.
5. a kind of preparation method of clostridium septicum alpha toxin mutant of any of claims 1 or 2 characterized by comprising will
The gene of the clostridium septicum alpha toxin mutant is encoded in host cell expression.
6. preparation method according to claim 5 characterized by comprising compiled using mammalian expression systems expression
The gene of the code clostridium septicum alpha toxin mutant；
Preferably, using the gene of clostridium septicum alpha toxin mutant described in expressing cho cell system expression；Chinese hamster ovary celI is preferred
CHO-S cell line.
7. preparation method according to claim 5 or 6, which is characterized in that the coding clostridium septicum alpha toxin is dashed forward
The gene cloning of variant is then introduced into host cell to expression vector, then screens and cultivate the expression clostridium septicum alpha toxin and dash forward
The host cell of variant, by the albumen of host cell expression it is purified after obtain the clostridium septicum alpha toxin mutant；
Preferably, the expression vector includes pcDNA3, preferably pcDNATM3.3-
8. gene, claim 5- described in clostridium septicum alpha toxin mutant of any of claims 1 or 2, claim 3 or 4
The clostridium septicum α that the preparation method or the preparation method of clostridium septicum alpha toxin mutant described in any one of 7 are prepared
Application of the Toxin mutants in following (x1)-(x5)；
(x1) clostridium septicum vaccine is prepared；
(x2) clostridium septicum alpha toxin antibody is prepared；
(x3) reagent and/or kit of preparation detection clostridium septicum alpha toxin；
(x4) reagent and/or kit of preparation detection clostridium septicum alpha toxin antibody；
(x5) clostridium septicum alpha toxin diagnostic antigen is prepared.
9. a kind of clostridium septicum vaccine, which is characterized in that comprising clostridium septicum alpha toxin mutant of any of claims 1 or 2 or
Gene described in claim 3 or 4.
10. clostridium septicum vaccine according to claim 9, which is characterized in that the clostridium septicum vaccine includes corrupt shuttle
Bacterium alpha toxin mutant；The clostridium septicum alpha toxin mutant has the amino acid sequence as shown in SEQ IDNO.3；The corruption
Perfringens alpha toxin mutant is lost to express to obtain by mammalian expression systems；
Preferably, the concentration of clostridium septicum alpha toxin mutant is 30-50 μ g/mL in the clostridium septicum vaccine；Preferably 35-
45μg/mL；More preferably 40 μ g/mL；
Preferably, the vaccine further includes auxiliary material, and the auxiliary material includes one of vaccine adjuvant, stabilizer and antibiotic or more
Kind；
Preferably, the vaccine adjuvant include aluminium hydroxide gel, Freund's complete adjuvant, incomplete Freund's adjuvant, white-oil adjuvant,
MF59 adjuvant or Montanide ISA series of adjuvants；It is preferable to use Montanide ISA series of adjuvants；More preferably use ISA
15A adjuvant.
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| 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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