CN110075288A - A kind of nontoxic c-type clostridium botulinum genetic engineering subunit vaccine and its production method - Google Patents
A kind of nontoxic c-type clostridium botulinum genetic engineering subunit vaccine and its production method Download PDFInfo
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Abstract
The present invention relates to a kind of nontoxic c-type clostridium botulinum genetic engineering subunit vaccine and its production methods, nontoxicity c-type clostridium botulinum subunit vaccine prepared by the present invention is the nontoxic region H using codon optimization, clostridium botulinum toxin (BoNTs) heavy chain for containing malt glycoprotein (MBP) labelC(BoHC) recombinant protein (rMBP-BoHC) production, i.e., guarantee the safety of vaccine to the maximum extent, and keep its validity.Meanwhile the antigen for going back vaccine can be expressed in the form of soluble, therefore have many advantages, such as that preparation process is simple, immunizing dose is low, immune efficacy is good, be the ideal candidates vaccine of the existing c-type clostridium botulinum vaccine upgrading in China.
Description
Technical field
The present invention relates to a kind of nontoxic c-type clostridium botulinum genetic engineering subunit vaccine and its production methods.Belong to for animals
Field of biological product.
Background technique
Clostridium botulinum (Clostridium botulinum) poisoning is one kind by clostridium botulinum toxin (Clostridium
Botulinum Neurotoxin, BoNTs) caused by fatal disease very serious, not only seriously endanger human health,
And weight huge economic loss is caused to animal husbandry.At present it is known that clostridium botulinum share 7 serotypes of A-G.Wherein, right
Maximum animal husbandry harm is c-type clostridium botulinum.There is livestock meat in many countries and regions, including Brazil, Europe and North America
The report of bacillus venenosus poisoning, and the death rate may be up to 99.92% or more (Gil L A F, Cunha C, Moreira
G,et al.Production and Evaluation of a Recombinant Chimeric Vaccine against
Clostridium botulinum Neurotoxin Types C and D[J].PLoS ONE,2013,8(7):
E69692.), this not only causes huge economic loss, but also is even more global public safety problem.
Since botulism has the characteristics that morbidity is anxious, the course of disease is short and the death rate is high, once morbidity, comes toward contact
It just dies suddenly because exotoxin is poisoned not as good as treatment, therefore immunity inoculation is the effective ways of the prevention and control disease.Current commodity
It is prepared by the natural toxin (toxoid) and standard adjuvant by inactivating for changing vaccine.Such vaccine is preventing caused by animal clostridium botulinum
Although achieving certain effect in terms of disease, this vaccine still exposes some defects, such as epidemic disease in use
Seedling is immune easily to cause animal local inflammation and toxic reaction；The toxin amount generated in vitro is unstable, and is related to during the preparation process
BoNTs (strongest biotoxin Hakami R M, Ruthel G, Stahl A M, et al.Gaining known to nature
ground:assays for therapeutics against botulinum neurotoxin[J].Trends in
Microbiology, 2010,18 (4): 164-172) inactivation, leak or inactivate that the bio-safeties such as to be not thorough hidden there are toxin
Suffer from.Therefore, the clostridium botulinum genetic engineering subunit vaccine that safety is good, Effective Antigens content is high, immunogenicity is strong is developed, is
Following developing direction.
The precursor toxin of clostridium botulinum toxin (BoNTs) is made of single polypeptide chain, living after the protease cutting of itself
Change, the toxin of activated form is made of two chains of light chain (L) and heavy chain (H).Wherein, light chain is a kind of metal-dependant albumen
Enzyme passes through N-terminal (the abbreviation H with heavy chainN) connection, play enzymatic activity.C-terminal (the abbreviation BoH of heavy chainC) it is toxin and nerve
First receptorbinding region, while being also a critically important nontoxic region (Ravichandran for possessing protective epitope
E,Al-Saleem FH,Ancharski DM,Elias MD,Singh AK et al.(2007)Trivalent vaccine
against botulinum toxin serotypes A,B,and E that can be administered by the
mucosal route.Infect Immun 75:3043-3054).It is existing the study found that in Escherichia coli or Pichia pastoris
The recombination BoH of middle generationCThe generation of neutralizing antibody can be induced, and prevent botulism (Gil L A F, Cunha C,
Moreira G,et al.Production and Evaluation of a Recombinant Chimeric Vaccine
against Clostridium botulinum Neurotoxin Types C and D[J].PLoS ONE,2013,8(7):
e69692.).However, the H recombinated aboveCUsually with the presence of insoluble inclusion bodies, carry out at denaturation and renaturation
Reason.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 difficult
It improves, this is to limit the principal element of its application, and can overcome the problems, such as this well using solubility expression mode.For this purpose,
How to construct solubility expression carrier and optimize the high-efficiency expression method of soluble protein, is that this field is ground always for a long time
The hot subject studied carefully.
Summary of the invention
Present invention aims at nontoxic c-type clostridium botulinum genetic engineering subunit vaccine is prepared, for preventing by c-type meat
Disease caused by malicious clostridium infects.
Technical solution of the present invention
1. a kind of nontoxic c-type clostridium botulinum genetic engineering subunit vaccine, it is characterised in that the vaccine contains using big
Recombination clostridium botulinum toxin (the rMBP-BoH that enterobacteria is expressed as host cell and in cytoplasmC)；The recombinant toxin is
By recombinantly expressing rMBP-BoHCEscherichia coli (Escherichia coli) BL21 (DE3) cell as production bacterial strain system
Standby, which is named as BL/BoM plants of escherichia coli, and delivers Chaoyang District, Beijing City on 03 08th, 2019
The culture presevation administration committee, China, Institute of Microorganism, Academia Sinica, institute 3 of North Star West Road 1 common micro-organisms center is protected
Hiding number: CGMCC No.17317；
The vaccine infects associated diseases by c-type clostridium botulinum for preventing.
2. a kind of nontoxic c-type clostridium botulinum genetic engineering subunit vaccine of the present invention, it is characterised in that the expression
RMBP-BoHC, which contains only the nontoxic of heavy chain compared with mature wild type Clostridium botulinum toxin (BoNTs)
Region, the end C- (abbreviation BoHC)；
The rMBP-BoHCAvirulence greatly reduces the bio-safety risk in vaccine production process.
3. a kind of nontoxic c-type clostridium botulinum genetic engineering subunit vaccine of the present invention, it is characterised in that the vaccine
In the rMBP-BoH that containsC, it is that BoH is realized as addition hydrotropy label by malt glycoprotein (MBP)CSolubility expression.
4. a kind of nontoxic c-type clostridium botulinum genetic engineering subunit vaccine of the present invention, it is characterised in that the expression
RMBP-BoHC, the gene order codon optimization of coding, it is easier to high expression and solvable is realized in Escherichia coli
Expression.
5. a kind of nontoxic c-type clostridium botulinum genetic engineering subunit vaccine of the present invention, it is characterised in that the expression
RMBP-BoHC, containing the label for being convenient for protein purification, preferably C-terminal contains 6 histidine (6*His) labels.
6. a kind of non-toxic c-type clostridium botulinum genetic engineering subunit vaccine of the present invention, it is characterised in that it is prepared
Method are as follows: pass through host cell expression rMBP-BoHC, with the expression rMBP-BoHCBL/BoM plants of conducts of Escherichia coli
Production of vaccine bacterial strain after fermented culture, inducing expression, bacterial cell disruption, soluble antigen separation and purification of protein, adds two-phase oil cream
Adjuvant is mixed.
7. a kind of nontoxic c-type clostridium botulinum genetic engineering subunit vaccine of the present invention, it is characterised in that for expressing
rMBP-BoHC" host cell ", " host cell " covers prokaryotic cell and/or eukaryocyte, can be production embrace a sub- shuttle
It is bacterium, C.perfringens, clostridium acetobutylicum, Bacillus cereus, Su Yunjin bud pole bacterium, Cao's shape bud pole bacterium, thermophilic molten
Proteins Spore bacillus, anthrax bud pole bacterium, Bacillus megaterium, bacillus subtilis, Escherichia coli or yeast cells；
The host cell is preferably e. coli host cell, especially e. coli bl21 (DE3) or Rosetta
(DE3)。
Beneficial effect of the present invention
Nontoxic c-type clostridium botulinum genetic engineering subunit vaccine prepared by the present invention is using codon optimization, contains
There is the nontoxic region H of clostridium botulinum toxin (BoNTs) of malt glycoprotein (MBP) labelC(BoHC) recombination clostridium botulinum toxin
(rMBP-BoHC) production, i.e., guarantee the safety of vaccine to the maximum extent, and keep its validity.Meanwhile going back the antigen of vaccine
It can be expressed in the form of soluble, therefore have many advantages, such as that preparation process is simple, immunizing dose is low, immune efficacy is good, be China
The ideal candidates vaccine of existing c-type clostridium botulinum vaccine upgrading.
1. for the first time by BoHCWith malt glycoprotein (MBP) amalgamation and expression, the solubility expression in Escherichia coli is realized,
Obtain the excellent recombination clostridium botulinum toxin rMBP-BoH of immunogenicityC, so as to avoid the cumbersome of inclusion body denaturation renaturation
Influence of the technique to antigen protein immunogenic reduces the preparation time and production cost of vaccine.
2. BoH will be encoded for the first timeCIt is carried out with the gene order of hydrotropy label protein malt glycoprotein (MBP) fusion protein close
Numeral optimization, it is easier to high efficient expression is realized in Escherichia coli.
It goes out again 3. preparing subunit vaccine using gene engineering method and substituting the pathogenic clostridium of traditional culture and prepare toxin
The method of detoxification living, greatly reduces the bio-safety risk in production process.
Microbial resources information of the present invention
Microorganism of the present invention is recombinant expression rMBP-BoHCEscherichia coli, which is named as expressing
rMBP-BoHCBL/BoM plants of escherichia coli, and delivered BeiChen West Road, Chaoyang District, BeiJing City 1 on 03 08th, 2019
Number No. 3 culture presevation administration committee, China, Institute of Microorganism, Academia Sinica common micro-organisms centers of institute, deposit number:
CGMCC No.17317；The vaccine is for preventing the botulism disease as caused by c-type clostridium botulinum.
Detailed description of the invention
Fig. 1: rMBP-BoHCM in the SDS-PAGE qualification result figure of expression1:Protein marker；1:BSA(1μg)；2:
BSA(2μg)；3: empty carrier cell lysate；4:15 DEG C, 16h induction cell lysate；5:37 DEG C, 4h induction cell split
Solve object；6: empty carrier cell cracking supernatant；7:15 DEG C, 16h induction cell cracking supernatant；8:37 DEG C, 4h induction cell split
Solve supernatant；9: empty carrier cell cracking precipitating；10:15 DEG C, 16h induction cell cracking precipitating；11:37 DEG C, 4h induction it is thin
Cellular lysate precipitating.
Fig. 2: rMBP-BoHCIn the Western blot qualification result figure of expression: M2:Western blot marker；1:
Empty carrier cell lysate；2:15 DEG C, 16h induction cell lysate；3:37 DEG C, 4h induction cell lysate；4:15℃,
The cell cracking supernatant of 16h induction；5:37 DEG C, 4h induction cell cracking supernatant；6:15 DEG C, the cell cracking of 16h induction it is heavy
It forms sediment；7:37 DEG C, 4h induction cell cracking precipitating.
Fig. 3: rMBP-BoHCPurifying figure in M1: albumen marker；The cell pyrolysis liquid of 1:15 DEG C of induction；2:15 DEG C lures
The cell cracking supernatant led；3: flowing through liquid；4: eluent；The eluent of 5:20mM Imidazole；6:50mM Imidazole
Eluent；The eluent of 7:300mM Imidazole.
The specific embodiment of the invention
1. recombinating clostridium botulinum toxin (rMBP-BoHC)
(1) term " MBP " means malt glycoprotein, which can be used as the use of hydrotropy label, " BoHC" mean meat poisoning shuttle
The C-terminal of verticillium toxin (BoNTs) heavy chain, the region be one of clostridium botulinum toxin it is critically important possess protective epitope
Nontoxic region."BoHC" also cover one or more clostridium botulinum toxins for modifying (including chemical modification and gene modification)
Heavy chain C-terminal.Term " gene modification " means missing, displacement or adds one or more amino acids basics.
(2) method of the invention can be used to generate any clostridium botulinum toxin or derivatives thereof, including such as meat poisoning shuttle
The polypeptide and its derivative of verticillium toxin (BoNTs) amino acid sequence SEQ ID No.1 (GenBank number: ABV57469.1).Art
Language " derivative " includes amino acid mutation such as addition, displacement, missing or truncates one or more amino acid residues.
rMBP-BoHCIt may include polypeptide sequence, the polypeptide sequence is and clostridium botulinum toxin mentioned above
(BoNTs) sequence has the homologue or derivative of at least 50% sequence identity.
rMBP-BoHCIt may include the derivative in SEQ ID No.1.Wherein the derivative has one or more points
Mutation and/or one or more additional amino acid residues.On the other hand, the recombination clostridium botulinum toxin may include
Polypeptide sequence, the sequence of the polypeptide sequence and SEQ ID No.1 has at least 30%, at least 50%, at least 60%, at least
70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%
Or bigger sequence identity.
Term used herein " sequence identity " refers to same between determining reference amino acid sequence and search sequence
Property, wherein sequence is so compared, to obtain the matching of highest rank, and calculating can be used in the sequence identity
The public technology of code or method calculate in machine program (such as BLASTP, BLASTN, FASTA).Homogeneity percentage value can
To be calculated within the scope of complete amino acid sequence or in some region of amino acid sequence.
(3)rMBP-BoHCAdditional amino acid residue can be contained in N-terminal or C-terminal or in interior location.Additional
One or more proteolytic cleavage sites can be distributed in amino acid residue with side.Additional amino acid sequence, which can be used as, to be examined
Mark label play a role and/or allow in conjunction with solid support, and example is His label.
(4) rMBP-BoH is encodedCNucleic acid molecules optionally can include regulating element according to this patent.As herein
Term " regulating element " used refers to the regulating element of gene expression, including transcription and translation, and including element such as TATA frame,
Promoter, enhancer, ribosome bind site etc..The regulating element may include one or more homologous regulating elements and/
Heterologous regulatory element." homologous regulating element " is the gene expression being related in the wild-type cell nucleic acid molecule or polypeptide
Regulating element." heterologous regulatory element " is the tune not being related in the gene expression of the wild-type cell nucleic acid molecule or polypeptide
Save element.In addition it is also possible to using the regulating element of inducible expression, such as inducible promoter.
Encode the rMBP-BoHCNucleic acid molecules can be designed to be conducive in host cell, particularly bacterial host
Cell is preferably high level expression in Bacillus coli cells, such as carries out codon optimization according to escherichia expression system.
On the other hand, the present invention can be used arbitrarily comprising encoding the rMBP-BoHCNucleic acid molecules expression vector, carrier can fit
In expressing the recombination fusion protein in vitro and/or in vivo.The carrier can be for instantaneous and/or stable gene table
The carrier reached can additionally comprise regulating element and/or selected marker, can be viral source, bacteriophage source or bacterium
Source.For example, the expression vector can be pET28a carrier.
Encode the rMBP-BoHCNucleic acid molecules or expression vector can pass through host cell expression.As used herein,
Term " host cell " covers the prokaryotic cell and/or eukaryocyte for being suitble to translate the nucleic acid molecules or the carrier.It is described
Host cell not only includes the not host cell of table clostridium botulinum toxin or its homologue, be also covered by expression clostridium botulinum toxin or
The host cell of homologue, such as wild type.As used herein, term " host cell " can be production and embrace sub- clostridium, perfringens
Clostridium, clostridium acetobutylicum, Bacillus cereus, Su Yunjin bud pole bacterium, Cao's shape bud pole bacterium, thermophilic molten proteins Spore bar
Bacterium, anthrax bud pole bacterium, Bacillus megaterium, bacillus subtilis, Escherichia coli or yeast cells.Preferably, host cell
It is e. coli host cell, especially e. coli bl21 (DE3) or Rosetta (DE3).The present invention expression
rMBP-BoHCEscherichia coli BL21 (DE3), be named as BL/BoM plants.
2. the building that BL/BoM plants of escherichia coli, expression and identification
(1) synthesis of the gene of coding recombination clostridium botulinum toxin: according to malt glycoprotein (MBP) and clostridium botulinum toxin
Nontoxic region HCNatural coding gene sequence (sequence 1), connect after codon optimization, and the 3 ' of tandem gene
End addition purifies amino acid label coded sequence used.This section of gene order (sequence 2) is synthesized with chemically synthesized method.
(2) building of clostridium botulinum toxin expression vector is recombinated
Using artificial synthesized gene as template, the target DNA band that PCR amplification arrives, warp are carried out using the primer pair of design
After recycling, with prokaryotic expression carrier simultaneously using being attached after double digested, the prokaryotic expression for obtaining inserting target gene is carried
Body.By the plasmid connected convert DH5 α competent cell, picking monoclonal into LB liquid medium containing kanamycin,
37 DEG C of shaken cultivations are stayed overnight, and through PCR and sequencing, finally determine positive strain, it is spare to extract plasmid.
(3) building of the engineering strain of expression recombination clostridium botulinum toxin
Obtained plasmid will be extracted, will be transformed into e. coli bl21 (DE3) competent cell, picking monoclonal is to containing
In the LB liquid medium of kanamycins, 37 DEG C of shaken cultivations are stayed overnight, and are identified through PCR, after target DNA fragment, are sent Sino-U.S.
Calm and peaceful company's sequencing, sequence is correctly determined as positive strain, and 50% isometric glycerol LB is added, and -70 DEG C freeze.
(4) expression and identification of clostridium botulinum toxin are recombinated
BL/BoM plants of escherichia coli are inoculated in 4mL LB liquid medium containing kanamycin, is placed in 37 DEG C
Shaken cultivation works as OD600When being 0.6~0.8, it is added after the IPTG solution of final concentration of 0.5mM and is respectively placed in 37 DEG C and 15 DEG C and lures
Lead culture 4h and 16h.Thalline were collected by centrifugation after the completion of bacterium solution culture, by every gram of thallus Wen Chongjia 10mL lysate [0.02mol/L
Tris buffer (pH value 7.2), 0.3mol/L NaCl] ratio be resuspended thallus, the ultrasonication thallus 15min in ice-water bath,
Broken condition are as follows: work 9s, interval 9s, ultrasonic power 400W.By broken bacterium solution in 4 DEG C, it is centrifuged with 12000r/min
10min collects supernatant.Take 30 μ L supernatants that 4 × SDS-PAGE sample-loading buffer of 10 μ L is added, 70 DEG C of effect 10min are carried out
12%SDS-PAGE electrophoresis, meanwhile, using anti-His antibody, Western blot identification is carried out to it, it is final to determine expression most
Optimal conditions.
2. recombinating the purifying of clostridium botulinum toxin
Fermented and cultured in 1L LB liquid medium containing kanamycin is inoculated in by BL/BoM plants of escherichia coli, 37
DEG C shaken cultivation OD600When being 0.6~0.8, according to the optimal expression condition of above-mentioned determination, carries out inducing expression and collect thallus
It is purified.
3. the preparation of non-toxic c-type clostridium botulinum genetic engineering subunit vaccine
(1) strain: seedling BL/BoM plants of escherichia coli that strain is expression recombination clostridium botulinum toxin.
(2) first order seed breeding and identification: a small amount of LB liquid medium of freeze-drying lactobacillus is dissolved, streak inoculation is in containing card
The LB solid plate of that mycin is set 37 DEG C and is cultivated 12~16 hours, and standard compliant single bacterium colony is chosen, and inoculation contains kanamycins
LB liquid medium, set 37 DEG C cultivate 8~12 hours, dispensed after being mixed with 50% glycerol equal proportion, set -25 DEG C or less guarantors
It deposits, through purely after the assay was approved, as seedling first order seed.
(3) secondary seed breeding and identification: taking first order seed, is inoculated with the LB Liquid Culture containing kanamycins by 1% amount
Base, sets 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 liquid medium, 37 DEG C of shaken cultivation OD600When being 0.6~0.8, according to the optimal expression condition of above-mentioned determination, induced
It expresses and collects thallus and purified.
(5) it breaks bacterium: thallus being collected with 5000r/min centrifugation 5min, adds 10ml lysate (pH value by every gram of thallus weight in wet base
7.2 0.02mol/L Tris buffers, 0.3mol/L NaCl) ratio thallus is resuspended, cryogenic high pressure homogeneous is used under the conditions of 4 DEG C
Machine is with pressure breaking thallus 3 times of 800bar.Lysate is centrifuged 30min through 4 DEG C of 10000r/min, collects supernatant.
(6) it purifies: according to the operation instructions of Ni-IDA affinity chromatography medium kit (Nanjing Jin Sirui), thallus being split
It is purified in solution supernatant in the destination protein of solubility expression, through 0.22 μm of aperture membrane filtration, -80 DEG C of preservations are standby.
(7) protein content detect: with BCA method detection protein content (PierceTM BCA Protein Assay Kit,
TG268883)。
(8) steriling test: by existing " Chinese veterinary pharmacopoeia " (the Chinese veterinary pharmacopoeia committee, Republic of China Veterinary Pharmacopoeia,
Two 〇 mono-
5 years versions three, Chinese agriculture publishing house, 2016, hereinafter referred to as " Chinese veterinary pharmacopoeia ") annex progress.Answer sterile life
It is long.
(9) vaccine formulation: two-phase oil adjuvant (such as 206 adjuvants) are imported in oily phase tank, at least 121 DEG C of high pressure sterilizations
30min is cooled to room temperature spare.According to determining the protein quantity as a result, qualification will be examined with PBS (7.2 0.01mol/L of pH value)
Purifying protein suitably dilute after mix.Water phase is added in emulsion tank, with 80~100r/min stirring, while pressing 1:1 (V/V)
Ratio, be slowly added to oily phase, after adding stir 20~30min.Sampling is tested after emulsification, is dispensed after qualified, final to make
It is the vaccine of 50 μ g/mL and 100 μ g/mL at concentration.Meanwhile being mixed with same dose of PBS and adjuvant, as control vaccine.
4. the inspection of nontoxic c-type clostridium botulinum genetic engineering subunit vaccine
(1) character
Appearance should be milky emulsion.
Dosage form 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,
It should be spread in cloud.
Stability is drawn vaccine 10mL and is added in centrifuge tube, is centrifuged 15min with 3000r/min, should not be demulsified, and manage
The water phase that bottom is precipitated should be not more than 0.5mL.
By " Chinese veterinary pharmacopoeia ", (annex is measured viscosity, should meet regulation.
(2) steriling test is tested by " Chinese veterinary pharmacopoeia " annex, answers asepsis growth.
(3) safety verification 1.5~2.0kg of weight healthy rabbits 4, each muscle or the vaccine that 50 μ g/mL are subcutaneously injected
4.0mL observes 10d.Work should be all good for.
(4) efficacy test
(1) serum neutralisation
The experimental animal for being 0 to c-type clostridium botulinum toxin serum neutralization titer is selected, specific as follows: weight 1.5~
Sheep similar in 2.0kg healthy rabbits and 1~3 years old weight, each neck subcutaneously or intramuscularly vaccinate the vaccine of optimal dose, often
4 rabbit of vaccine inoculation or sheep of kind dosage.21d after inoculation, blood sampling, separation serum is spare, and carries out second and be immunized.Two
Exempt from rear 14d, take a blood sample, separation serum is spare.Take every animal blood serum 0.4mL and 0.8mL c-type clostridium botulinum toxin (containing 4 respectively
A and above mouse MLD), 37 DEG C of effect 40min of postposition are mixed, are then injected intravenously 16~18g mouse 2,0.3mL/ is only.Together
When respectively compared with batches mouse 2, injecting 1MLD toxin identical with toxin serum mixture respectively.1d is observed, determines knot
Fruit.
It is all dead to compare mouse, serum neutralization titer reaches 1 or more to c-type clostridium botulinum toxin, and (animal is immunized in 0.1mL
In serum and 1 and the above MLD toxin), that is, be judged to qualification.
(2) Immunization method 1.5~2.0kg of weight healthy rabbits 6, take its 4, each neck is subcutaneously or intramuscularly injected
The vaccine 2.0mL of 50 μ g/mL, remaining 2 injection control vaccines.21d after inoculation, blood sampling, separation serum is spare, and carries out second
It is secondary immune.After second of immune 14d, 4 immune group rabbit and 2 control group rabbit are taken, are injected intravenously the c-type of 10MLD respectively
Clostridium botulinum toxin observes 5d.
It is all dead to compare rabbit, immune animal at least protects 3, that is, is judged to qualification.
Embodiment
Following embodiment is more preferably to illustrate technical solution of the present invention, but be not construed as limiting to technical solution of the present invention.
Embodiment 1
--- building, expression and the identification that BL/BoM plants of escherichia coli
1. encoding rMBP-BoHCGene synthesis
The application is according to the nontoxic region H according to malt glycoprotein (MBP) and clostridium botulinum toxinC(BoHC) natural volume
Code gene order (sequence 1), connects after codon optimization, adds 6 × His label used at 3 ' ends of tandem gene
Coded sequence.With chemically synthesized method, this section of gene order, GMBPBoH have been synthesizedC.Specific nucleic acid sequence such as SEQ ID
Shown in No.2, amino acid sequence is as shown in SEQ ID No.3.
Sequence 1 (clostridium botulinum toxin heavy chain amino acid sequence)
(the coding recombination clostridium botulinum toxin (rMBP-BoH of sequence 2C) gene nucleotide series)
(recombination clostridium botulinum toxin (the rMBP-BoH of sequence 3C) amino acid sequence)
2. recombinating the building of clostridium botulinum toxin expression vector
With artificial synthesized GMBPHCFor template, PCR amplification is carried out using primer pair 1F/1R (4/ sequence 5 of sequence).
Wherein upstream primer 1F sequence are as follows:
5 '-ccgtctagag gtaccaaaac tgaag-3 ' 25 (sequence 4), 5 ' ends introduce restriction enzyme Xba I
Site and protectiveness base；
Downstream primer 1R sequence are as follows:
5 '-ccgctcgagt tagtggtgat gatg-3 ' 24 (sequence 5), 5 ' ends introduce restriction enzyme Xho I
Site and protectiveness base.
PCR system are as follows:Buffer (Mg2+plus) 10 μ L, dNTPs 4 μ L, each 1 μ of upstream and downstream primer
L,1 μ L of HS polymerase, 2 μ L of DNA profiling supplement ddH2O is to 50 μ L systems.PCR reaction condition are as follows: 98 DEG C pre-
It is denaturalized 1min；98 DEG C of denaturation 10s, 56 DEG C of annealing 30s, 72 DEG C of extension 2min20s, totally 33 recycle；Last 72 DEG C of extensions
10min。
Expand obtained target DNA band, double digested using I/Xho of Xba I after recovered, digestion identical as process
The pET28a carrier of digestion connects, and the plasmid connected is converted Top10 competent cell, that is mould to card is contained for picking monoclonal
In the LB liquid medium of element, 37 DEG C of shaken cultivations are stayed overnight, and are extracted plasmid and are carried out PCR and double digestion identification, qualification result is sun
Property plasmid send the sequencing of calm and peaceful company, Sino-U.S., double digestion result and the correct plasmid of sequencing result are named as pET28a-
GMBPBoHC.The plasmid connected is converted into DH5 α competent cell, picking monoclonal to LB liquid containing kanamycin is trained
It supports in base, 37 DEG C of shaken cultivations are stayed overnight, and it is spare to extract plasmid.
3. expressing rMBP-BoHCEngineering strain building
Obtained plasmid will be extracted, will be transformed into e. coli bl21 (DE3) competent cell, picking monoclonal is to containing
In the LB liquid medium of kanamycins, 37 DEG C of shaken cultivations are stayed overnight, and are identified through PCR, after target DNA fragment, are named as
BL/BoM plants of escherichia coli, and 50% isometric glycerol LB is added, -70 DEG C freeze, and the bacterial strain is in 03 month 2019
Deliver within 08th China, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica culture presevation management committee
Member's meeting common micro-organisms center, deposit number: CGMCC No.17317.
Embodiment 2
——rMBP-BoHCExpression and identification
1.rMBP-BoHCExpression be inoculated in 3mL for BL/BoM plants of genetic engineering bacterium escherichia coli and contain card that is mould
In the LB liquid medium of element, 37 DEG C of shaken cultivations are placed in, OD is worked as600When being 0.6~0.8, it is added final concentration of 0.5mM's
IPTG solution is respectively placed in 37 DEG C and 15 DEG C of Fiber differentiations 4h and 16h.Thalline were collected by centrifugation after the completion of bacterium solution culture, by every gram of bacterium
Body temperature adds the ratio of 10mL lysate [0.02mol/L Tris buffer (pH value 7.2), 0.3mol/L NaCl] that bacterium is resuspended again
Body, ultrasonication thallus 30min, broken condition in ice-water bath are as follows: work 9s, interval 9s, ultrasonic power 400W.It will be crushed
Bacterium solution afterwards is centrifuged 10min in 4 DEG C, with 12000r/min, collects supernatant.Take 30 μ L supernatants that 4 × SDS-PAGE of 10 μ L is added
Sample-loading buffer, 70 DEG C of effect 10min carry out 12%SDS-PAGE electrophoresis, the result is shown in Figure 1.It will be seen from figure 1 that at 15 DEG C
Under conditions of 37 DEG C, rMBP-BoHC has expression, and the ratio of the solubility expression under 15 DEG C of inductive conditions is higher, gray scale
Scanning the result shows that, the rMBP-BoH expressed under this conditionCSolvable sex ratio reaches 50% or so.Therefore, we determined that
rMBP-BoHCMost suitable inducing expression condition be 15 DEG C, inducing expression 16h.
2.rMBP-BoHCWestern blot identification take in above-mentioned steps the rMBP-BoHC under different inductive conditions, adopt
With anti-His antibody, Western blot identification is carried out to it, as a result sees Fig. 2.As can be seen from Figure 2, the identification knot of Western blot
Fruit is consistent with PAGE gel electrophoresis result, due to being in the destination protein of solubility expression, space structure and wild type poison
Element is closest.Therefore, comprehensive SDS-PAGE and Western blot qualification result, we further determine that destination protein most
Suitable inducing expression condition is 15 DEG C, inducing expression 16h.
Embodiment 3
——rMBP-BoHCPurifying
BL/BoM plants of genetic engineering bacterium escherichia coli are inoculated in 1L LB liquid medium containing kanamycin
Fermented and cultured, 37 DEG C of shaken cultivations to OD600When being 0.6~0.8, temperature is adjusted to 15 DEG C, is added final concentration of 0.5mM's
IPTG solution Fiber differentiation 4h.Thalline were collected by centrifugation after the completion of bacterium solution culture, collects thallus with 5000r/min centrifugation 5min, presses
Every gram of thallus weight in wet base adds the ratio weight of 10ml lysate (7.2 0.02mol/L Tris buffer of pH value, 0.3mol/L NaCl)
Thallus is hanged, uses cryogenic high pressure homogenizer with pressure breaking thallus 3 times of 800bar under the conditions of 4 DEG C.Lysate is through 4 DEG C of 10000r/
Min is centrifuged 30min, supernatant is collected, according to the operation instructions of Ni-IDA affinity chromatography medium kit, on cellular lysate
It is purified in clear in solubility expression rMBP-BoHC, through 0.22 μm of aperture membrane filtration, the as rMBP- of preliminary purification
BoHC。
Embodiment 4
——rMBP-BoHCMeasurement to mouse virulence
According to " Republic of China Veterinary Pharmacopoeia " (version in 2015) three, (the Chinese veterinary pharmacopoeia committee, the Chinese people are total
With state's veterinary drug allusion quotation, two 〇 First Five-Year Plans years version three, Chinese agriculture publishing house, 2016, hereinafter referred to as " Chinese veterinary pharmacopoeia ") in regulation,
The method of tail vein injection is selected to detect toxin fusion protein to the virulence of mouse.Meanwhile stomach and liver meat enzymic digestion soup is set and is made
It is negative control and Clostridium botulinum culture supernatant as positive control.The results show that working as rMBP-BoHCDosage of inoculation reach
When 100 μ g, all mouse are strong living and have no adverse reaction, and the Clostridium botulinum culture supernatant of 0.02 μ L can lead to mouse 5/
5 is dead.Should the result shows that, rMBP-BoHCIn Mice Body be it is nontoxic, be confirmed as avirulent fusion protein.
Embodiment 5
--- the preparation of non-toxic c-type clostridium botulinum genetic engineering subunit vaccine
(1) strain: seedling is expression rMBP-BoH with strainCBL/BoM plants of escherichia coli.
(2) first order seed breeding and identification: a small amount of LB liquid medium of freeze-drying lactobacillus is dissolved, streak inoculation is in containing card
The LB solid plate of that mycin is set 37 DEG C and is cultivated 12~16 hours, and standard compliant single bacterium colony is chosen, and inoculation contains kanamycins
LB liquid medium, set 37 DEG C cultivate 8~12 hours, dispensed after being mixed with 50% glycerol equal proportion, set -25 DEG C or less guarantors
It deposits, through purely examining qualification.
(3) secondary seed breeding and identification: taking first order seed, is inoculated with the LB Liquid Culture containing kanamycins by 1% amount
Base, sets 37 DEG C of shaken cultivations 8~12 hours.
(4) prepared by antigen for vaccine: qualified secondary seed is taken, by 2% inoculation of culture medium total amount containing kanamycins
LB liquid medium, 37 DEG C of cultivation temperature.As culture OD600When value is 0.6~0.8, temperature is adjusted to 15 DEG C, is added eventually
Concentration is the IPTG Fiber differentiation 16h of 0.5mM.
(5) it breaks bacterium: thallus being collected with 5000r/min centrifugation 5min, adds 10ml lysate (pH value by every gram of thallus weight in wet base
7.2 0.02mol/L Tris buffers, 0.3mol/L NaCl) ratio thallus is resuspended, cryogenic high pressure homogeneous is used under the conditions of 4 DEG C
Machine is with pressure breaking thallus 3 times of 800bar.Lysate is centrifuged 30min through 4 DEG C of 10000r/min, collects supernatant.
(6) it purifies: according to the operation instructions of Ni-IDA affinity chromatography medium kit (Nanjing Jin Sirui), thallus being split
Solve the rMBP-BoH in supernatant in solubility expressionCIt is purified, through 0.22 μm of aperture membrane filtration, -80 DEG C of preservations are standby.
(7) protein content detects: detecting rMBP-BoH with BCA methodCContent (PierceTM BCA Protein Assay
Kit, TG268883).
(8) steriling test: by existing " Chinese veterinary pharmacopoeia " (the Chinese veterinary pharmacopoeia committee, Republic of China Veterinary Pharmacopoeia,
Two 〇 First Five-Year Plans year version three, Chinese agriculture publishing house, 2016, hereinafter referred to as " Chinese veterinary pharmacopoeia ") annex progress, result is sterile life
It is long.
(9) vaccine formulation: two-phase oil adjuvant (such as 206 adjuvants) are imported in oily phase tank, at least 121 DEG C of high pressure sterilizations
30min is cooled to room temperature spare.According to determining the protein quantity as a result, qualification will be examined with PBS (7.2 0.01mol/L of pH value)
Purifying rMBP-BoHCIt is mixed after appropriate dilution.Water phase is added in emulsion tank, with 80~100r/min stirring, while pressing 1:1
(V/V) ratio is slowly added to oily phase, and 20~30min is stirred after adding.Sampling is tested after emulsification, is dispensed after qualified, most
The vaccine that concentration is 50 μ g/mL and 100 μ g/mL is made eventually.Meanwhile being mixed with same dose of PBS and adjuvant, as control epidemic disease
Seedling.
Embodiment 6
--- the inspection of nontoxic c-type clostridium botulinum genetic engineering subunit vaccine
(1) character
Appearance milky white emulsion.
Dosage form takes a cleaning suction pipe, draws a small amount of vaccine drop in cleaning cold water surface, spreads in cloud, be in oil-in-water
Water-in type (W/O/W).
Stability is drawn vaccine 10mL and is added in centrifuge tube, is centrifuged 15min with 3000r/min, is not demulsified, and tube bottom
The water phase of precipitation is 0.3mL.
Viscosity is measured by " Chinese veterinary pharmacopoeia " (annex), meets regulation.
(2) steriling test is tested by " Chinese veterinary pharmacopoeia " annex, asepsis growth.
(3) safety verification 1.5~2.0kg of weight healthy rabbits 4, each muscle or the vaccine that 50 μ g/mL are subcutaneously injected
4.0mL observes 10d, all strong to live.
(4) efficacy test
(1) serum neutralisation
The experimental animal for being 0 to c-type clostridium botulinum toxin serum neutralization titer is selected, concrete operations are as follows: selecting weight
Sheep similar in 1.5~2.0kg healthy rabbits 4 and 1~3 years old weight each 8.It wherein, is 50 μ g/mL with 2mL antigen protein
Vaccine 4 rabbit are immunized through intramuscular injection path；With 1mL (immunizing dose be 100 μ g/ only) and 2mL, (immunizing dose is respectively
200 μ g/ are only) antigen protein be 100 μ g/mL vaccine 4 sheep are immunized respectively through intramuscular injection path, 21d after inoculation is adopted
Blood, separation serum is spare, and carries out second and be immunized.Two exempt from rear 14d, blood sampling, and separation serum is spare.Every animal blood is taken respectively
Clear 0.4mL and 0.8mL c-type clostridium botulinum toxin (containing 4 and the above mouse MLD), mix 37 DEG C of effect 40min of postposition, then
Intravenous injection 16~18g mouse 2,0.3mL/ is only.It is each simultaneously to criticize mouse 2 with same, 1MLD is injected respectively and toxin serum is mixed
The identical toxin of object is closed to compare.1d is observed, determines result.
After measured, after primary immunization, 4/4 rabbit (immunizing dose is 100 μ g/), 4/4 high dose immunised sheep
(immunizing dose be 200 μ g/ only) and 1/4 low dosage immunised sheep (immunizing dose is 100 μ g/) serum to c-type meat
The neutralization titer of malicious clostridial toxin reaches 1 (in 0.1mL immune serum and c-type clostridium botulinum toxin of 1 MLD).Two exempt from
Afterwards, every rabbit anteserum can reach 4~8MLD (in 0.1mL immune serum to the neutralize antibody titers of clostridium botulinum toxin
With the c-type clostridium botulinum toxin of 4~8 MLD)；High dose immunised sheep (immunizing dose is 200 μ g/) serum is to c-type meat
The neutralization titer of malicious clostridial toxin reaches 3~5MLD, and (in 0.1mL immune serum and the c-type clostridium botulinum of 3~5 MLD is malicious
Element)；Low dosage immunised sheep (immunizing dose is 100 μ g/) serum reaches 1 to the neutralization titer of c-type clostridium botulinum toxin
~3MLD (in 0.1mL immune serum and c-type clostridium botulinum toxin of 1~3 MLD).
(2) Immunization method
With 1.5~2.0kg of weight healthy rabbits 6, its 4 are taken, with the vaccine that 2mL antigen protein is 50 μ g/mL through flesh
4 rabbit are immunized in meat injecting pathway, and remaining 2 rabbit inject control vaccine.21d after inoculation carries out second and is immunized.After two exempt from
14d takes 4 immune group rabbit and 2 control group rabbit, is injected intravenously the c-type clostridium botulinum toxin of 10 MLD, control respectively
Group rabbit is in for 24 hours interior 2/2 dead, immune group rabbit still 4/4 survival after 5d.
About the multi-joint dry powder inactivated vaccine regulation of sheep clostridium in " Chinese veterinary pharmacopoeia ", rabbit or sheep serum are to c-type meat poisoning shuttle
The neutralization titer of verticillium toxin reaches 1 (in 0.1mL immune serum and c-type clostridium botulinum toxin of 1 MLD), that is, is judged to close
Lattice.Therefore, vaccine produced by the invention, antigenic content down to 100~200 μ g/ only in the case where, after primary immunization rabbit and
Sheep serum has reached existing to the Immunization protecting effect that the neutralization titer of c-type clostridium botulinum toxin and two exempt from rear rabbit
The defined standard of row " Chinese veterinary pharmacopoeia ".
Above the experimental results showed that, nontoxic region clostridium botulinum toxin (BoNTs) containing malt glycoprotein (MBP) label
HCRecombinant protein (rMBP-BoHC) there is good immunogenicity, the nontoxic c-type clostridium botulinum gene prepared by the recombinant protein
Engineering subunit vaccine is the ideal candidates vaccine of the existing c-type clostridium botulinum vaccine upgrading in China.
Sequence table
<110>China Veterinery Drug Inspection Office
<120>a kind of nontoxic c-type clostridium botulinum genetic engineering subunit vaccine and its production method
<160> 5
<170> SIPOSequenceListing 1.0
<210> 8
<211> 433
<212> PRT
<213>clostridium botulinum toxin heavy chain (Clostridium botulinum heavy chain)
<400> 8
Met Asp Ile Ile Asn Glu Tyr Phe Asn Asn Ile Asn Asp Ser Lys Ile
1 5 10 15
Leu Ser Leu Gln Asn Arg Lys Asn Thr Leu Val Asp Thr Ser Gly Tyr
20 25 30
Asn Ala Glu Val Ser Glu Glu Gly Asp Val Gln Leu Asn Pro Ile Phe
35 40 45
Pro Phe Asp Phe Lys Leu Gly Ser Ser Gly Glu Asp Arg Gly Lys Val
50 55 60
Ile Val Thr Gln Asn Glu Asn Ile Val Tyr Asn Ser Met Tyr Glu Ser
65 70 75 80
Phe Ser Ile Ser Phe Trp Ile Arg Ile Asn Lys Trp Val Ser Asn Leu
85 90 95
Pro Gly Tyr Thr Ile Ile Asp Ser Val Lys Asn Asn Ser Gly Trp Ser
100 105 110
Ile Gly Ile Ile Ser Asn Phe Leu Val Phe Thr Leu Lys Gln Asn Glu
115 120 125
Asp Ser Glu Gln Ser Ile Asn Phe Ser Tyr Asp Ile Ser Asn Asn Ala
130 135 140
Pro Gly Tyr Asn Lys Trp Phe Phe Val Thr Val Thr Asn Asn Met Met
145 150 155 160
Gly Asn Met Lys Ile Tyr Ile Asn Gly Lys Leu Ile Asp Thr Ile Lys
165 170 175
Val Lys Glu Leu Thr Glu Glu Glu Glu Phe Ser Lys Thr Ile Thr Phe
180 185 190
Glu Ile Asn Lys Ile Pro Asp Thr Gly Leu Ile Thr Ser Asp Ser Asp
195 200 205
Asn Ile Asn Met Trp Ile Arg Asp Phe Tyr Ile Phe Ala Lys Glu Leu
210 215 220
Asp Gly Lys Asp Ile Asn Ile Leu Phe Asn Ser Leu Gln Tyr Thr Asn
225 230 235 240
Val Val Lys Asp Tyr Trp Gly Asn Asp Leu Arg Tyr Asn Lys Glu Tyr
245 250 255
Tyr Met Val Asn Ile Asp Tyr Leu Asn Arg Tyr Met Tyr Ala Asn Ser
260 265 270
Arg Gln Ile Val Phe Asn Thr Arg Arg Asn Asn Asn Asp Phe Asn Glu
275 280 285
Gly Tyr Lys Ile Ile Ile Lys Arg Ile Arg Gly Asn Thr Asn Asp Thr
290 295 300
Arg Val Arg Gly Gly Asp Ile Leu Tyr Phe Asp Met Thr Ile Asn Asn
305 310 315 320
Lys Ala Tyr Asn Leu Phe Met Lys Asn Glu Thr Met Tyr Ala Asp Asn
325 330 335
His Ser Thr Glu Asp Ile Tyr Ala Ile Gly Leu Arg Glu Gln Thr Lys
340 345 350
Asp Ile Asn Asp Asn Ile Ile Phe Gln Ile Gln Pro Met Asn Asn Thr
355 360 365
Tyr Tyr Tyr Ala Ser Gln Ile Phe Lys Ser Asn Phe Asn Gly Glu Asn
370 375 380
Ile Ser Gly Ile Cys Ser Ile Gly Tyr Phe Arg Leu Gly Gly Asp Trp
385 390 395 400
Tyr Arg His Asn Tyr Leu Val Pro Thr Val Lys Gln Gly Asn Tyr Ala
405 410 415
Ser Leu Leu Glu Ser Thr Ser Thr His Trp Gly Phe Val Pro Val Ser
420 425 430
Glu
<210> 2
<211> 2367
<212> DNA
<213>clostridium botulinum toxin (rMBP-BoHC) is recombinated
<400> 2
tctagaggta ccaaaactga agaaggtaaa ctggtaatct ggattaacgg cgataaaggc 60
tataacggtc tcgctgaagt cggtaagaaa ttcgagaaag ataccggaat taaagtcacc 120
gttgagcatc cggataaact ggaagagaaa ttcccacagg ttgcggcaac tggcgatggc 180
cctgacatta tcttctgggc acacgaccgc tttggtggct acgctcaatc tggcctgttg 240
gctgaaatca ccccggacaa agcgttccag gacaagctgt atccgtttac ctgggatgcc 300
gtacgttaca acggcaagct gattgcttac ccgatcgctg ttgaagcgtt atcgctgatt 360
tataacaaag atctgctgcc gaacccgcca aaaacctggg aagagatccc ggcgctggat 420
aaagaactga aagcgaaagg taagagcgcg ctgatgttca acctgcaaga accgtacttc 480
acctggccgc tgattgctgc tgacgggggt tatgcgttca agtatgaaaa cggcaagtac 540
gacattaaag acgtgggcgt ggataacgct ggcgcgaaag cgggtctgac cttcctggtt 600
gacctgatta aaaacaaaca catgaatgca gacaccgatt actccatcgc agaagctgcc 660
tttaataaag gcgaaacagc gatgaccatc aacggcccgt gggcatggtc caacatcgac 720
accagcaaag tgaattatgg tgtaacggta ctgccgacct tcaagggtca accatccaaa 780
ccgttcgttg gcgtgctgag cgcaggtatt aacgccgcca gtccgaacaa agagctggcg 840
aaagagttcc tcgaaaacta tctgctgact gatgaaggtc tggaagcggt taataaagac 900
aaaccgctgg gtgccgtagc gctgaagtct tacgaggaag agttggcgaa agatccacgt 960
attgccgcca ccatggaaaa cgcccagaaa ggtgaaatca tgccgaacat cccgcagatg 1020
tccgctttct ggtatgccgt gcgtactgcg gtgatcaacg ccgccagcgg tcgtcagact 1080
gtcgatgaag ccctgaaaga cgcgcagact ggtaccgatt acgatatccc aacgaccaag 1140
cttgtatccg aagaaggcga cgttcagctg aatccgatct ttccgtttga cttcaagctg 1200
ggctctagcg gtgaggatcg tggcaaagtt atcgtgaccc agaacgagaa tatcgtttac 1260
aacagcatgt atgagagctt ctccatcagc ttctggattc gcattaacaa gtgggtttct 1320
aacctgccag gttatactat cattgactcc gttaagaaca acagcggttg gtctattggc 1380
atcatttcta actttctggt attcaccctg aaacagaacg aggactccga gcagtctatc 1440
aacttctctt acgacatttc taacaacgct ccgggttaca acaaatggtt cttcgtgacc 1500
gtaactaaca acatgatggg caacatgaag atctacatca acggtaaact gattgatacc 1560
atcaaggtta aggagctgac cggcattaac ttctccaaga ctattacctt tgagatcaac 1620
aagattccgg acactggtct gattacctct gactccgaca atatcaacat gtggattcgt 1680
gatttctata tctttgcgaa agagctggat ggcaaagaca tcaacatcct gttcaacagc 1740
ctgcaataca ccaacgtggt gaaagactat tggggtaacg atctgcgtta caacaaagaa 1800
tactacatgg tgaacatcga ctacctgaac cgctatatgt acgcaaacag ccgtcagatt 1860
gtattcaaca ctcgtcgcaa caacaatgac tttaacgagg gctacaagat catcatcaaa 1920
cgcatccgtg gcaataccaa cgatacccgt gtgcgtggtg gcgacatcct gtactttgat 1980
atgaccatca acaacaaagc gtataacctg ttcatgaaga acgaaactat gtatgctgat 2040
aaccactcca ccgaggacat ctacgccatt ggtctgcgtg agcagaccaa ggacatcaac 2100
gacaacatca tcttccagat tcagccgatg aacaacacct actactatgc gagccaaatc 2160
ttcaagagca actttaacgg tgagaacatc tctggtatct gctccattgg cacctaccgt 2220
ttccgcctgg gtggtgactg gtatcgtcac aactatctgg ttccgaccgt taaacagggt 2280
aactacgcat ctctgctgga gtccacctcc acccactggg gtttcgttcc ggtgagcgag 2340
catcaccatc atcaccacta actcgag 2367
<210> 3
<211> 789
<212> PRT
<213>clostridium botulinum toxin (rMBP-BoHC) is recombinated
<400> 3
Ser Arg Gly Thr Lys Thr Glu Glu Gly Lys Leu Val Ile Trp Ile Asn
1 5 10 15
Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu
20 25 30
Lys Asp Thr Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu
35 40 45
Glu Lys Phe Pro Glu Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile
50 55 60
Phe Trp Ala His Asp Arg Phe Gly Gly Tyr Ala Glu Ser Gly Leu Leu
65 70 75 80
Ala Glu Ile Thr Pro Asp Lys Ala Phe Glu Glu Asp Lys Leu Tyr Pro
85 90 95
Phe Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro
100 105 110
Ile Ala Val Glu Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro
115 120 125
Asn Pro Pro Lys Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu
130 135 140
Lys Ala Lys Gly Lys Ser Ala Leu Met Phe Asn Leu Glu Glu Pro Tyr
145 150 155 160
Phe Thr Trp Pro Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr
165 170 175
Glu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly
180 185 190
Ala Lys Ala Gly Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His
195 200 205
Met Asn Ala Asp Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys
210 215 220
Gly Glu Thr Ala Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile
225 230 235 240
Asp Thr Ser Lys Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys
245 250 255
Gly Glu Pro Ser Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn
260 265 270
Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr
275 280 285
Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu
290 295 300
Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro
305 310 315 320
Arg Ile Ala Ala Thr Met Glu Asn Ala Glu Glu Lys Gly Glu Ile Met
325 330 335
Pro Asn Ile Pro Glu Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala
340 345 350
Val Ile Asn Ala Ala Ser Gly Arg Glu Glu Thr Val Asp Glu Ala Leu
355 360 365
Lys Asp Ala Glu Thr Gly Thr Asp Tyr Asp Ile Pro Thr Thr Lys Leu
370 375 380
Val Ser Glu Glu Gly Asp Val Glu Glu Leu Asn Pro Ile Phe Pro Phe
385 390 395 400
Asp Phe Lys Leu Gly Ser Ser Gly Glu Asp Arg Gly Lys Val Ile Val
405 410 415
Thr Glu Asn Glu Asn Ile Val Tyr Asn Ser Met Tyr Glu Ser Phe Ser
420 425 430
Ile Ser Phe Trp Ile Arg Ile Asn Lys Trp Val Ser Asn Leu Pro Gly
435 440 445
Tyr Thr Ile Ile Asp Ser Val Lys Asn Asn Ser Gly Trp Ser Ile Gly
450 455 460
Ile Ile Ser Asn Phe Leu Val Phe Thr Leu Lys Glu Asn Glu Asp Ser
465 470 475 480
Glu Glu Glu Ser Ile Asn Phe Ser Tyr Asp Ile Ser Asn Asn Ala Pro
485 490 495
Gly Tyr Asn Lys Trp Phe Phe Val Thr Val Thr Asn Asn Met Met Gly
500 505 510
Asn Met Lys Ile Tyr Ile Asn Gly Lys Leu Ile Asp Thr Ile Lys Val
515 520 525
Lys Glu Leu Thr Gly Ile Asn Phe Ser Lys Thr Ile Thr Phe Glu Ile
530 535 540
Asn Lys Ile Pro Asp Thr Gly Leu Ile Thr Ser Asp Ser Asp Asn Ile
545 550 555 560
Asn Met Trp Ile Arg Asp Phe Tyr Ile Phe Ala Lys Glu Leu Asp Gly
565 570 575
Lys Asp Ile Asn Ile Leu Phe Asn Ser Leu Glu Tyr Thr Asn Val Val
580 585 590
Lys Asp Tyr Trp Gly Asn Asp Leu Arg Tyr Asn Lys Glu Tyr Tyr Met
595 600 605
Val Asn Ile Asp Tyr Leu Asn Arg Tyr Met Tyr Ala Asn Ser Arg Glu
610 615 620
Ile Val Phe Asn Thr Arg Arg Asn Asn Asn Asp Phe Asn Glu Gly Tyr
625 630 635 640
Lys Ile Ile Ile Lys Arg Ile Arg Gly Asn Thr Asn Asp Thr Arg Val
645 650 655
Arg Gly Gly Asp Ile Leu Tyr Phe Asp Met Thr Ile Asn Asn Lys Ala
660 665 670
Tyr Asn Leu Phe Met Lys Asn Glu Thr Met Tyr Ala Asp Asn His Ser
675 680 685
Thr Glu Asp Ile Tyr Ala Ile Gly Leu Arg Glu Glu Thr Lys Asp Ile
690 695 700
Asn Asp Asn Ile Ile Phe Glu Ile Glu Pro Met Asn Asn Thr Tyr Tyr
705 710 715 720
Tyr Ala Ser Glu Ile Phe Lys Ser Asn Phe Asn Gly Glu Asn Ile Ser
725 730 735
Gly Ile Cys Ser Ile Gly Thr Tyr Arg Phe Arg Leu Gly Gly Asp Trp
740 745 750
Tyr Arg His Asn Tyr Leu Val Pro Thr Val Lys Glu Gly Asn Tyr Ala
755 760 765
Ser Leu Leu Glu Ser Thr Ser Thr His Trp Gly Phe Val Pro Val Ser
770 775 780
Glu His His His His
785
<210> 4
<211> 25
<212> DNA
<213>artificial synthesized (1 F)
<400> 4
ccgtctagag gtaccaaaac tgaag 25
<210> 5
<211> 24
<212> DNA
<213>artificial synthesized (1 R)
<400> 5
ccgctcgagt tagtggtgat gatg 24
Claims (7)
1. a kind of nontoxic c-type clostridium botulinum genetic engineering subunit vaccine, it is characterised in that the vaccine contains using large intestine bar
Recombination clostridium botulinum toxin (the rMBP-BoH that bacterium is expressed as host cell and in cytoplasmC)；The recombinant toxin is by weight
Group expression rMBP-BoHCEscherichia coli (Escherichia coli) BL21 (DE3) cell as production bacterial strain preparation,
The bacterial strain is named as BL/BoM plants of escherichia coli, and delivers Chaoyang District, Beijing City North Star west on 03 08th, 2019
The culture presevation administration committee, China, Institute of Microorganism, Academia Sinica, institute 3 of road 1 common micro-organisms center, deposit number:
CGMCC No.17317；
The vaccine infects associated diseases by c-type clostridium botulinum for preventing.
2. a kind of nontoxic c-type clostridium botulinum genetic engineering subunit vaccine as described in claim 1, it is characterised in that the expression
RMBP-BoHC, which contains only the nontoxic of heavy chain compared with mature wild type Clostridium botulinum toxin (BoNTs)
Region, the end C- (abbreviation BoHC)；
The rMBP-BoHCAvirulence greatly reduces the bio-safety risk in vaccine production process.
3. a kind of nontoxic c-type clostridium botulinum genetic engineering subunit vaccine as described in claim 1, it is characterised in that the vaccine
In the rMBP-BoH that containsC, it is that BoH is realized as addition hydrotropy label by malt glycoprotein (MBP)CSolubility expression.
4. a kind of nontoxic c-type clostridium botulinum genetic engineering subunit vaccine described in claim 1, it is characterised in that the expression
rMBP-BoHC, the gene order codon optimization of coding, it is easier to high expression and solvable table are realized in Escherichia coli
It reaches.
5. a kind of nontoxic c-type clostridium botulinum genetic engineering subunit vaccine described in claim 1, it is characterised in that the expression
rMBP-BoHC, containing the label for being convenient for protein purification, preferably C-terminal contains 6 histidine (6*His) labels.
6. a kind of nontoxic c-type clostridium botulinum genetic engineering subunit vaccine described in claim 1, it is characterised in that preparation method
Are as follows: by host cell expression, with the expression rMBP-BoHCBL/BoM plants of Escherichia coli be used as production of vaccine bacterium
Plant, after fermented culture, inducing expression, bacterial cell disruption, soluble antigen separation and purification of protein, adjuvant is added to be mixed.
7. a kind of nontoxic c-type clostridium botulinum genetic engineering subunit vaccine described in claim 1, it is characterised in that for expressing
rMBP-BoHC" host cell ", " host cell " covers prokaryotic cell and/or eukaryocyte, can be production embrace a sub- shuttle
It is bacterium, C.perfringens, clostridium acetobutylicum, Bacillus cereus, Su Yunjin bud pole bacterium, Cao's shape bud pole bacterium, thermophilic molten
Proteins Spore bacillus, anthrax bud pole bacterium, Bacillus megaterium, bacillus subtilis, Escherichia coli or yeast cells；
The host cell is preferably e. coli host cell, especially e. coli bl21 (DE3) or Rosetta
(DE3)。
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Cited By (1)
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| CN105749266A (en) * | 2016-04-26 | 2016-07-13 | 齐鲁动物保健品有限公司 | Mink hemorrhagic pneumonia and botulism combined inactivate vaccine and preparing method thereof |
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| US20030215468A1 (en) * | 1995-03-16 | 2003-11-20 | Allergan, Inc., Allergan Botox Limited | Soluble recombinant botulinum toxin proteins |
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| US5919665A (en) * | 1989-10-31 | 1999-07-06 | Ophidian Pharmaceuticals, Inc. | Vaccine for clostridium botulinum neurotoxin |
| US20030215468A1 (en) * | 1995-03-16 | 2003-11-20 | Allergan, Inc., Allergan Botox Limited | Soluble recombinant botulinum toxin proteins |
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