CN101016336A - A-type kreotoxin B cell antigen epitope peptide and use thereof - Google Patents
A-type kreotoxin B cell antigen epitope peptide and use thereof Download PDFInfo
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Abstract
The invention discloses a cell antigen epitope peptide of A-typed botulism toxin B as core immune antigen, which is characterized by the following: protecting botulism toxin from infecting; identifying A-typed specific antibody of botulism toxin due to characteristic antigen mark; distinguishing A-type from other patterns in the serum; making the short peptide possess the same or similar function with full toxin antigen and recombinant toxin antigen without allergic reaction; overcoming the serum crossing problem among full toxin antigen or recombinant antigen; diagnosing the infection of botulism toxin to prepare new peptide vaccine; fitting for targeting and sieving botulism toxin resistant drug.
Description
Technical field
The present invention relates to biotoxin B cell antigen epitope peptide, relate in particular to botulinus toxin B cell antigen epitope peptide, also relate to the purposes of above-mentioned epitope peptide.
Background technology
Botulinum neurotoxin (Clostridium Botulinum Neurotoxins, BoNT, be called for short botulinus toxin) be the extracellular toxin that under anaerobic produces by clostridium botulinum, 7 serotypes (A-G) are arranged, be the strongest protein of present known virulence, A wherein, B, the E BOTULINUM TOXIN TYPE A A is to cause human common type of poisoning, and clinical observation shows that botulinum toxin type A can cause more serious illness and cause higher mortality ratio than other serotype.
The precursor of botulinum toxin type A is that molecular weight is the nontoxic single chain protein of 150kD; be cut into through tropina enzyme or external proteolytic enzyme and have double chain form and just have toxicity; be that light chain (L) is connected by a disulfide linkage with heavy chain (H); the H chain is the land of botulinus toxin; be that to enter neurocyte necessary; itself and neurocyte toxoreceptor bonded position are the fragments (Hc antigen) of the about 50kD of C end molecule amount; studies show that Hc antigen is the main protection antigen of botulinus toxin; contain neutrality B cell epitope; it can substitute botulinus toxin standard class toxin to a certain extent; induce the protection antibody of generation, produce effective immune protective effect at natural toxin alive.Simultaneously, as main toxin antigenic structure, Hc antigen is also being brought into play important effect in the development of preparations such as the Infect And Diagnose of botulinus toxin and therapeutic antibodies.
At present, aspect vaccine development, the acquisition of the important protective antigen of botulinus toxin, method one is the product poison cultivation of pass through Clostridium botulinum of report in 1988 such as Siegel, extracts botulinus toxin from culture, deactivation prepares toxoid antigen.So not only need to be with the poison operation, and can't remove compositions such as formaldehyde and tropina fully, have security hidden danger (Siegel, LS.J Clin Microbiol.1988 November; 26 (11): 2351-2356); Method two, nineteen ninety-fives such as Michael A reports be primarily aimed at Hc antigen and fragment thereof, the using gene engineering technique method, by the structure of protokaryon and yeast expression system, recombinant expressed and purifying, preparation recombinant toxin antigen.Because Hc antigen is a large protein, its gene structure A+T% content is up to 76%, the codon bias is very high, and recombinant expressed level is lower, needs to improve expression amount by the genetic modification method, recombinant expressed and higher (the Michael A of cost purifying, Clayton J, M.Clayton, et al.J.Infection andImmunity, 1995,63 (7): 2738-2742).Consider that the core texture that antigen plays a decisive role in the induction of immunity reaction in vivo is an epitope, so dominant antigen epi-position or comprise the small peptide of epitope and their combination, or identical functions similar can be had to complete antigen protein.Under normal conditions, epi-position length is from several to dozens of amino acid, can the application experiment method, avoid the structure of botulinum toxin complex, directly obtain epitope, epitope peptide is the small peptide that contains one or several epitope sequence, and its unique characteristics are arranged: molecular weight is little, simple in structure, be easy to synthetic preparation; Importantly, have antigenicity and immunogenicity, can stimulate the B cell to produce specific antibody as the definite functions of antigen core texture; The holotoxin antigen of comparing, epitope peptide uses safer almost without any toxicity.The using value that contains the small peptide of epi-position also is embodied in: the epi-position small peptide at botulinum toxin type A can carry out random combination or series design with the epi-position small peptide at other serotype botulinus toxin, can be applied to the preparation of the botulinus toxin vaccine of different protection domains at an easy rate; Same, if with antigen or the combination of epi-position small peptide at other pathogenic agent, can further widen its vaccine use.
Aspect the botulinus toxin Infect And Diagnose, mainly adopt double antibody sandwich method to detect the antigenic method of botulinus toxin, because the detection sensitivity requirement mainly is applicable to serious poisoner, and inapplicable to previous infection and low dosage the infected.And infect the very practical double antigens sandwich antibody testing method of judgement at other most of pathogen antigens, in detecting, the botulinus toxin specific antibody has certain problem.Mainly be the serotype complexity because of botulinus toxin, the antigenic amino acid identity of toxin is higher, has tangible antigen-antibody cross reaction (Ferreira J, Maslanka S, Johnson E, et al, J AOAC Int.2003:86:314-31.) each other.
Epitope or the epi-position small peptide authentication method that adopts mainly contains following several at present: one, adopt the method for peptide section scanning (peptide scan) or gene partitioned representation: Markt.T in 1996 and Dertzbaugh etc. are by a series of eclipsed protein fragments of gene recombination method partitioned representation botulinum toxin type A Hc, the sequence fragment H455-661 and H1150-1289 (the Mark T of BoNT/A generation protective immunity have been determined, Dertzbaugh, and Michael WW.J.Vaccine, 1996,14 (16): 1538-1544.), exploratory development is carried out in its application in the botulinus toxin vaccine construction, and applied for United States Patent (USP) 20030185850; Atassi was by a series of overlapped 5 amino acid whose ten nonapeptides of external chemosynthesis in 1996, adopt antitoxic serum to screen the possible epi-position of BoNT/A-Hc, the result shows that the peptide sequence of the antitoxic serum identification in different immunity source has certain difference (Atassi MZ, Dolimbek BZ, Hayakari M, et al.J Protein Chem 1996 Oct; 15 (7): 691-700).Two, utilize display technique of bacteriophage or protein chip technology, wash in a pan sieve by many wheels, final isolation identification obtains mimic epitopes: calendar year 2001 Wu HC uses the toxin neutralization monoclonal antibody screens botulinum toxin type A from phage random peptide library simulation neutralizing epitope CXDC (Wu HC, Yeh CT, Tarn LJ, et al.Appl Environ Microbiol 2001 Jul; 67 (7): 3201-7), utilization display technique of bacteriophage such as calendar year 2001 Mullaney BP have carried out the neutralizing epitope drawing to BoNT/A, infer that there are a plurality of dominant antigen epi-positions in toxin, wherein the Sphingolipids,sialo binding site is at Glu1202, His1252, Trp1265, the receptor protein land is positioned at 1115-1223 sequence (Mullaney BP, PallaviciniMG, Marks JD.Infect Immun.2001 Oct; 69 (10): 6511-4), for the toxitherapy medicinal design provides possible target spot.Three, a series of monoclonal antibodies of preparation at antigen molecule, determine the scope that epi-position exists by competition analysis: employing monoclonal antibodies such as Toru in 1997 carry out epi-position and draw, determined neutralizing epitope sequence (the Toru Kubota of BoNT/E, Toshihiro Watanabe, Noriko Yokosawa, et al.Applied andEnvironmental, Microbiology, 1997,63 (4): 1214-1218).But these methods are bothersome, and effort and required cycle are longer, and are unsuitable for the research of complicated epi-position, thereby restricted the development of botulinus toxin study on prevention.The development of information biology, and the determining of numerous protein three-dimensional crystalline structure that comprises botulinum toxin type A for the analysis of epitope provides new clue, also provide thinking and approach for novel peptide vaccine research etc.
Summary of the invention
At having the uncertain of botulinum toxin type A epitope now; and the deficiency of botulinus toxin vaccine research and detection diagnostic techniques; the invention provides one group of small peptide that contains the botulinum toxin type A protective epitope, the aminoacid sequence of these small peptides is shown in sequence in the sequence table 1,2 and 3.
These small peptides and holotoxin antigen, recombinant toxin antigen have same or analogous function; the effective immunoprotection effect of excitating organism; can effectively discern the existence that botulinum toxin type A infects the back specific antibody, have that preparation is simple, cost is low, have no side effect, with the achiasmate characteristics of other serotype botulinus toxin antigen.
The present invention also provides and has comprised in above-mentioned three kinds of botulinum toxin type A B cell antigen epitope peptides at least two kinds multi-component antigen epitope peptide, also disclose the multi-component antigen epitope peptide that comprises above-mentioned three kinds of botulinum toxin type A B cell antigen epitope peptides, also disclose the multi-component antigen epitope peptide that comprises with the above-mentioned three kinds of botulinum toxin type A B cell antigen epitope peptides of equal proportion blended.
The present invention above-mentioned small peptide is provided simultaneously and have identical sequence and the protein of constitutional features, polypeptide and composition thereof in botulinus toxin new generation vaccine Development Application.Be tested and appraised and obtain effective botulinum toxin type A protective epitope or epitope peptide, new more accurate, effective cAg motif or small peptide is provided, it is applied to the preparation of botulinus toxin new peptides vaccine as important antigen component.
Protein, polypeptide and composition thereof the application in the botulinus toxin Infect And Diagnose that the present invention provides these small peptides simultaneously and had same characteristic features.Be tested and appraised and obtain effective botulinum toxin type A epitope or epi-position small peptide, new special cAg motif or the small peptide of type is provided, utilize itself and type specific antibody to catch the bonded function simultaneously, it is applied to the detection of the specific antibody that botulinum toxin type A infects as important antigen component, the botulinus toxin Infect And Diagnose is determined to the standard of serotype.
The present invention analyzes epi-position small peptide and A type Botulinum Antitoxin horse serum specific combination by the ELISA experiment.By elisa plate, BSA is as negative control with the epitope peptide bag, and after the BSA sealing, the horse serum that adds anti-A type botulinus toxin carries out combination; The washing back adds the anti-horse IgG of enzyme mark rabbit, hatches after scouring, carries out the substrate colour developing then, detects OD
450The result shows that epitope peptide of the present invention all can combine specifically with botulinum toxin type A toxinicide horse serum.
The present invention these small peptides are provided simultaneously and have the polypeptide of same characteristic features and composition in the purposes of botulinus toxin as resisting botulinus toxin drug screening target.Because small peptide of the present invention is the immunizing antigen of the core of botulinus toxin, also be the characteristic antigen sign of botulinus toxin, therefore these small peptides can be carried out the screening of resisting botulinus toxin medicine as target.
It is template that the present invention provides the crystalline structure with botulinum toxin type A simultaneously, the comprehensive analysis method of applying biological information science and protein structure analytical Calculation etc., and screening obtains the method for B cell antigen epi-position or epi-position small peptide.
Therefore the present invention is tested and appraised and obtains effective botulinum toxin type A protective epitope or epitope peptide; new more accurate, effective cAg motif or small peptide is provided; simultaneously it being applied to the preparation of botulinus toxin new peptides vaccine as important antigen component, is one of main innovate point of the present invention.The present invention is tested and appraised and obtains effective botulinum toxin type A epitope or epi-position small peptide, new special cAg motif or the small peptide of type is provided, utilize itself and type specific antibody to catch the bonded function simultaneously, it is applied to the detection of the specific antibody of botulinum toxin type A infection as important antigen component, the botulinus toxin Infect And Diagnose is determined to the standard of serotype, is two of main innovate point of the present invention.The present invention is a template with the crystalline structure of botulinum toxin type A, the comprehensive analysis method of applying biological information science and protein structure analytical Calculation etc., and screening obtains B cell antigen epi-position or epi-position small peptide, also is innovative point of the present invention.
Description of drawings
Fig. 1 is the specific combination of epi-position small peptide and botulinum toxin type A toxinicide horse serum
Fig. 2 is the cross coupled experiment of epi-position small peptide and different serotypes toxinicide horse serum
Fig. 3 is the immunoprotection experiment of single epitope peptide vaccine
Fig. 4 is the immunoprotection experiment of combination epi-position peptide vaccine
Embodiment
Determining of embodiment 1 botulinum toxin type A epitope peptide
One, material:
SEPHADEX G-15; SEPHAROSE affinity post; HPLC is available from Pharmacia company;
Insight II software, SGI graphics workstation are Neotrident company products.
Two, method and result:
1.A the screening of BOTULINUM TOXIN TYPE A A Hc fragment B cell antigen epi-position
RC Stevens﹠amp in 1998; DB Lacy has obtained the crystalline structure (PDBstucture:3BTA, 2.3 ) of the double-stranded botulinum toxin type A be made up of 1285 amino-acid residues by the analysis of high resolving power multidimensional crystal X grating spectrum; The present invention utilizes the three-dimensional crystalline structure of botulinum toxin type A among the Protein Data Bank PDB (http://www.rcsb.org/pdb/home/home.do), use Insight II software and SGI graphics workstation, carry out the analysis of botulinum toxin type A B cell antigen epi-position, select continuous at least three amino-acid residue exposed areas greater than 30%, and its pairing space structure is that the sequence of corner or random coil structure is possible B cell antigen epi-position, and the partial results of the epitopic features analysis of single amino acids sees Table 1.
Table 1 botulinum toxin type A Characterization of antigenic epitopes result
| monomer | area | fraction/SAS | structure |
| SER A884 ASN A885 LEU A918 GLU A919 SER A920 SER A1041 ASN A1042 LYS A1186 ASN A1187 | 64.355 111.117 82.383 113.383 57.455 72.583 132.387 188.055 154.142 | 0.479 0.741 0.517 0.652 0.462 0.587 0.851 0.894 0.935 | TURN TURN TURN TURN TURN TURN TURN TURN TURN |
2. contain the design and the preparation of the small peptide of protective epitope
According to above-mentioned epi-position The selection result; contain the design of the small peptide of protective epitope; different epi-positions are effectively connected; and between epi-position, introduce suitable amino acid intervening sequence; prolong epitope regions; consequence devised 3 epitope peptides, length is about 20 amino acid, the aminoacid sequence of epitope peptide 1#, 2# and 3# is respectively shown in sequence in the sequence table 1,2 and 3.
The synthetic employing solid-phase synthesis of epitope peptide, in order to increase the biologic activity of epitope peptide, peptide sequence N end adopts the acetylize sealing in building-up process, can strengthen the stability of polypeptide, prevents that the body endoproteinase from separating, and prolongs the intravital transformation period.The C end has been introduced D type amino acid, and it can not influence the space structure of polypeptide, can improve intravital stability and activity equally.Preliminary synthetic product is through SEPHADEXG-15 or SEPHAROSE affinity column purifying.Purified product is used HPLC (high performanceliquid chromatography) and is done amino acid analysis.
One, material:
Anti-A, B, E BOTULINUM TOXIN TYPE A A horse serum, the anti-horse IgG of enzyme mark rabbit (Chinese biological goods calibrating institute)
Two, method and result:
1. the elisa assay of epi-position small peptide and A type Botulinum Antitoxin horse serum specific combination
Result: show that as accompanying drawing 13 epitope peptides of design synthetic all can combine specifically with botulinum toxin type A toxinicide horse serum.
2. the cross coupled of epitope peptide and different serotypes Botulinum Antitoxin horse serum experiment
The 1# epitope peptide of selection and botulinum toxin type A toxinicide horse serum specific combination carries out the cross coupled experiment between the different serotypes, and checking is infected in the main serotype antibody test the people no cross reaction.Epitope peptide 1# (10 μ g/ hole) bag is by elisa plate, and coating buffer is NaHCO3 pH8.6, bag by BSA as negative control, 4 ℃ of bag quilts that spend the night.Remove coating buffer, add 37 ℃ of sealing 1h of 100 μ l 3%BSA (0.02mol/L PBS, pH7.2 preparation), add the horse serum (dilution in 1: 3000) of anti-A, B, E BOTULINUM TOXIN TYPE A A respectively, do contrast with irrelevant antiserum(antisera), 100 μ l/ holes, 37 ℃ in conjunction with 1h; Wash with PBST (0.05%Tween 20); Add the anti-horse IgG of enzyme mark rabbit (dilution in 1: 1000) 100 μ l/ holes, incubated at room 30 minutes; With PBST (0.05%Tween 20) washing, drain raffinate, add A, the colour developing of B liquid substrate, room temperature reaction 3-5 minute, add 2N H2SO4 termination reaction, detect OD
450
Result such as accompanying drawing 2 show, the 1# epitope peptide can with botulinum toxin type A antitoxic serum specific combination, and do not intersect with other serotype antitoxic serum, also find in to the sampling Detection of clinical sausage poisoning patients serum's sample adopting enzyme linked immunosorbent assay (ELISA) dual-antigen sandwich method, it is consistent with botulinus toxin standard class toxin detection result of determination to adopt epitope peptide to detect, the 1# epitope peptide can replace botulinus toxin standard class toxin or recombinant antigen, infects the characteristic antigen of differentiating and diagnosing as the botulinus toxin different serotypes.
The preparation of embodiment 3 epitope peptide vaccines and immunoprotection effect thereof
One, material:
Epitope peptide is pressed the foregoing description preparation;
BALB/C mice is available from Military Medical Science Institute's Experimental Animal Center.
Two, method and result
1. the preparation of single or polycomponent epitope peptide vaccine
Single epitope peptide antigen: epitope peptide 1#, 2# and the 3# of purity＞85%, with 0.02mol/LPBS pH7.2 dissolving, being mixed with concentration is the epitope peptide antigen of 0.8mg/ml respectively.
Polycomponent epitope peptide antigen: epitope peptide 1#, 2# and the 3# of purity＞85%, even with 1: 1: 1 mixed, with 0.02mol/L PBS pH7.2 dissolving, be mixed with every kind of epitope peptide concentration and still be the epitope peptide antigen of 0.8mg/ml.
The epitope peptide vaccine: single or polycomponent epitope peptide antigen respectively with isopyknic Al (OH)
3Adjuvant mixes, and is mixed with single epitope peptide vaccine or polycomponent epitope peptide vaccine.
2. the immunity of epitope peptide vaccine and antibody test
7-8 BALB/C mice in age in week, female, be divided into four groups of A, B, C, D at random, 8 every group.
Group result is as follows:
A normal group: injections of antigens (physiological saline of abdominal injection same dose) not
B positive controls: botulinum toxin type A standard class toxin immunity
C negative control group: irrelevant antigen immune
D experimental group: a: single epitope peptide immunity (1# or 2# or 3#)
B: epitope peptide combination immunity (1#+2#+3#)
Take the immunization ways of abdominal injection, every injection 200 μ l (only being equivalent to antigen 80 μ g/).Booster immunization every other week, dosage is exempted from head.Respectively at blood sampling in 4 days after the 3rd, 4 immunity, ELISA detected the variation of tiring of specific antibody in the serum.
The result shows that the specific antibody positive rate of two experimental group after immune 3 times is respectively 87.5% (7/8) and 100% (8/8), and the specific antibody positive rate after immune 4 times is 100%.
3. the malicious protectiveness of attacking of epitope peptide vaccine immunity is tested
All experiment mices are attacked poison, abdominal injection 10 with the botulinum toxin type A toxin of living behind the booster immunization 4 times
3LD
55/ only; observe the protection situation of each group respectively; each experimental group protection situation such as accompanying drawing 3; what accompanying drawing 3 showed is the malicious back of attacking of epitope peptide immune mouse survival condition; as can be seen from the figure 8 of negative control group mouse are all dead in 18h; standard class toxin immunity group is protected fully, compares with negative control group, and single epitope peptide immune mouse all can protect 10 to some extent
3LD
50Botulinum toxin type A attack poison, different immunoprotection degree may be relevant with different epi-position epi-positions that small peptide comprises role size in immunity identification, the survival rate that accompanying drawing 3 shows the epitope peptide immune mouses more not immune group has tangible prolongation effect.The combination immune result of accompanying drawing 4 epitope peptides shows that epitope peptide has tangible stack immanoprotection action, compares with single epitope peptide, and the combination immunity has better immune protective effect, proves that there are a plurality of dominant antigen epi-positions in botulinus toxin.Though the polycomponent epitope peptide of this test is only with epitope peptide 1#, 2#, 3#, mixed with 1: 1: 1 experimentizes, but the result infers epitope peptide 1#, 2#, the 3# polycomponent epitope peptide with different mixed by experiment, or among epitope peptide 1#, 2#, the 3# any two in varing proportions blended polycomponent epitope peptide also can obtain similar results.Only,, or, can further improve immune protective effect, prepare botulinus toxin new generation vaccine efficiently by reorganization connection structure multi-epitope series connection vaccine by other immunogen component of suitable interpolation based on small peptide provided by the invention.
4. the safety experiment of epitope peptide vaccine
7-8 BALB/C mice in age in week, female, with each 8 of said components difference abdominal injections, consumption is 0.4mg/ only (immunizing dose 5 times), the observation experiment mouse has or not abnormal response in three weeks, the experiment mice of safety examination any abnormal response do not occur at experimental session, illustrates that this epitope peptide vaccine can not produce any untoward reaction.
Sequence table
＜110〉Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL
＜120〉botulinum toxin type A B cell antigen epitope peptide and uses thereof
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Asn Trp Tyr Asn Arg Gln Ile Glu Arg Ser Ser Arg Thr Leu Gly Cys
1 5 10 15
Ser
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Arg Thr Leu Gly Cys Ser Trp Glu Phe Ile Pro Val Asp Asp Gly Trp
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Claims (10)
1.A BOTULINUM TOXIN TYPE A A B cell antigen epitope peptide is characterized in that having the aminoacid sequence shown in the sequence 1,2 or 3 in the sequence table.
2. polycomponent botulinum toxin type A B cell antigen epitope peptide is characterized in that comprising at least two kinds in the described three kinds of botulinum toxin type A B cell antigen epitope peptides of claim 1.
3. according to the described multi-component antigen epitope peptide of claim 2, it is characterized in that comprising the described three kinds of botulinum toxin type A B cell antigen epitope peptides of claim 1.
4. according to the described multi-component antigen epitope peptide of claim 3, it is characterized in that the described three kinds of botulinum toxin type A B cell antigen epitope peptides of claim 1 mix with equal proportion.
5. claim 1 or 2 described botulinum toxin type A B cell antigen epitope peptides or the multi-component antigen epitope peptide purposes in the specific antibody diagnostic reagent that the preparation botulinum toxin type A infects.
6. claim 1 or 2 described botulinum toxin type A B cell antigen epitope peptides or the multi-component antigen epitope peptide purposes in preparation botulinus toxin new peptides vaccine.
7. claim 1 or 2 described botulinum toxin type A B cell antigen epitope peptides or multi-component antigen epitope peptide are as the purposes of resisting botulinus toxin drug screening target.
8. the polypeptide or the purposes of protein in the specific antibody diagnostic reagent that the preparation botulinum toxin type A infects that contain the described botulinum toxin type A B cell antigen of claim 1 epitope peptide.
9. the polypeptide or the purposes of protein in preparation botulinus toxin new peptides vaccine that contain the described botulinum toxin type A B cell antigen of claim 1 epitope peptide.
10. contain the polypeptide of the described botulinum toxin type A B cell antigen of claim 1 epitope peptide or protein purposes as resisting botulinus toxin drug screening target.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107490680A (en) * | 2016-06-09 | 2017-12-19 | 常州博闻迪医药科技有限公司 | A kind of Blood glycated haemoglobin enzyme-linked immune detection method |
| CN109374899A (en) * | 2018-08-24 | 2019-02-22 | 上海飞晟生物科技有限公司 | It is a kind of for detecting the ELISA kit and its detection method of anti-MDA5 antibody |
| CN111153969A (en) * | 2020-01-13 | 2020-05-15 | 中国人民解放军陆军军医大学 | Botulinum toxin antigen protein, recombinant vector and application thereof |
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2007
- 2007-03-07 CN CN 200710064227 patent/CN101016336A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107490680A (en) * | 2016-06-09 | 2017-12-19 | 常州博闻迪医药科技有限公司 | A kind of Blood glycated haemoglobin enzyme-linked immune detection method |
| CN109374899A (en) * | 2018-08-24 | 2019-02-22 | 上海飞晟生物科技有限公司 | It is a kind of for detecting the ELISA kit and its detection method of anti-MDA5 antibody |
| CN111153969A (en) * | 2020-01-13 | 2020-05-15 | 中国人民解放军陆军军医大学 | Botulinum toxin antigen protein, recombinant vector and application thereof |
| CN111153969B (en) * | 2020-01-13 | 2022-06-07 | 中国人民解放军陆军军医大学 | Botulinum toxin antigen protein, recombinant vector and application thereof |
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