CN105198992B - A kind of preparation method of anti-Vibrio vulnificus haemolysis fibroin (VVH) antibody in people source and application - Google Patents
A kind of preparation method of anti-Vibrio vulnificus haemolysis fibroin (VVH) antibody in people source and application Download PDFInfo
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Abstract
The invention discloses a kind of preparation method of anti-Vibrio vulnificus haemolysis fibroin (VVH) antibody in people source and applications.Anti- Vibrio vulnificus haemolysis fibroin (VVH) antibody in people source of the present invention is human antibody, has the ability for neutralizing Vibrio vulnificus haemolysis fibroin VVH.The antibody of anti-VVH provided by the present invention includes light chain variable region and heavy chain variable region, the amino acid sequence of the heavy chain variable region be in sequence table sequence 3 from the 135th 260 amino acids residue of amino terminal, the amino acid sequence of the light chain variable region be in sequence table sequence 3 from the 1st 113 amino acids residue of amino terminal.The present invention is using immobilization Vibrio vulnificus VVH as antigen, with the natural Large phage library in people source of structure, successfully screen the anti-Vibrio vulnificus VVH antibody in people source, it is identified through ELISA, the antibody of the anti-VVH of the present invention has specificity well, has broad application prospects in the treatment of vibrio infection.
Description
Technical field
The invention belongs to biotechnologies, and in particular to a kind of anti-Vibrio vulnificus haemolysis fibroin (VVH) antibody in people source
Preparation method and application.
Background technology
Vibrio vulnificus is that a kind of pathogenic mermaid more outstanding in offshore sea waters suffers from morbid vibrio altogether, is naturally occurring in coastal waters
In seawater and marine flora and fauna, human body can be entered through gastrointestinal tract mucous or cutaneous open wound, lead to cellulitis, septic
The serious infection symptoms such as shock, multiple organ failure use antibiotic active treatment, still there is the very high death rate (＞
50%), therefore the health to human body or even existence and sea fishery etc. have high risks.It is directed to wound arc both at home and abroad at present
The treatment of bacterium is still based on antibiotic traditional therapy, the problems such as the appearance of antibody-resistant bacterium and medicament residue so that biology
Treating the research of new method becomes current hotspot, especially in recent years, the anti-infectious immunity treatment based on monoclonal antibody
Progress is rapid, but also having become the important content of modern biotechnology pharmacy for the antibody mediated immunity treatment of Vibrio vulnificus.
Vibrio vulnificus it is pathogenic related to a variety of virulence factors, wherein Vibrio vulnificus hemolysin (VVH) is a kind of to generally acknowledge
The virulence factor by Vibrio vulnificus structural gene vvhA coding, discharged when being Vibrio vulnificus invasion human body to extracellular unique
A kind of after birth perforation toxin, can induce mammalian cell to necrose, apoptosis, key effect is played in pathogenic course.
The detection antibody preparation for VVH is had been developed that at present, for Vibrio vulnificus toxin protein, nonmetallic protease, outer membrane egg
It is white that virulence factors neutrality antibody is waited also to come out, the small of infection lethal dose Vibrio vulnificus can be effectively treated to a certain extent
Mouse improves survival rate, and partial antibody vaccine has been applied to the prevention of fish disease in sea fishery, and has obtained preferable prevention effect
Fruit.But these Vibrio vulnificus neutralizing antibodies are mouse sources or other animal derived so far, belong to human body heterologous protein, easily make human body
Heterologous antigenic reaction is generated, thus limits its further application in human body.And high-affinity, high antibacterial activity
The development of humanized antibody then largely compensates for this defect, has become the main of the following anti-infection bio treatment
Developing direction.
Phage antibody library technique is to produce the main method of full humanized antibody at present, it can be by repertoire antibody base
Because being illustrated in phage surface, and the selection index system of analog antibody immune system in vitro, resist to being presented on phage surface
Body is screened with immobilization antigen in vitro.Display technique of bacteriophage realizes the important breakthrough of human antibody technology of preparing,
Animal and cell fusion need not be immunized, required gene is directly screened using antigen.Using screening of phage antibody library technology, at present
It is existing (such as to be exhaled for various bacteria (such as anti-streptococcus pneumonia, tubercle bacillus, yersinia pestis, Klebsiella Pneumoniae) and virus
Inhale road coronavirus, dengue fever virus etc.) ScFv human single chain variable fragments antibodies come out, but there is not yet anti-Vibrio vulnificus humanization is anti-
The research of body is reported.
Invention content
It is an object of the present invention to provide a kind of antibody of anti-Vibrio vulnificus VVH.
The antibody of anti-Vibrio vulnificus VVH provided by the invention includes light chain variable region and heavy chain variable region；The heavy chain can
Sequence 1 from amino terminal 135-260 amino acids residues, the light chain variable in becoming the amino acid sequence in area as sequence table
The amino acid sequence in area be in sequence table sequence 1 from amino terminal 1-113 amino acids residues.
In above-mentioned antibody, the antibody of the anti-Vibrio vulnificus VVH in antibody behaviour source of the anti-Vibrio vulnificus VVH, the anti-wound
The antibody for hindering vibrios VVH is single-chain antibody.
In above-mentioned antibody, the antibody be it is following 1) or 2) shown in protein：
1) protein that amino acid sequence forms shown in sequence in sequence table 1；
2) by the amino acid residue sequence of sequence in sequence table 1 by one or several amino acid residues substitution and/or
It lacks and ors add and the protein with the same function derived from 1).
It is a further object to provide the non-coding DNA molecules of above-mentioned antibody.
The present invention provides the 405- that the nucleotides sequence of the heavy chain variable region of above-mentioned antibody is classified as sequence 2 in sequence table
780 nucleic acid molecules；The nucleotides sequence of the light chain variable region of the antibody is classified as 1-339 cores of sequence 2 in sequence table
Thuja acid molecule.
In above-mentioned non-coding DNA molecules, the non-coding DNA molecules are following 1) -3) in any DNA molecular：
1) its nucleotide sequence is the sequence 2 in sequence table；
2) DNA molecular that can be limited under strict conditions with sequence in sequence table 2 hybridizes and the DNA of encoding such antibodies divides
Son；
3) there is 90% or more homology and the DNA molecular of encoding such antibodies with DNA molecular 1).
It is a still further object of the present invention to provide the recombinant expression carrier containing above-mentioned non-coding DNA molecules or expression cassette or turn
Gene cell system or recombinant bacterium.
Above-mentioned antibody is in following a)-d) in it is any in application also belong to protection scope of the present invention：
A) product of specific binding Vibrio vulnificus VVH is prepared；
B) product of the free VVH antigens of specific binding is prepared；
C) product for the treatment of or auxiliary treatment Vibrio vulnificus is prepared；
D) it is used as Vibrio vulnificus vaccine.
Final object of the present invention be to provide it is a kind for the treatment of or the anti-vibrio infection of auxiliary treatment product.
The active constituent of the product for the treatment of provided by the invention or the anti-vibrio infection of auxiliary treatment is above-mentioned antibody.
In above application or above-mentioned antibody, the product is drug.
It to unique cytotoxin VVH of extracellular release is target antigen that the present invention, which selects Vibrio vulnificus, utilizes natural great Rong
Measure the anti-Vibrio vulnificus VVH antibody in people source prepared by Large human naive scFv phage library screening technique, antibody structure and type with it is existing
The Vibrio vulnificus of report detects and therapeutic antibodies differ, which has higher neutralization Vibrio vulnificus VVH
Ability, compared with domestic and international existing antibody, this antibody is complete human antibody, and it is anti-will not theoretically to induce apparent allergy
It answers；And circulating half-life in vivo is short, penetration power is strong, will not have residual contamination as antibiotics drug, in Vibrio vulnificus infection
It has broad application prospects in treatment.
Description of the drawings
Fig. 1 is the Competitive assays rate of different anti-VVH phage antibodies and free antigen.
Fig. 2 is the neutralization activity of anti-VVH phage antibodies.
Specific implementation mode
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
In document, " Qiao Yuanyuan, Wang Yan, Chen Xiaosui, Wang Yuxiao change ice to naive phage antibody library in following embodiments
The structure of Large phage library and identification, Chinese microbiology and Journal of Immunology, 2004,24 (3), in 194-197.
It is disclosed in state's PLA's Navy General Hospital ", the public can obtain from General Hospital of the PLA Navy.
In document, " Wang Yan, Wang Yuxiao, Chen Xiaosui change ice, Liu Xiaolin, for big to BS1365 cells in following embodiments
The structure of the expression vector of capacity phage antibody library and identification, China Immunology Journal, 2003,19 (2), 93-96. Chinese
It is disclosed in people's liberation army Navy General Hospital ", the public can obtain from General Hospital of the PLA Navy.
In document, " Wang Yan, Liu Qunying change ice to Escherichia coli HB2151 in following embodiments, and Chen Yuping, Zhu Yingchun are old
Know fringe, anti-tnf-alpha people's single-chain antibody, Journal of Immunology, 1999,15 (4) are cloned from semi-synthetic antibody library:87-89. " in disclose
It crosses, the public can obtain from General Hospital of the PLA Navy.
The VVH antigens for screening in following embodiments are in document " Wei Jie, Guo Jianwei, the Vibrio vulnificus haemolysis such as horse piebald horse
The high efficient expression of plain gene and identification Journal of the Fourth Military Medical University, 2008,29 (14):1249-1252. " and document " Fu Kaifei,
The foundation of the anti-Vibrio vulnificus monoclonal antibody hybridoma cell lines of the such as Ma Cong, Guo Jianwei and Preliminary Identification cells and molecular immune
Learn magazine, 2012,28 (3):It is disclosed in 279-281. ", the public can obtain from Navy General Hospital clinical laboratory.
The solvent of super nutritive meat soup (Super broth, SB) culture solution in following embodiments be water, solute and its
It is a concentration of in SB culture solutions：Tryptone 30g/L, yeast powder 20g/L, 3- (N- morpholinoes) propane sulfonic acid 10g/L.
The preparation method of embodiment 1, anti-VVH antibody
One, the preparation of large capacity Large human naive scFv phage library
Bibliography " Sblattero D, Bradbury A.Exploiting recombination in single
bacteria to make large phage antibody libraries.Nat Biotechnol,2000；18:In 74. "
Method prepare large capacity Large human naive scFv phage library, be as follows：
1,7 × 10 are taken out from naive phage antibody library12The phage particle superinfection of cfu (clonogenic unit)
BS1365 cells, infection multiplicity (multiplicity of infection, MOI 100/1, specific steps reference literature " king
A kind of jade, Wang Yuxiao, Chen Xiaosui change ice, and Liu Xiao woodss is used for structure and the identification of the expression vector of Large phage library, in
State's Journal of Immunology, 2003,19 (2), the method in 93-96. ", 37 DEG C of water-bath 1h so that more copy carriers enter it is same thin
Born of the same parents, the positioning recombination mediated by cre-loxp make the random exchange pairing of light and heavy chain between same intracellular different carriers, obtain
To infection system.
2,2 × YT culture solution (solvents containing ampicillin (100ug/ml) are added into the infection system of above-mentioned steps 1
It is for water, solute and its mass fraction in culture solution：Peptone 1.6%, yeast extract 1% and sodium chloride
0.5%) to 400mL, 37 DEG C of shaken cultivations to exponential phase (OD=0.5).
3, (pfu=1.7 × 10 5ml helper virus VCSM13 are added in the infection system after being cultivated to step 212)(Merck
ST200251), 30 DEG C of low temperature induction overnight incubations, next day centrifuge supernatant, and PEG8000 (being purchased from Promege companies) recycling precipitates,
And phage titre is measured, then the XL1-Blue bacterium (1000ml) of exponential phase are infected with the ratio of MOI≤1
(Stratagen, article No.:200228), 37 DEG C of static 30min.
4, it takes 10 μ l to be laid on the culture plate containing ampicillin and calculates storage capacity, ampicillin, which is added, keeps its final concentration of
100g/ml adds (pfu=1.7 × 10 10ml helper phage VCSM1312), 30 DEG C of low temperature induction overnight incubations, next day returns
Supernatant is received, PEG8000 recycling precipitations obtain large capacity recombinant phages antibody work library (large capacity people source phage antibody
Library), and calculate the storage capacity and titre in large capacity recombinant phages antibody work library.
The result shows that：The storage capacity of large capacity Large human naive scFv phage library is 7 × 1010, titre is 3 × 1012cfu/ml。
Two, the screening, preparation of anti-VVH phage antibodies and ELISA detections
1, the screening of anti-VVH phage antibodies
" Wang Yan, Liu Qunying change the such as ice from humanized's phage antibody library to the screening reference literature of anti-VVH phage antibodies
Isolate 1 plant of anti-HBsAg Fab clone's China Immunology Journal containing unusual sequences, 1998,14 (1):115-119. " and
" such as Wang Yuxiao, Qiao Yuanyuan, Zhou Lijun screen anti-tnf-alpha people single-chain antibody cells and molecule to document from high-capacity antibody library
Biology magazine, 2008,24 (9):878-880. " in method.It is as follows：
(1) VVH antigens are diluted to 50 μ g/ml with the carbonate buffer solution (PH9.6) of 0.05mol/L, obtain VVH antigens
Solution.
(2) the immune pipe of 1ml VVH antigenic solutions coating is taken, 4 DEG C overnight, closes 2h with 2.5% skimmed milk power, is added
Large capacity Large human naive scFv phage library (about 3 × 10 prepared by 1ml above-mentioned steps one12Cfu), 37 DEG C of incubation 2h.
(3) product of step (2) is washed 15 times (2-4 wheel washing 20 times or more) with PBST, distillation water washing 1 time,
Then the fresh XL1-Blue bacterium direct infections of 1ml, 37 DEG C of incubation 15min are added, then are transferred to 10ml super nutritive meat soups
In (Super broth, SB) culture solution (containing 20 μ g/ml ampicillins).
(4) 1ml glycine elutions buffer solution (0.1mol/L HCl, glycine tune pH value are added into the product of step (3)
To 2.2, it is that 0.1%), after 37 DEG C stand 15min, elution recycling obtains eluent that BSA, which is added, to its mass fraction.
(5) eluent of above-mentioned steps (4) is neutralized to pH7.0 with 2mol/L Tris, the XL1-Blue of 10ml is added
Bacterium, 37 DEG C of standing 15min, adds the SB culture solutions of 10ml.
(6) bacterium solution that appropriate step (5) obtains is laid on the culture plate containing ampicillin, measures titre, remaining bacterium solution
Be added containing ampicillin culture solution to 100ml after 37 DEG C of 3~4h of culture, add helper virus VCSM13 (2 ×
1012Cfu), 30 DEG C of shaken cultivations are stayed overnight.Next day recycles supernatant, and PEG8000 precipitations obtain secondary phage antibody library.
(7) above-mentioned secondary phage antibody library is carried out to the screening of next round again, and keeps VVH coatings anti-in next round screening
Former concentration is successively decreased 10 μ g/ml by wheel.The titre in secondary library after often wheel screens is measured, and calculates the rate of recovery of phage antibody,
It is enriched with index as specific VVH phage antibodies.
It is as shown in table 1 that phage antibody library respectively takes turns the selection result：As can be seen from Table 1：Specific VVH phage antibodies
Concentration effect it is good, enrichment times 100.
Table 1, phage antibody library respectively take turns the selection result
Note:The rate of recovery=recycling bacteriophage/input bacteriophage
(8) 85 clones of random picking from the culture plate colony that third, fourth round screening are enriched with, train respectively at 2 × YT
Overnight incubation in nutrient solution, next day take 50 μ l bacterium solutions to be added in 2ml SB culture solutions, and 37 DEG C of cultures add auxiliary to exponential phase
(pfu=1.7 × 10 helper phage VCSM1312), 30 DEG C of overnight incubations collect supernatant, respectively obtain anti-VVH phage antibodies.
2, the ELISA detections of anti-VVH phage antibodies
The anti-VVH phage antibodies solution that above-mentioned steps 1 are obtained respectively carries out ELISA detections, while with ovalbumin
(OVA) (Sigma companies are purchased from) and bovine serum albumin(BSA) (BSA) (being purchased from Sigma companies) identifies its antigen-antibody as a contrast
The specificity of reaction.It is as follows：
(1) it is coated with：50 μ L VVH antigenic solutions (1 μ g/ml) are added in 96 hole elisa Plates, 37 DEG C of coatings are overnight.
(2) it closes：3% defatted milk-PBS closes 2h；
(3) add antibody：Anti- VVH phage antibodies to be measured 50 μ L, 37 DEG C of incubation 1h prepared by above-mentioned steps 2 is added；PBST
Washing is three times；
(4) add secondary antibody：The anti-M13 mouse monoclonal antibodies of HRP- (being purchased from GE company 27-9420-01) of 50 μ L of Xiang Kongzhong additions, 37 DEG C
It is incubated 3h；
(5) it develops the color：50 μ L substrates developing solutions are added per hole and (are purchased from Shanghai Mai Yueer biotech companies, article No.：
1A001)：Carry out colour developing 5min.Survey OD values at A490nm.
It will develop the color for positive clone again respectively with ovalbumin (OVA), bovine serum albumin(BSA) (BSA) and ferritin
(Fer) (Sigma companies are purchased from) and are used as antigen, carry out specific detection according to the method described above, experiment is set to be repeated three times.
The result shows that：In above-mentioned steps 2 in 85 clones of picking, there can be 22 with the protein bound positive colonies of VVH,
Positive rate 25.9%.Wherein 3 combine the ELISA testing results of active high clone as shown in Figure 2.
Table 2, phage antibody are detected from different antigen reactive ELISA
Three, the acquisition of soluble anti-VVH antibody and ELISA are identified
1, the acquisition of soluble anti-VVH antibody
22 specific anti-VVH phage antibodies of the positive for being identified two ELISA of above-mentioned steps with 2 × YT culture solutions
Carry out 108Dilution postoperative infection Escherichia coli HB2151 again, 37 DEG C of incubation 15min, inoculated and cultured disk, and picking individual colonies are put into
Overnight incubation in 2 × YT culture solutions, the bacterium solution that next day takes 50 μ l to be incubated overnight are added in the SB culture solutions of 1.5mL, 37 DEG C of cultures
To exponential phase, IPTG (1mmol/L) (being purchased from Promege companies) is added, 30 DEG C of overnight incubations, anti-VVH's is single-stranded anti-
Body by it is free it is soluble in the form of express in non-inhibited phenotype bacterium HB2151, the supernatant cultivated is soluble anti-VVH anti-
Body.
2, the ELISA identifications of soluble anti-VVH antibody
The anti-VVH antibody of solubility of above-mentioned acquisition is subjected to ELISA detections, while pure with ovalbumin (OA) and ox blood
Albumen (BSA) is as a contrast.For ELISA detection method with 3 in above-mentioned step 2, the antibody in step (3) is soluble anti-VVH
Antibody, the secondary antibody in step (4) are the anti-V5 antibody (Invitrogen, R961-25) of HRP-.Survey OD values at A490.Experiment sets three
Secondary repetition.
ELISA testing results show to share 12 strain clones in 22 positive colonies active, have specific binding activity with VVH
Clone have 8.The anti-VVH antibody of solubility of active high clone is combined to be respectively designated as antibody strain 1, antibody by wherein 3
Strain 2 and antibody strain 3.Antibody strain 1, antibody strain 2 and antibody strain 3 and different antigen reactive ELISA testing results are as shown in table 3.
Table 3, soluble antibody and different antigen reactive ELISA testing results
Four, the Competitive assays of antibody and neutralization activity measure
1, the preferable antibody strain 1 of activity, antibody strain 2 and the antibody strain 3 that selecting step three obtains, use Competitive assays respectively
ELISA method measures its neutralization activity.It is as follows：
It by VVH antigenic solutions (1 μ g/ml) coated elisa plate, is closed with 3% defatted milk, 50 μ l is added per hole and combine activity
Preferable anti-VVH antibody, while being separately added into the free VVH antigens (diluted concentration 1 of 50 μ l various concentrations:100、1:500、
1:5000) it is, control with OVA antigens, 37 DEG C of incubations 1h, PBST wash rear and HRP- antiphagins antibody (1 three times:2000 is dilute
Release) reaction, it is developed the color with OPD, H2SO4After termination, OD values at 490nm are measured.Control group is anti-VVH antibody+unrelated free antigen
Group；Experimental group is anti-VVH antibody+Competitive assays VVH antigen groups.Neutralization activity inhibiting rate (%)=(control group A 490- experimental groups
A490)/control group A 490 × 100%.
The results are shown in Figure 1：The anti-VVH antibody of 3 plants of solubilities obtained can specifically bind free VVH antigens, production
Raw depression effect, depression effect increase with the increase of free antigen, and dosage is proportionate.The antibody strain 1 for selecting effect good
Carry out neutralization activity measurement.
2, VVH human antibodies neutralization activity detects
Neutralization activity is carried out using Competitive assays ELISA method to the phage antibody of antibody strain 1 to be detected.Specific steps
It is as follows：
(1) routine immunization method is used to prepare the anti-Vibrio vulnificus serum of mouse, after cracking thalline using sonioation method, by thalline
Albumen and equivalent complete Freund's adjuvant mixing, conventional intraperitoneal injection are immunized BALB/c mouse (8 week old, 180 μ g/ are only), exempt from every 3 weeks
Epidemic disease 1 time is immunized 4 times altogether, and 3d eyeballs take blood after final immunization, detaches serum, is saved backup for -20 DEG C after detection potency；
(2) it by VVH antigen coat ELISA Plates, is closed with 3% defatted milk, the bacteriophage of the antibody strain 1 of 50ul is added per hole
Antibody, while being separately added into 50ul gradient dilutions (1:100,1:500,1:2500,1:12500) the anti-Vibrio vulnificus blood of mouse
Clearly, 37 DEG C incubation 1h, PBST wash 3 times after with HRP- sheep anti-mouse iggs (1:2000 dilutions) reaction, it is developed the color with OPD, H2SO4It terminates
Afterwards, OD values at 490nm are measured.Unrelated phage antibody (TNF-α antibody)+anti-Vibrio vulnificus serum is as negative control.It neutralizes
Maximum inhibition (%)=(negative control A490- laboratory sample A490)/negative control A490 × 100%.
The results are shown in Figure 2：After the anti-Vibrio vulnificus serum doubling dilution of mouse, the neutralization activity of confrontation VVH antibody inhibits
Rate is respectively 43.90%, 38.67%, 21.23% and 5.53%, and then unrestraint effect when anti-TNF-α antibody is added.Explanation
The anti-VVH antibody of the present invention can specifically bind Vibrio vulnificus VVH.
Five, the gene sequencing of the anti-antibody variable regions VVH
It chooses the high and 1 Song Nuosai companies of antibody strain with neutralization activity of specific binding activity and carries out gene order survey
It is fixed, sequence analysis is carried out to the variable region of the clone using PCGENE softwares.
The result shows that：The anti-VVH antibody (antibody strain 1) that the present invention obtains is human antibody, the light chain of moderate resistance VVH antibody
Variable region gene comes from VKII subgroups, and heavy chain variable region gene comes from VH3 subgroups, the amino acid sequence such as sequence of the anti-VVH antibody
Shown in row 1, wherein being heavy chain variable region from amino terminal 135-260, from amino terminal, 1-113 are light chain variable
Area；It encodes in the nucleotide sequence such as sequence table of the anti-VVH antibody shown in sequence 2, wherein 405-780 is heavy chain variable region
Coding gene sequence, 1-339 are the coding gene sequence of light chain variable region.
Claims (11)
1. a kind of antibody of anti-Vibrio vulnificus VVH comprising light chain variable region and heavy chain variable region, the ammonia of the heavy chain variable region
Base acid sequence be in sequence table sequence 1 from amino terminal 135-260 amino acids residues, the amino of the light chain variable region
Acid sequence be in sequence table sequence 1 from amino terminal 1-113 amino acids residues.
2. antibody according to claim 1, it is characterised in that：The anti-wound in antibody behaviour source of the anti-Vibrio vulnificus VVH
The antibody of the antibody of vibrios VVH, the anti-Vibrio vulnificus VVH is single-chain antibody.
3. antibody according to claim 1 or 2, it is characterised in that：The antibody is the ammonia shown in sequence in sequence table 1
The protein of base acid sequence composition.
4. the non-coding DNA molecules of any antibody in claim 1-3.
5. non-coding DNA molecules according to claim 4, it is characterised in that：The nucleotide of the heavy chain variable region of the antibody
Sequence is 403-780 nucleic acid molecules of sequence 2 in sequence table；The nucleotides sequence of the light chain variable region of the antibody is classified as
1-339 nucleic acid molecules of sequence 2 in sequence table.
6. non-coding DNA molecules according to claim 4 or 5, it is characterised in that：The nucleotides sequence of the non-coding DNA molecules
Row are as shown in the sequence 2 in sequence table.
7. thin containing the recombinant expression carrier of any non-coding DNA molecules or expression cassette or transgenosis in claim 4-6
Born of the same parents system or recombinant bacterium.
8. in claim 1-3 any antibody it is following a) or b) in application：
A) product for the treatment of or auxiliary treatment Vibrio vulnificus is prepared；
B) Vibrio vulnificus vaccine is prepared.
9. application according to claim 8, it is characterised in that：The product is drug.
10. the product of a kind for the treatment of or the anti-vibrio infection of auxiliary treatment, active constituent is any described in claim 1-3
Antibody.
11. product according to claim 10, it is characterised in that：The product is drug.
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| CN102171248B (en) * | 2008-10-01 | 2015-07-15 | 安进研发（慕尼黑）股份有限公司 | Cross-species-specific PSMAxCD3 bispecific single chain antibody |
| CN101818200A (en) * | 2010-03-04 | 2010-09-01 | 宁波博奥生物工程有限公司 | Wound vibrio quantitative detecting test strip |
| US8420072B2 (en) * | 2011-03-24 | 2013-04-16 | The United States Of America, As Represented By The Secretary Of Agriculture | Vaccination of sex reversed hybrid tilapia (Oreochromis niloticus x O. aureus) with an inactivated Vibrio vulnificus vaccine |
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