CN102719466A - Recombinant plasmid and subunit vaccine of extracellular protease recombinant protein of Aeromonas hydrophila prepared from same plasmid - Google Patents
Recombinant plasmid and subunit vaccine of extracellular protease recombinant protein of Aeromonas hydrophila prepared from same plasmid Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and relates to a recombinant plasmid and a subunit vaccine of extracellular protease recombinant protein of Aeromonas hydrophila, the subunit vaccine being prepared therefrom the recombinant plasmid. The recombinant plasmid pET32a-epr2-3 uses pET32a as an original plasmid, an epr3 gene of the Aeromonas hydrophila is inserted between a restriction site KpnI and a restriction site EcoRI of the plasmid pET32a, and an epr2 gene of the Aeromonas hydrophila is inserted between the restriction site EcoRI and a restriction site SalI. The subunit vaccine of extracellular protease recombinant protein of Aeromonas hydrophila comprises a recombinant protein epr2-3 obtained by inducing an Escherichia coli transfected with the recombinant plasmid pET32a-epr2-3 to express and an adjuvant. Experiments show that the subunit vaccine is relatively high in security, simple in production technology, and undemanding in preservation and transportation conditions, can stimulate immune response of a body in all respects, and has a wide range of development and application prospects.
Description
Technical field
The invention belongs to biological technical field, relate to the Aeromonas hydrophila extracellular protease recombinant protein subunit vaccine of a kind of recombinant plasmid and preparation thereof.
Background technology
The applications well in existing more than 40 year of gene clone technology; It comprises connecting in external manual work with the carrier DNA that the self-replicating ability is arranged from different biological genes; Be built into new recombinant DNA; Send into then and go in the receptor biological to express, thereby produce the transfer of genetic material and state and reconfigure.
Subunit vaccine has many characteristics: (1) subunit vaccine is stable, expense is low, produce easily and use; (2) being one type of new generation vaccine, is the vaccine that the component of the former existence of protective immunity that pathogenic bacterium are main is processed.(3) even remove in the pathogenic agent and excite the protective immunity deleterious composition that has nothing to do, strengthened the immunogenicity of vaccine effectively and reduced spinoff.
Aeromonas hydrophila (Ah) is one of important threat of China's culture fishery, and it is the conditioned pathogen that people, animal and hydrocoles suffer from altogether.This bacterium extensively is present in the water surrounding, is the main pathogenic bacterium of multiple aquatic animal, also can cause human diarrhoea or septicemia etc., and food safety is had threat, and is to suppress patient's function of immune system and liver function.Pathogenic numerous with it virulence factor of this bacterium is closely related; Wherein extracellular protease is one of modal virulence factor; It has phagolysis; Can stimulate body to produce specific antibody, closely related with the immanoprotection action of pathogenic and the body of Ah, be the focus of studying the Aeromonas hydrophila vaccine always.And the recombinant subunit vaccine that the virulence gene extracellular protease that utilizes Ah prepares does not still have relevant report at present.
Summary of the invention
The objective of the invention is to develop a kind of recombinant subunit vaccine that can effectively control the Aeromonas hydrophila infection, the prevention and control that can be Aeromonas hydrophila provide strong support.
The object of the invention can be realized through following technical scheme:
A kind of recombinant plasmid pET32a-epr2-3; This recombinant plasmid is the plasmid that sets out with pET32a; Insert the epr3 gene of Aeromonas hydrophila between its Kpn I and the EcoR I restriction enzyme site; Insert the epr2 gene of Aeromonas hydrophila between EcoR I and Sal I restriction enzyme site, wherein said epr3 gene GenBank accession number is EU275147.1, and epr2 gene GenBank accession number is CP000462.1.
Described recombinant plasmid pET32a-epr2-3 preferably prepares through following method:
(1) clone of epr2 and epr3 gene: the DNA that with the preserving number is the Aeromonas hydrophila J-1 strain of CGMCC NO.3220 is a template; Pass through pcr amplification with primer Epr2-1/Epr2-2 and primer Epr3-1/Epr3-2 respectively; Obtain epr2 gene and epr3 gene; It is inserted the pMD-18T plasmid respectively, obtain pMD-18T-epr2 and pMD-18T-epr3;
(2) structure of recombinant plasmid pET32a-epr3: utilize Kpn I and EcoR I that pMD-18T-epr3 and pET32a are carried out double digestion respectively; The epr3 gene enzyme that obtains is cut between the Kpn I and EcoR I restriction enzyme site of product insertion pET32a, obtained recombinant plasmid pET32a-epr3;
(3) structure of recombinant plasmid pET32a-epr2-3: utilize EcoR I and Sal I that pMD-18T-epr2 and pET32a-epr3 are carried out double digestion respectively; Enzyme is cut between the EcoR I and Sal I restriction enzyme site of product epr2 gene insertion recombinant plasmid pET32a-epr3, obtained recombinant plasmid pET32a-epr2-3.
The host of containing described recombinant plasmid pET32a-epr2-3.
Described host's preferred bacterium or cell, further preferred e. coli bl21.
A kind of Aeromonas hydrophila extracellular protease recombinant protein Epr2-3, described Aeromonas hydrophila extracellular protease recombinant protein is secreted by the described recombination bacillus coli BL21 that contains recombinant plasmid pET32a-epr2-3.
Described Aeromonas hydrophila extracellular protease recombinant protein Epr2-3; The recombination bacillus coli BL21 that preferably will contain recombinant plasmid pET32a-epr2-3 is inoculated in the LB substratum, adds 0.1M IPTG inducible protein after the shaking culture and expresses, and continues to cultivate the centrifugal acquisition thalline in back; Cleer and peaceful inclusion body in the centrifugal acquisition after the ultrasonication; Inclusion body is carried out the sex change dissolving, get both more respectively, collect gained and be recombinant protein Epr2-3 with His affinity column purification of recombinant proteins.
The application of described recombinant plasmid pET32a-epr2-3 in preparation Aeromonas hydrophila extracellular protease recombinant protein subunit vaccine.
The application of described Aeromonas hydrophila extracellular protease recombinant protein Epr2-3 in preparation Aeromonas hydrophila extracellular protease recombinant protein subunit vaccine.
A kind of Aeromonas hydrophila extracellular protease recombinant protein subunit vaccine contains described Aeromonas hydrophila extracellular protease recombinant protein Epr2-3 and adjuvant.
Preferred Freund's complete adjuvant of described adjuvant or DC-Chol adjuvant.
Beneficial effect:
The present invention has successfully made up the recombinant subunit vaccine of expressing the Ah extracellular protease first, and it has broken through the limitation of conventional inactivated vaccine of Ah and less toxic vaccine, has the replicability of less toxic vaccine and the security of inactivated vaccine concurrently, passes through safety evaluation easily.
Evidence, construction expression Ah recombinant subunit vaccine of the present invention have produced the antibody to Ah, and its antibody titers is significantly higher than, and inactivated vaccine produces.
The serotype of Aeromonas hydrophila is numerous, and there is limitation in the immune effect of inactivated vaccine.And extracellular protease Epr2, Epr3 are modal virulence factor and protective antigens among the different serotypes hypotype Ah.Extracellular protease gene epr2, epr3 tandem expression are obtained recombinant protein Epr2-3, can be as pathogenic hydrophila gingivalis different serotypes common protective antigen, the induced animal body produces high antibody of tiring.This subunit vaccine production technique is simple, and the storage and transport conditional request is not high, and animal is not had any detrimentally affect, can not cause the diffusion of bacterium, is a kind of ideal genetic engineering subunit vaccine, aspect the preventing and treating of Ah, has broad application prospects.
Description of drawings
Fig. 1 tandem expression recombinant plasmid pET32a-epr2-3 structural representation
Ori is a promotor, and Amp goes up antibiotic resistance gene for carrier pET32a, and epr2, epr3 are goal gene, and KpnI, EcoRI, SalI are restriction enzyme site.
Fig. 2 tandem expression recombinant plasmid pET32a-epr2-3 enzyme is cut detection
1:DL5000DNA Marker 2:pET32a-epr2-3 enzyme is cut the SDS protein electrophoresis of product 3:pET32a-epr2-34:DL10000DNA Marker Fig. 3 recombinant protein Epr2-3
1: albumen Marker MP1022: the intestinal bacteria 3 that transform pET32a: the intestinal bacteria that transform recombinant plasmid pET32a-epr2-3
Fig. 4 western blot identifies the reactionogenicity of recombinant protein Epr2-3
M: dye albumen Marker 1 in advance: the intestinal bacteria 2 that transform recombinant plasmid pET32a-epr2-3: the intestinal bacteria that transform pET32a
Fig. 5 indirect ELISA is measured the antibody titers of immune serum
Can find out that recombinant protein Epr2-3 mice immunized serum antibody titer organizes apparently higher than other, can better stimulate body to produce immunoreation.
Fig. 6 recombinant protein cooperates with different adjuvants measures immunogenicity
The vaccine that Freund's complete adjuvant and DC-Chol cooperate recombinant protein Epr2-3 to the immune protective rate of mouse up to 95%, far above 65% of deactivation group.
Biomaterial preservation information
Aeromonas hydrophila J-1 strain; Classification called after Aeromonas hydrophila Aeromonas hydrophila; This bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on August 6th, 2009; The address is a Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, and its preserving number is CGMCC NO.3220.
Embodiment
1.epr2, the amplification of epr3 gene, clone, evaluation
1.1PCR primer design and synthetic
According to two pairs of Auele Specific Primers to epr2 (GenBank CP000462.1) and epr3 (GenBank EU275147.1) of gene order design of Ah standard strain J-1, holding respectively at 5 ' of primer, the Chinese has the digestion with restriction enzyme site.Because it is synthetic by Shanghai invitrogen biotech firm.
Epr2-1：5′-CCG?GAA?TTC?ATG?ACA?CTC?TTG?CTC?ACC?ACC?C-3′（SEQ?ID?No.1），
Epr2-2：5′-GTC?GAC?CTA?GGA?GGC?GGG?CAG?CC-3′（SEQ?ID?No.2），
Epr3-1：5′-GGT?ACC?ATG?AAA?GCG?ACT?CCC?ATT?GCC?C-3′（SEQ?ID?No.3），
Epr3-2：5′-CCG?GAA?TTC?TCA?GTT?TTC?GCT?CGG?CGT?ATT?CTC-3′（SEQ?ID?No.4）
1.2PCR
Get 2*Taq PCR Mix 12.5 μ l, epr2-12 μ l, epr2-22 μ l, Aeromonas hydrophila J-1 strain (CGMCC NO.3220) bacterium liquid 4 μ l mend to TV 25 μ l with aseptic ultrapure water.On the PCR appearance, react.Loop parameter is 95 ℃ of preparatory sex change 5min, 95 ℃ of sex change 30s, and 57 ℃ of annealing 30s, 72 ℃ are extended 30s, carry out 30 circulations altogether; 72 ℃ are extended 5min then.Working method to epr3-1 is the same, and loop parameter is 95 ℃ of preparatory sex change 5min, 95 ℃ of sex change 30s, and 55 ℃ of annealing 30s, 72 ℃ are extended 1min, carry out 30 circulations altogether; 72 ℃ are extended 5min then.The PCR product carries out 1.2% (g/ml) agarose gel electrophoresis.
1.3PCR product reclaims and purifying
The PCR product downcuts the sepharose piece that contains the purpose band under uv lamp behind agarose gel electrophoresis, reclaim purification kit fast with DNA and reclaim the purpose fragment in the gel.The concrete operations step is undertaken by the specification sheets that Geneaid company gel reclaims test kit.
1.4PCR the enzyme of product is cut purifying
The endonuclease reaction TV is 50 μ l, and behind the PCR, epr2 reclaims fragment 41 μ l, 10 * T buffer, 5 μ l, EcoRI2 μ l, SalI2 μ l.37 ℃ of effect 3.0h carry out agarose gel electrophoresis then and reclaim test kit and enzyme is cut after product reclaim, and method is the same.Epr3 reclaims fragment 41 μ l, 10 * T buffer, 5 μ l, and KpnI2 μ l, EcoRI2 μ l, all the other operate same epr2.1.5pMD-18T cut purifying with the enzyme of pET32a
The endonuclease reaction TV is 50 μ l, respectively pMD-18T and pET32a two plasmids is carried out enzyme and cuts purifying, plasmid 41 μ l, 10 * Tbuffeer, 5 μ l, EcoRI 2 μ l, SalI2 μ l.37 ℃ of effect 3.0h carry out agarose gel electrophoresis then and reclaim test kit and enzyme is cut after product reclaim, and method is the same.With KpnI and EcoRI two plasmids are carried out enzyme with same method and cut and reclaim product.
1.6 purpose fragment epr2, epr3 are connected with pET32a's
PCR product 6.0 μ L are received in the enzyme switchback of epr3, and product 2.0 μ L, 10 * T4 ligase enzyme damping fluid, 1.0 μ L are received in the switchback of carrier pMD-18T enzyme; T4DNA ligase enzyme 1.0 μ L; Spend the night 16 ℃ of connections behind each product of mixing, obtain pMD-18T-epr3, obtain pMD-18T-epr2 with same procedure.Connect product Transformed E .coli DH5 α respectively with two.Extract pMD-18T-epr3; And pMD-18T-epr3 is carried out enzyme with EcoRI and KpnI cut, method is the same, again enzyme is cut the purpose fragment cloning that is obtained and advances in the pET32a expression vector; Transformed E .coli BL21 then obtains pET32a-epr-3. and it is carried out enzyme cuts and identify and dna sequencing.The pMD-18T-epr2 that obtains before then extracting; And pMD-18T-epr2 is carried out enzyme with EcoRI and SalI cut, method is the same, enzyme is cut gained purpose fragment cloning go into pET32a-epr-3; Transformed E .coli BL21 finally obtains recombinant plasmid pET32a-epr2-3 (Fig. 1) then.
1.7 preparation and the conversion of bacillus coli DH 5 alpha and BL21 competence bacterium
PET32a-epr2-3 transforms BL21 competence bacterium, obtains transforming the recombination bacillus coli of recombinant plasmid pET32a-epr2-3.
Preparation of DH5 α that uses in the above-mentioned steps and BL21 competence bacterium and the conversion process that is connected product see molecular cloning for details.
1.8 the extraction of recombinant plasmid and evaluation
Extract recombinant plasmid pET32a-epr2-3 and, in PCR and double digestion evaluation (Fig. 2), can see the purpose band with plasmid extraction kit with PCR and double digestion evaluation.
1.9 goal gene epr2-3 sequential analysis
Examining order is assisted to accomplish by invitrogen company.With NCBI BLAST epr2 in measured sequence and the GeneBank DB (GenBank CP000462.1) and epr3 (GenBank EU275147.1) are carried out homology relatively; The base sequence that has shown gene that we were cloned into and Aeromonas hydrophila J-1 pnca gene is in full accord, and the gene that is cloned into meets the requirement of carrier reading frame fully.
2.1 the expression of recombinant protein Epr2-3
The recombination bacillus coli bacterium liquid that is positive through evaluation among the embodiment 1 is inoculated in the fresh LB liquid nutrient medium of 5mL 37 ℃ of shaking culture 14-16h.Get fresh bacterium liquid and be re-seeded in the fresh LB liquid nutrient medium of 200mL, 37 ℃ of 200rpm shaking culture 4h are to OD600=0.6; Add 0.1M IPTG to final concentration 1mmol/L, 4h is cultivated in continuous oscillation; Take out in bacterium liquid to the 1.5 μ L centrifuge tube, 4 ℃ of centrifugal 2min of 13000rpm, the thalline after centrifugal is resuspended with 40 μ LPBS (ph=7.2), and adds 10 μ L, 5 * SDS sample-loading buffer; Heated and boiled to 100 ℃ 10min, the centrifugal 2min of 13000rpm.Get 10 μ L lysates and carry out the SDS electrophoresis, first 80V electrophoresis 30min, 120V then.Coomassie brilliant blue G250 dyeing 2h after the electrophoresis, each hour decolouring once has altogether 3 times then.Adopt Biorad2000 that albumin glue is taken pictures, can see purpose band (Fig. 3), Marker contrasts with standard protein, its size of decidable.
2.2 the purifying of recombinant protein Epr2-3
2.1 4 ℃ of shaking culture 10min of the positive thalline of middle cultivation gained, PBS is resuspended and be stored in-80 ℃.Frozen thalline needs freeze thawing 3 times before resuspended with PBS, carry out the ultrasonic disruption of 5s then with 300W power, and 15s carries out 60 circulations at interval.After the UW, 4 ℃ of following centrifugal 10min of 4000 * g, visible proteic supernatant of epr2-3 and the insoluble inclusion body of being dissolved with.Inclusion body reacts 2h in 4 ℃ of following sex change liquid, and the centrifugal 15min of 12000 * g under 4 ℃.Resuspended with 20mL PBS damping fluid, use after the fragmentation of ultrasonic disruption appearance centrifugal more then.Get cleer and peaceful inclusion body deposition respectively and carry out the SDS-PAGE electrophoresis and detect, and with His affinity column purification of recombinant proteins, and measure with BIO-RAD (Smart Spec TM3000) and to collect proteic concentration.
2.3.western blot identifies the reactionogenicity of recombinant protein Epr2-3
Recombinant protein Epr2-3 behind the purifying carries out the SDS-PAGE electrophoresis, after electrophoresis finishes, SDS-PAGE glue is cut, and puts into film transfer printing damping fluid behind the mark and soaks 30min; Get smaller pvdf membrane, methyl alcohol soaks to be put into film transfer printing damping fluid behind the 15s and soaks 15min; Get 2 of smaller filter paper and put into film transfer printing damping fluid immersion several minutes.Successively with filter paper, pvdf membrane, SDS-PAGE glue, filter paper is overlapping places on the half-dried transfer groove, catch up with bubble after the adjustment electric current to 0.8mA/cm2, transfer printing 2h.The transfer printing back pvdf membrane that finishes seals with 5% skimmed milk, puts that 50rpm spends the night in 37 ℃ of shaking tables.Film is put into the mid-37 ℃ of shaking table 50rpm effect 1h of confining liquid that contain anti-(the full bacterium serum of mouse source Ah J-1 strain) that contain the 1:1000 dilution; Film taken out with TBST give a baby a bath on the third day after its birth time each 5min; Again film is put into the confining liquid that contains ELIAS secondary antibody (sheep anti-mouse igg of HRP mark) that contains the 1:10000 dilution, similarity condition effect 1h; Give a baby a bath on the third day after its birth time each 5min again with TBST; Film is put into sedimentation type TMB single-component substrate colour developing liquid, room temperature lucifuge effect 10min.The negative control group that is provided with is carried out the abduction delivering products therefrom for empty carrier pET32a is transformed into BL21.Result (Fig. 4) shows that target protein Epr2-3 has the expression of greater efficiency, in negative control and blank, does not then have target protein to occur.
3.1 measure the medium lethal dose(LD&-{50}) of ICR mouse with Aeromonas hydrophila J-1 strain
80 age, the big female mouse of ICR was divided into 8 groups, 10 every group at random all around.With Aeromonas hydrophila J-1 strain (CGMCC NO.3220) be seeded in the LB liquid nutrient medium and 28 ℃ of shaking culture to logarithmic phase.Collect thalline and be 2.5 * 10 with being diluted to concentration in 10mMPBS (ph=7.2) solution it
3CFU/ml.1-7 group mouse is write down death toll with the injection of 0.2mL bacterium.The 8th group of injection PBS is as the blank group.Twice of this experiment repetition.The result sees table 1.
Table 1
3.2 indirect ELISA is measured the antibody titers of immune serum
40 4 age in week the big female mouse of ICR by random assignment to 4 group, 10 every group.The recombinant protein Epr2-3 of the 1st group of intramuscular injection 100ug/mL and the vaccine 0.2mL that cooperates by 1: 1 volume ratio with freund's adjuvant; (preparation method is referring to " aeromonas hydrophila inactivated vaccine is made machine examination test professional etiquette journey to the 2nd group of injection aeromonas hydrophila inactivated vaccine "; No. the 06th, 2001 new veterinary drug card words) as positive controls 0.2ml, these two groups of mouse carry out booster shot after two weeks.3,4 groups are group that causes death (injection 0.2ml PBS) and blank control group.The 0th, 6,13,20,27,34,41 and 48 days every group randomly draw 2 mouse and carry out eye socket blood sampling, separation of serum is measured the intravital antibody titers of mouse that immunity is crossed with indirect ELISA.
Result (Fig. 5) shows: the mice serum antibody titer of reorganization Epr2-3 protein immunization is apparently higher than other three groups.
3.3 recombinant protein and different adjuvants are cooperated its immanoprotection action to mouse of mensuration
90 4 age in week the big female mouse of ICR by random assignment to 9 group, 10 every group.20 μ g recombinant protein Epr2-3 are diluted to 100 μ l, it is cooperated by 1: 1 volume ratio with each adjuvant.The 1-6 group cooperates ISA206, recombinant protein Epr2-3 to cooperate DC-Chol, recombinant protein Epr2-3 to cooperate astragalus polysaccharide adjuvant for recombinant protein Epr2-3 cooperates Freund's complete adjuvant, recombinant protein Epr2-3 respectively; Recombinant protein Epr2-3 cooperates white oil, recombinant protein Epr2-3 to cooperate white lake, and every kind of adjuvant using dosage is 100 μ l.The 7th group by 100 μ l 2.0 * 10
8The aeromonas hydrophila inactivated vaccine of CFU cooperates Freund's complete adjuvant, and 8,9 groups are respectively cause death group (injecting 200 μ l PBS) and blank control group.Observe the variation of mouse every day, and the record mortality ratio, observed continuously 10 days; 10 days not dead yet mouse impose euthansia.
Result (Fig. 6) shows: the vaccine that Freund's complete adjuvant and DC-Chol cooperate recombinant protein Epr2-3 to the immune protective rate of mouse up to 95%, far above 65% of deactivation group.
In sum, the subunit vaccine of the extracellular protease recombinant protein that this patent is related can produce higher antibody of tiring in the induced animal body, and immune animal is produced good protective action, is a kind of ideal novel subunit vaccine.
Claims (10)
1. recombinant plasmid pET32a-epr2-3; It is characterized in that this recombinant plasmid is the plasmid that sets out with pET32a; Insert the epr3 gene of Aeromonas hydrophila between its Kpn I and the EcoR I restriction enzyme site; Insert the epr2 gene of Aeromonas hydrophila between EcoR I and Sal I restriction enzyme site, wherein said epr3 gene GenBank accession number is EU275147.1, and epr2 gene GenBank accession number is CP000462.1.
2. recombinant plasmid pET32a-epr2-3 according to claim 1 is characterized in that this plasmid prepares through following method:
(1) clone of epr2 and epr3 gene: the DNA that with the preserving number is the Aeromonas hydrophila J-1 strain of CGMCC NO.3220 is a template; Pass through pcr amplification with primer Epr2-1/Epr2-2 and primer Epr3-1/Epr3-2 respectively; Obtain epr2 gene and epr3 gene; It is inserted the pMD-18T plasmid respectively, obtain pMD-18T-epr2 and pMD-18T-epr3;
(2) structure of recombinant plasmid pET32a-epr3: utilize Kpn I and EcoRI that pMD-18T-epr3 and pET32a are carried out double digestion respectively; The epr3 gene enzyme that obtains is cut between the Kpn I and EcoRI restriction enzyme site of product insertion pET32a, obtained recombinant plasmid pET32a-epr3;
(3) structure of recombinant plasmid pET32a-epr2-3: utilize EcoRI and SalI that pMD-18T-epr2 and pET32a-epr3 are carried out double digestion respectively; The enzyme that obtains is cut between the EcoR I and Sal I restriction enzyme site of product epr2 gene insertion recombinant plasmid pET32a-epr3, obtained recombinant plasmid pET32a-epr2-3.
3. the host of containing the described recombinant plasmid pET32a-epr2-3 of claim 1.
4. host according to claim 3 is characterized in that described host is bacterium or cell, preferred e. coli bl21.
5. Aeromonas hydrophila extracellular protease recombinant protein Epr2-3 is characterized in that described Aeromonas hydrophila extracellular protease recombinant protein is secreted by the described recombination bacillus coli BL21 that contains recombinant plasmid pET32a-epr2-3 of claim 4.
6. Aeromonas hydrophila extracellular protease recombinant protein Epr2-3 according to claim 5; It is characterized in that the recombination bacillus coli BL21 that will contain recombinant plasmid pET32a-epr2-3 is inoculated in the LB substratum; Adding 0.1 MIPTG inducible protein after the shaking culture expresses; Continue to cultivate the centrifugal acquisition thalline in back, cleer and peaceful inclusion body in the centrifugal acquisition after the ultrasonication carries out the sex change dissolving to inclusion body; Get both more respectively with His affinity column purification of recombinant proteins, collect gained and be recombinant protein.
7. the application of the described recombinant plasmid pET32a-epr2-3 of claim 1 in preparation Aeromonas hydrophila extracellular protease recombinant protein subunit vaccine.
8. the application of the described Aeromonas hydrophila extracellular protease of claim 5 recombinant protein Epr2-3 in preparation Aeromonas hydrophila extracellular protease recombinant protein subunit vaccine.
9. an Aeromonas hydrophila extracellular protease recombinant protein subunit vaccine is characterized in that containing the described Aeromonas hydrophila extracellular protease of claim 5 recombinant protein Epr2-3 and adjuvant.
10. Aeromonas hydrophila extracellular protease recombinant protein subunit vaccine according to claim 9 is characterized in that described adjuvant is Freund's complete adjuvant or DC-Chol adjuvant.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104001164A (en) * | 2014-04-23 | 2014-08-27 | 杭州师范大学 | Aeromonas hydrophila heat shock protein subunit vaccine and preparation method thereof |
| CN104623648A (en) * | 2015-01-13 | 2015-05-20 | 广东海大畜牧兽医研究院有限公司 | Process capable of improving immunity efficacy of bacteria inactivated vaccine |
| CN109568572A (en) * | 2018-12-02 | 2019-04-05 | 河南师范大学 | A kind of preparation method and applications of Aeromonas Multivalent DNA Vaccine |
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| CN101942505A (en) * | 2010-04-28 | 2011-01-12 | 南京农业大学 | Pathogenic aeromonas hydrophila assay kit |
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| CN101942505A (en) * | 2010-04-28 | 2011-01-12 | 南京农业大学 | Pathogenic aeromonas hydrophila assay kit |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104001164A (en) * | 2014-04-23 | 2014-08-27 | 杭州师范大学 | Aeromonas hydrophila heat shock protein subunit vaccine and preparation method thereof |
| CN104623648A (en) * | 2015-01-13 | 2015-05-20 | 广东海大畜牧兽医研究院有限公司 | Process capable of improving immunity efficacy of bacteria inactivated vaccine |
| CN104623648B (en) * | 2015-01-13 | 2018-05-18 | 广东海大畜牧兽医研究院有限公司 | A kind of technique for improving bacteria inactivation vaccine immunity effect |
| CN109568572A (en) * | 2018-12-02 | 2019-04-05 | 河南师范大学 | A kind of preparation method and applications of Aeromonas Multivalent DNA Vaccine |
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