CN106834308A - The preparation method and applications of Rofe source of fish Streptococcusagalactiae recombinant C BP protein vaccines - Google Patents
The preparation method and applications of Rofe source of fish Streptococcusagalactiae recombinant C BP protein vaccines Download PDFInfo
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- CN106834308A CN106834308A CN201710070035.6A CN201710070035A CN106834308A CN 106834308 A CN106834308 A CN 106834308A CN 201710070035 A CN201710070035 A CN 201710070035A CN 106834308 A CN106834308 A CN 106834308A
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- 238000002360 preparation method Methods 0.000 title claims abstract description 24
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
- A61K39/092—Streptococcus
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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Abstract
The invention discloses a kind of preparation method and applications of Rofe source of fish Streptococcusagalactiae recombinant C BP protein vaccines.The recombinant C BP protein vaccines are prepared by Rofe source of fish Streptococcusagalactiae recombinant C BP albumen.The amino acid sequence of the Rofe source of fish Streptococcusagalactiae recombinant C BP albumen is as shown in sequence 2.The present invention is with Rofe source of fish Streptococcusagalactiae as research object; Cbp genes are cloned into from the Streptococcusagalactiae genome of the Rofe source of fish; construction of expression vector is connected with pET28a (+) carrier; prokaryotic expression and purifying are carried out in E.coliBL21 (DE3); obtain a kind of high specificity, with immune protection effectiveness for 56.35% ± 8.09% recombinant C BP protein vaccines, be Tilapia mossambica Streptococcusagalactiae disease immune protection lay a good foundation.
Description
Technical field
The invention belongs to molecular vaccinology technical field.More particularly, to a kind of restructuring of Rofe source of fish Streptococcusagalactiae
The preparation method and applications of CBP protein vaccines.
Background technology
Streptococcusagalactiae (Streptococcus agalactiae) is that a kind of people and animals fish suffers from gram-positive bacteria altogether, can be drawn
Play neonatal meningitis, bovine mastitis and fish meningoencephalitis.Streptococcusagalactiae can infect various seawater, freshwater fish and cause
Death, heavy economic losses is caused to China or even World Aquaculture.Therefore, what research Streptococcusagalactiae was related prevents and treats skill
Art has critically important realistic meaning.
At present, some patents related to hammer bacteria vaccine are had at home.Application number：200580013779.X, invention
Title：It is dense with the bacterium culture that streptococcus agalactiae vaccine discloses a kind of intact killed cells of β hemolytic Streptococcus agalacties
Combination-vaccine prepared by contracting extract is imitated by injecting and soaking two ways to the immunoprotection of the anti-Streptococcusagalactiae of Tilapia mossambica
Fruit is respectively 80% and 35%.Application number：200780025577.6, denomination of invention：Combination vaccine against streptococcus disclose one
Kind of combined vaccine, distinguish the ratio (based on cell: cell) of streptococcus and Streptococcus iniae in bacterin preparation for 20: 1 when difficulty or
When 40: 1, can obtain anti-difficulty distinguish streptococcic 64% relative protection ratio and anti-Streptococcus iniae 71% relative protection ratio.
Application number：201080047156.5, denomination of invention：Streptococcal combi-vaccine, discloses a kind of combination-vaccine, comprising agalasisa hammer
The combination-vaccine of the serotype Ia cells of bacterium bion 1 and serotype III cells provides biological for the anti-Streptococcusagalactiae of Tilapia mossambica
The protective rate of the 92% of 88.9% protective rate of the serotype Ia of type 1 and the anti-serotype III of Tilapia mossambica Streptococcusagalactiae bion 1.
Application number：201010207289.6, denomination of invention：The preparation method of bivalent inactivated vaccine of tilapia streptococcus, discloses one kind
Tilapia mossambica Streptococcusagalactiae and Streptococcus iniae mix the preparation process of bigeminy vaccine in equal volume.Application number：
201210337267.0, denomination of invention：A kind of vaccine of prevention Tilapia mossambica streptococcosis, discloses a kind of full bacterium of Streptococcusagalactiae
By immersion, injection and after oral immunity Tilapia mossambica, highest can reach 60%, 90% and 75% immune guarantor to inactivated vaccine respectively
Shield rate.Application number：201410121138.7, apply for title：A kind of oral vaccine of Streptococcusagalactiae and preparation method thereof, builds
A kind of recombinant attenuated salmonella for expressing Streptococcusagalactiae sip albumen, most high available after Tilapia mossambica is fed by its spice
63% anti-Streptococcusagalactiae immune protective rate.
But, the streptococcus agalactiae vaccine that above disclosure is used is the full bacterium inactivated vaccine of single or bigeminy, is prepared
Vaccine immunity effect it is directly related with the immunogenicity of vaccine strains, and Streptococcusagalactiae only consistent to serotype has
Effect, does not have effect to Streptococcus iniae.
The content of the invention
The technical problem to be solved in the present invention is the defect and deficiency for overcoming above-mentioned prior art, there is provided a kind of Rofe source of fish
The recombinant C BP albumen of Streptococcusagalactiae Cbp gene codes, and Tilapia mossambica Streptococcusagalactiae is prevented and treated as recombinant protein vaccine
The anti-Streptococcusagalactiae different serotypes of Tilapia mossambica can be produced specific protectiveness by disease.The preparation of the recombinant protein vaccine
Method is simple, safe preparation process, and vaccine reaches 56.35% to the immune protective rate of the anti-Streptococcusagalactiae of Tilapia mossambica ±
8.09%.
It is an object of the invention to provide a kind of preparation method of Rofe source of fish Streptococcusagalactiae recombinant C BP protein vaccines.
Another object of the present invention is to provide the application of the Rofe source of fish Streptococcusagalactiae recombinant C BP protein vaccines.
Above-mentioned purpose of the present invention is achieved through the following technical solutions：
A kind of Rofe source of fish Streptococcusagalactiae Cbp genes, its nucleotide sequence is as shown in sequence 1.
Described Rofe source of fish Streptococcusagalactiae Cbp genes are cloned from the Streptococcusagalactiae of the Rofe source of fish and obtained.
A kind of recombinant C BP albumen of Rofe source of fish Streptococcusagalactiae Cbp gene codes, its amino acid sequence such as institute of sequence 2
Show.
The recombinant C BP albumen is obtained by above-mentioned Rofe source of fish Streptococcusagalactiae Cbp gene codes.
In addition, the preparation method of above-mentioned Rofe source of fish Streptococcusagalactiae recombinant C BP albumen, comprises the following steps：
S1. primer pair CBP-F and CBP-R are utilized, the matter containing restructuring in Cbp gene clonings to prokaryotic expression system, will be obtained
The engineering bacteria of grain pET28a-CBP；
S2. the engineering bacteria containing recombinant plasmid pET28a-CBP is enlarged culture, purifying is reclaimed and obtains recombinant C BP eggs
In vain；
Wherein, as shown in sequence 3, the sequence of primer CBP-R is as shown in sequence 4 for the sequence of the primer CBP-F；It is described
The sequence of Cbp genes is as shown in sequence 1.
In addition, it is further preferred that the specific method of step S1 is：
S11. Cbp genes are cloned：With Rofe source of fish Streptococcusagalactiae genomic DNA as template, with primer CBP-F and CBP-
R enters performing PCR clone's Cbp genes；
S12. CBP clone strains are built：The Cbp genes of clone are connected to pET28a carriers, and Transformed E .coli DH5 α
Competent cell, the positive CBP bacterial strains containing recombinant plasmid pET28a-CBP are obtained using card that penicillin screening；
S13. CBP expression bacterial strains are built：Recombinant plasmid pET28a-CBP Transformed E .coli BL21 (DE3) competence is thin
Born of the same parents, through card, that penicillin resistance is screened, and picking single bacterium colony enters performing PCR identification, filters out positive colony cell, as the matter containing restructuring
The engineering bacteria of grain pET28a-CBP.
Wherein, it is highly preferred that PCR reaction systems are described in step S11：The 25 μ l reaction systems (genes of template DNA containing 50ng
Group DNA), 2.5 μ 10 × PCR of l Buffer (Mg+), 2 μ l dNTP Mixture (2.5mM), 1 μ l CBP-F, 1 μ l CBP-R,
0.125 μ l rTaq (5U/ μ l), plus sterile purified water is to 25 μ l.
It is highly preferred that PCR conditions are described in step S11：94 DEG C of predegeneration 3min；Then 94 DEG C are denatured 30s, 53 DEG C of annealing
35s, 72 DEG C extend 30 circulations of effect under 2min；Last 72 DEG C of extensions 8min；4 DEG C of insulations.
It is highly preferred that the system connected described in step S12 is：20 μ l systems include 3 μ l PCR primers, 1 μ l Vector, 2
μ l 10 × Buffer, 1 μ l Enzyme Mix A, 13 μ l ddH2O.(note：Linked system used carrier, enzyme, buffer solution come
From Si Dansai biotech companies ' even kit of complying with one's wishes ')
It is highly preferred that the specific method of step S12 is as follows：By after the product recovery purifying of PCR described in step S11 with
PET28a carriers are connected, and 30min is connected at 37 DEG C；10 μ l connection products are transferred to 100 μ l bacillus coli DH 5 alpha competent cells,
Ice bath 30min, 42 DEG C of heat shock 60s, ice bath 5min；It is subsequently adding 500 μ l LB fluid nutrient mediums, 37 DEG C of concussion and cultivate 45min；
4000rpm is centrifuged 5min, stays 100 μ l to blow and beat precipitation bacterium solution, mixed liquor is added and contains 50 μ g/ml cards that penicillin (Kan+)
On LB solid mediums, in 37 DEG C of overnight incubations after coating is uniform；10 single bacterium colonies identification on random picking flat board, identification knot
Fruit is CBP clone strains for positive single bacterium colony.
It is highly preferred that the specific method of step S13 is as follows：5 μ l plasmids pET28a are transferred to 100 μ l e. coli bl21s
(DE3) competent cell, ice bath 30min, 42 DEG C of heat shock 60s, ice bath 5min；It is subsequently adding 500 μ l LB fluid nutrient mediums, 37
DEG C concussion and cultivate 45min；4000rpm is centrifuged 5min, stays 100 μ l to blow and beat precipitation bacterium solution, mixed liquor is added and contains 50 μ g/ml cards
On the LB solid mediums of that penicillin (Kan+), in 37 DEG C of overnight incubations after coating is uniform；10 on random picking flat board
Single bacterium colony identifies that qualification result is positive single bacterium colony as CBP expression bacterial strains.
It is further preferred that the specific method of step S2 is as follows：
S21., engineering bacteria single bacterium colony containing recombinant plasmid pET28a-CBP is inoculated in the LB liquid containing kanamycins
In culture medium, concussion and cultivate 10h；
S22. by bacterium solution LB fluid nutrient mediums of the addition containing kanamycins, Amplification Culture 2h is 0.6 to OD600；
S23. in bacterium solution add IPTG Fiber differentiations after, using imidazole elution purify reclaim, the restructuring for being purified
CBP albumen, as described Rofe source of fish Streptococcusagalactiae recombinant C BP albumen, its amino acid sequence is as shown in sequence 2.
Wherein, more specifically preferably, the specific method of step S2 is as follows：
S21. E.coli BL21 (DE3) single bacterium colony containing recombinant plasmid pET28a-CBP is inoculated in 5ml and contains 50 μ
In the LB fluid nutrient mediums of g/ml kanamycins, 200r/min concussion and cultivates 10h at 37 DEG C；
S22. in 5ml bacterium solutions addition 500ml being contained into the LB fluid nutrient mediums of 50 μ g/ml kanamycins, expand training at 1: 100
2h is supported, 0.6 is about to OD600；
S23. to the IPTG that final concentration of 1mmol/l is added in bacterium solution, 16 DEG C of concussion and cultivates are overnight；It is collected by centrifugation and overnight lures
The thalline led, Lysis Buffer are resuspended, and ultrasonication 30min, is collected by centrifugation supernatant after ice bath 30min；
S24. the recombinant C BP albumen purified with Ni-NTA in supernatant, is first washed with the eluent containing 20mmol/l imidazoles
Decontamination, then with containing 100mmol/l imidazoles elution recombinant protein；The recombinant protein of wash-out is super with 10KDa super filter tubes
Filter, removes the imidazoles in eluent, the recombinant C BP albumen for being purified, as described Rofe source of fish Streptococcusagalactiae recombinant C BP
Albumen, its amino acid sequence is as shown in sequence 2.
In addition, above-mentioned Rofe source of fish Streptococcusagalactiae recombinant C BP albumen as or prepare Rofe source of fish Streptococcusagalactiae
Application in terms of recombinant C BP protein vaccines, also within protection scope of the present invention.
A kind of preparation method of Rofe source of fish Streptococcusagalactiae recombinant C BP protein vaccines, is by Rofe source of fish agalasisa hammer
Bacterium recombinant C BP albumen sterilizing PBS be diluted to 1mg/ml, with Freund completely or Freund's incomplete adjuvant be well mixed by 1: 1, be prepared into
Subunit vaccine.
Further, the vaccine that 1ml is prepared is taken, 5min is centrifuged under 3000 × g, vaccine then can use without lamination
In follow-up immunization.
The Rofe source of fish Streptococcusagalactiae recombinant C BP protein vaccines that the above method is prepared, and the Rofe source of fish
Application of the Streptococcusagalactiae recombinant C BP protein vaccines in terms of the medicine or preparation that prepare preventing and treating Tilapia mossambica Streptococcusagalactiae disease,
Also all should be within protection scope of the present invention.
The invention has the advantages that：
The invention provides a kind of preparation method of Rofe source of fish Streptococcusagalactiae recombinant C BP protein vaccines, gained restructuring
CBP protein vaccines can be used to prevent and treat Tilapia mossambica Streptococcusagalactiae disease, and the anti-Streptococcusagalactiae different serotypes of Tilapia mossambica can be produced
The specific protectiveness of life, vaccine reaches 56.35% ± 8.09% to the immune protective rate of the anti-Streptococcusagalactiae of Tilapia mossambica.
Compared to existing technology, recombinant C BP subunit vaccines of the invention have obvious advantage, in the prior art to Rofe
The preventing and treating of fish Streptococcusagalactiae still relies primarily on conventional medicament, and new anti-Streptococcusagalactiae disease vaccine opens in cultivating tilapia
Hair is study hotspot in recent years, but focuses primarily upon full bacterium inactivated vaccine and attenuated live vaccine；Although both vaccines are made
Preparation Method is easy, but use of the inactivated vaccine in aquiculture animal needs heavy dose of, multiple immunity inoculation, and attenuated live
Storage and the traffic condition requirement of vaccine are high, and the term of validity is shorter, and malicious dangerous in the presence of returning；Therefore both vaccines may draw
Play larger toxicity.Subunit vaccine is the vaccine as made by having immunocompetent pathogenic mycoprotein, in the present invention
Recombinant C BP subunit vaccines only contain a kind of pathogenic bacteria protein, thus can eliminate the antibody that many irrelevant antigens are induced, and reduce
Side reaction that vaccine brings and the other diseases caused by vaccine, security reliability.Also, with full bacterium inactivated vaccine and attenuation
Live vaccine is compared, and recombinant C BP vaccines of the invention do not exist serotype specificity, can resist the agalasisa hammer of different serotypes
Bacterium, the scope of application is wider.Therefore, had a good application prospect in terms of Tilapia mossambica Streptococcusagalactiae is prevented.
And, protein vaccine preparation method of the invention uses prokaryotic expression system, and preparation method is simple, and flow is succinctly fast
Speed, safe preparation process, express low cost, be suitable to large-scale production and application.
Brief description of the drawings
Fig. 1 is Rofe source of fish Streptococcusagalactiae Cbp gene PCR amplified productions；Wherein, swimming lane M：DL2000 Marker；Swimming
Road 1：Pcr amplification product.
Fig. 2 is Rofe source of fish Streptococcusagalactiae CBP protein purifications SDS-PAGE figures；M is albumen Marker；Lane 1 is egg
Bai Chaosheng liquid；Lane 2 is efflux；Lane 3 is the wash-out result of Wash buffer；Lane 4 is 50mM imidazole concentrations
The wash-out result of Elution buffer；Lane 5-9 are the wash-out result of the Elution buffer of 100mM imidazole concentrations；
Lane 10-11 are respectively the wash-out result of the Elution buffer of 200mM, 250mM imidazole concentration.
Fig. 3 is the immunogenicity that Western Blot detect CBP albumen；M is albumen Marker；
Fig. 4 is to attack malicious survival curve after recombinating the immune Tilapia mossambica of CBP protein vaccines.Abscissa is to attack number of days after poison；It is vertical
Coordinate is survival rate.
Fig. 5 is restructuring CBP albumen Elisa detection serum antibody titers；Abscissa is the serum of the 2nd, 4 weeks after just exempting from；It is vertical
Coordinate is antibody titer, and antibody titer produces the maximum dilution multiple of positive findings to represent.
Specific embodiment
The present invention, but embodiment are further illustrated below in conjunction with Figure of description and specific embodiment not to the present invention
Limit in any form.Unless stated otherwise, reagent, the method and apparatus that the present invention is used are for the art is routinely tried
Agent, method and apparatus.
Unless stated otherwise, following examples agents useful for same and material are purchased in market.
Embodiment 1 prepares Rofe source of fish Streptococcusagalactiae recombinant C BP albumen
1st, design of primers：
With Rofe source of fish Streptococcusagalactiae as template, design a pair primer CBP-F and CBP- comprising restriction enzyme
R, sequence is as follows：
Primer CBP-F (sequence is as shown in sequence 3)：
5’-GGGGGGTCTCTAGTGGATCAAACTACATCGGTTCAA-3’
Primer CBP-R ((sequence is as shown in sequence 4))：
5’-GCCGGGTCTCGTGGGAATTTCAATATAGCGACGAAT-3’
2nd, Cbp gene clonings
(1) PCR amplifications Cbp genes
Extract Rofe source of fish Streptococcusagalactiae genomic DNA, with genomic DNA as template, with above-mentioned primer pair CBP-F and
CBP-R enters performing PCR reaction.
PCR reaction systems are：25 μ l reaction systems template DNA containing 50ng (genomic DNA), 2.5 10 × PCR of μ l
The μ l of Buffer (Mg+), 2 μ l dNTP Mixture (2.5mM), 1 CBP-F, 1 μ l CBP-R, 0.125 μ l rTaq (5U/ μ l),
Plus sterile purified water is to 25 μ l.
PCR conditions are：94 DEG C of predegeneration 3min；Then 94 DEG C of denaturation 30s, 53 DEG C of annealing 35s, make under 72 DEG C of extension 2min
With 30 circulations；Last 72 DEG C of extensions 8min；4 DEG C of insulations.
Pcr amplification product electrophoretogram is as shown in Figure 1.M represents DL2000 DNA marker；1 represents pcr amplification product.
Cbp gene nucleotide series are as shown in sequence 1.
(2) CBP clone strains are built
It is connected with pET28a carriers after above-mentioned PCR primer recovery purifying, 30min is connected at 37 DEG C.
Linked system is：20 μ l systems include 3 μ l PCR primers, 1 μ l Vector, 2 μ l 10 × Buffer, 1 μ l
Enzyme Mix A, 13 μ l ddH2O.(note：It is public that linked system used carrier, enzyme, buffer solution are all from Si Dansai biotechnologys
Department's ' even kit of complying with one's wishes ')
10 μ l connection products are transferred to 100 μ l bacillus coli DH 5 alpha competent cells, ice bath 30min, 42 DEG C of heat shock 60s,
Ice bath 5min；It is subsequently adding 500 μ l LB fluid nutrient mediums, 37 DEG C of concussion and cultivate 45h；4000rpm is centrifuged 5min, stays 100 μ l to blow
Precipitation bacterium solution is beaten, mixed liquor is added on the LB solid mediums containing 50 μ g/ml cards that penicillin (Kan+), after coating is uniform
In 37 DEG C of overnight incubations.
10 single bacterium colonies identification on random picking flat board, qualification result is positive single bacterium colony as CBP clone strains.
3rd, CBP expression bacterial strains are built
CBP clone strain plasmids are extracted, recombinant plasmid pET28a-CBP Transformed E .coli BL21 (DE3) competence is thin
Born of the same parents, through card, that penicillin resistance is screened, and picking single bacterium colony enters performing PCR identification, filters out positive colony cell, as the matter containing restructuring
The engineering bacteria of grain pET28a-CBP.
4th, expression of purification of Recombinant CBP albumen --- the recombinant plasmid pET28a-CBP in E.coli BL21 (DE3) and pure
Change：
(1) E.coli BL21 (DE3) single bacterium colony containing recombinant plasmid pET28a-CBP is inoculated in 5ml and contains 50 μ g/
In the LB fluid nutrient mediums of ml kanamycins, 5ml bacterium solutions addition 500ml is contained 50 by 200r/min concussion and cultivates 10h at 37 DEG C
In the LB fluid nutrient mediums of μ g/ml kanamycins, 1: 100 Amplification Culture 2h is about 0.6 to OD600.It is dense eventually to being added in bacterium solution
The IPTG for 1mmol/l is spent, 16 DEG C of concussion and cultivates are overnight.The thalline for overnight inducing is collected by centrifugation, Lysis Buffer are resuspended, ice
Ultrasonication 30min after bath 30min, is collected by centrifugation supernatant.The recombinant C BP albumen purified with Ni-NTA in supernatant, first with containing
The eluent for having 20mmol/l imidazoles washes away impurity, then with the elution recombinant protein containing 100mmol/l imidazoles.Wash-out
Recombinant protein 10KDa super filter tube ultrafiltration, removes the imidazoles in eluent, the recombinant C BP albumen for being purified, as described sieve
Non- source of fish Streptococcusagalactiae recombinant C BP albumen, its amino acid sequence is as shown in sequence 2.
(2) protein purification result is as shown in Figure 2.M is albumen Marker；Lane 1 is albumen ultrasound liquid；Lane 2 is
Efflux；Lane 3 is the wash-out result of Wash buffer；Lane 4 is washed for the Elution buffer's of 50mM imidazole concentrations
De- result；Lane 5-9 are the wash-out result of the Elution buffer of 100mM imidazole concentrations；Lane 10-11 are respectively
The wash-out result of the Elution buffer of 200mM, 250mM imidazole concentration.
5th, Western Blot detections
Using the rabbit anteserum of infection Streptococcusagalactiae THN bacterial strains as primary antibody, the immunogenicity of recombinant protein c BP is detected,
Concrete operations are as follows：
(1) separation gel is cut to suitable size after electrophoresis terminates, is put into transferring film buffer solution and is soaked 10min.
(2) film process：Cut out in advance with an equal amount of filter paper of separation gel and film, be put into transferring film buffer solution and soak
10min。
(3) transferring film：Stack metafiltration paper, film, gel, metafiltration paper successively from bottom to up on cathode electrode plate, it is ensured that its
Accurate align bubble-free, then with 100mA constant current transferring films 1h.
(4) film is washed with TBST, steady shake, 5min × 3 time.
(5) coating buffer is added, 4 DEG C overnight.
(6) coating buffer is abandoned, film, 5min × 3 time is washed with TBST.
(7) primary antibody (rabbit anteserum after dilution), 37 DEG C of incubation 3h are added.
(8) primary antibody is abandoned, film is washed respectively with TBST, steady shake, 5min × 3 time.
(9) secondary antibody goat-anti rabbit (with TBS by 1: 5000 dilution) is added, steady shake is incubated at room temperature 2h.
(10) secondary antibody is abandoned, film is washed respectively with TBST, steady shake, 5min × 3 time.
(11) DAB colour developings：Film is put into lucifuge in the DAB nitrite ions of Fresh develops the color when there is band and be put into
ddH2O terminating reactions.
Result at purpose band size as shown in figure 3, have band, it was demonstrated that recombinant protein c BP has good immunogene
Property.
Embodiment 2 prepares Rofe source of fish Streptococcusagalactiae recombinant C BP protein vaccines
The recombinant C BP albumen sterilizing PBS of the purifying that embodiment 1 is obtained is diluted to 1mg/ml, with Freund completely and not
Freund's complete adjuvant is well mixed by 1: 1, is prepared into subunit vaccine.
The vaccine that 1ml is prepared is taken, 5min is centrifuged under 3000 × g, vaccine then can be used to subsequently exempt from without lamination
Epidemic disease.
The Rofe source of fish Streptococcusagalactiae recombinant C BP protein vaccine immunity tests of embodiment 3
1st, immunoprotection experiment
(1) bolti is purchased from Guangzhou central trench aquaculture Development Co., Ltd, and specification is 30 ± 5.0g, experiment
It is preceding temporarily to support two weeks.
(2) during immunization experiment, Tilapia mossambica is randomly divided into 3 groups, i.e. PBS groups, PBS+ adjuvants group, CBP vaccine groups.Every group 15
Tail, every group set three it is parallel.
Recombinant antigen CBP that CBP groups and the other μ l of intraperitoneal injection 100 of adjuvant component mix in equal volume with Freund's adjuvant and
PBS, PBS group inject the sterilizing PBS of 100 μ l.
2nd, immune programme for children
Tilapia mossambica is immunized twice, initial immunity uses Freund's complete adjuvant, booster immunization is incomplete using Freund
Adjuvant subunit vaccine.
During initial immunity, per the μ l vaccines of tail fish intraperitoneal injection 100.After two weeks, carried out per the μ l vaccines of tail fish intraperitoneal injection 100
Booster immunization.
The the 2nd and 4 week after just exempting from, every group is selected 3 tail fishes to take a blood sample, and after room temperature places 2h, 4 DEG C, 8000 × rpm centrifugation 10min are received
Collection supernatant, -80 DEG C of preservations after packing, for the measure of immune indexes.
3rd, immune protective rate is determined
(1) booster immunization carries out bacterium challenge viral dosage after two weeks with LD50 dosage, per tail fish intraperitoneal injection 100ul agalasisas
Streptococcus bacterium solution (1 × 108CFU/ml).After attacking poison, the situation of observation Tilapia mossambica, pulls dead fish out, and record 14 days in time daily
Interior death condition.Continuous Observation 14 days, stops observation, and experiment terminates.
The relative protection ratio (RPS) of Tilapia mossambica is calculated according to following formula：
RPS=(1- immune groups cumulative mortality/control group cumulative mortality) × 100%.
(2) Tilapia mossambica death condition is as shown in Figure 4 after attacking poison.
Result shows that 4th week after initial immunity is carried out attacking poison with Streptococcusagalactiae.Tilapia mossambica opens on the 1st day from after attacking poison
Beginning occurs as soon as death, and death is concentrated mainly on first 3 days after attacking poison.
Additionally, attacking malicious Tilapia mossambica shows some clinical symptoms for suffering from streptococcosis, such as exophthalmos and muddiness, peel off solely
Trip and body are curled.
Recombinant C BP albumen can obtain 56.35% for injecting immune as anti-Tilapia mossambica Streptococcusagalactiae disease vaccine ±
8.09% immune protective rate.
4th, Elisa detection antibodies titre
Detection method
(1) it is coated with：Recombinant C BP albumen to 200 μ g/ml is diluted with coating buffer, is added in 96 hole elisa Plates, 100 μ l/ holes, 4
DEG C coating overnight.
(2) wash：The coating buffer in 96 holes is discarded, is cleaned with less salt Buffer 5 times, 3min × 5 time, every time 250 μ l/
Hole, after liquid-transfering gun is gently blown and beaten, vibration is relaxed with the speed of 200rpm, is patted dry.
(3) close：1%BSA is added to be closed, 2h, is closed in 250 μ l/ holes by 22 DEG C.Washed by above-mentioned condition, clapped
It is dry.
(4) primary antibody reaction：The Tilapia mossambica antiserum that will be gathered PBS is by 2 times of stepwise dilutions (such as extension rate：2、22、23、
24、25、26、27、28Deng), added by 100 μ l/ holes, 22 DEG C, it is incubated 3h.Washed by above-mentioned condition, patted dry.
(5) secondary antibody reaction：The anti-fish IgM of mouse is diluted 10 times with PBS, is added by 100 μ l/ holes, 22 DEG C, be incubated 2h；Washing,
Pat dry.
(6) three anti-reflective should：The antibody that sheep anti-mouse igg-HRP is marked dilutes 5000 times with PBS, is added by 100 μ l/ holes,
22 DEG C of incubation 2h；Washing, pats dry.
(7) develop the color：Tmb substrate solution is added by 100 μ l/ holes, incubation at room temperature；After 10min, reaction terminating liquid 50 is added
μ l/ holes terminating reaction.
(8) reading：This 96 ELISA Plate is placed in carries out reading on ELIASA, determine the light absorption value at 450nm, i.e. OD450。
Otherwise it is feminine gender if the reading of test group is designated as the positive more than or equal to the twice of control group.Antibody titer with
The maximum dilution multiple of positive findings is represented.
Result is as shown in Figure 5.There is different degrees of growth in Post-immunisation serum antibody titer, and reaches peak in 4th week
Value, is 1: 8192.
Claims (10)
1. a kind of Rofe source of fish Streptococcusagalactiae Cbp genes, it is characterised in that its nucleotide sequence is as shown in sequence 1.
2. a kind of Rofe source of fish Streptococcusagalactiae recombinant C BP albumen, it is characterised in that its amino acid sequence is as shown in sequence 2.
3. Rofe source of fish Streptococcusagalactiae recombinant C BP albumen according to claim 1, it is characterised in that the recombinant C BP eggs
It is in vain to be obtained by the Streptococcusagalactiae Cbp gene codes of the Rofe source of fish described in claim 1.
4. the preparation method of Rofe source of fish Streptococcusagalactiae recombinant C BP albumen described in Claims 2 or 3, it is characterised in that including
Following steps：
S1. primer pair CBP-F and CBP-R are utilized, in CBP gene clonings to prokaryotic expression system, will be obtained containing recombinant plasmid
The engineering bacteria of pET28a-CBP；
S2. the engineering bacteria containing recombinant plasmid pET28a-CBP is enlarged culture, purifying is reclaimed and obtains recombinant C BP albumen；
Wherein, as shown in sequence 3, the sequence of primer CBP-R is as shown in sequence 4 for the sequence of the primer CBP-F；The Cbp bases
The sequence of cause is as shown in sequence 1.
5. preparation method according to claim 4, it is characterised in that the specific method of step S1 is：
S11. Cbp genes are cloned：With Rofe source of fish Streptococcusagalactiae genomic DNA as template, entered with primer CBP-F and CBP-R
Performing PCR clones Cbp genes；
S12. CBP clone strains are built：The Cbp genes of clone are connected to pET28a carriers, and Transformed E .coli DH5 α experience
State cell, the positive CBP bacterial strains containing recombinant plasmid pET28a-CBP are obtained using card that penicillin screening；
S13. CBP expression bacterial strains are built：By recombinant plasmid pET28a-CBP Transformed E .coli BL21 (DE3) competent cell, warp
Block the screening of that penicillin resistance, picking single bacterium colony enters performing PCR identification, positive colony cell filtered out, as containing recombinant plasmid
The engineering bacteria of pET28a-CBP.
6. preparation method according to claim 4, it is characterised in that the specific method of step S2 is as follows：
S21., engineering bacteria single bacterium colony containing recombinant plasmid pET28a-CBP is inoculated in the LB Liquid Cultures containing kanamycins
In base, concussion and cultivate 10h；
S22. by bacterium solution LB fluid nutrient mediums of the addition containing kanamycins, Amplification Culture 2h is 0.6 to OD600；
S23. in bacterium solution add IPTG Fiber differentiations after, using imidazole elution purify reclaim, the recombinant C BP eggs for being purified
In vain, as described Rofe source of fish Streptococcusagalactiae recombinant C BP albumen.
7. Rofe source of fish Streptococcusagalactiae recombinant C BP albumen described in Claims 2 or 3 as or prepare Rofe source of fish agalasisa chain
Application in terms of coccus recombinant C BP protein vaccines.
8. a kind of preparation method of Rofe source of fish Streptococcusagalactiae recombinant C BP protein vaccines, it is characterised in that be by the Rofe source of fish
Streptococcusagalactiae recombinant C BP albumen sterilizing PBS is diluted to 1mg/ml, or Freund's incomplete adjuvant complete with Freund mixes by 1: 1
It is even, it is prepared into subunit vaccine.
9. the Rofe source of fish Streptococcusagalactiae recombinant C BP protein vaccines that method is prepared according to claim 8.
10. Rofe source of fish Streptococcusagalactiae recombinant C BP protein vaccines described in claim 9 are preparing preventing and treating Tilapia mossambica agalasisa hammer
Application in terms of the medicine or preparation of bacterium disease.
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