CN105861521B - The preparation method and applications of Rofe source of fish Streptococcusagalactiae recombination GroEL protein vaccine - Google Patents
The preparation method and applications of Rofe source of fish Streptococcusagalactiae recombination GroEL protein vaccine Download PDFInfo
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- CN105861521B CN105861521B CN201610314912.5A CN201610314912A CN105861521B CN 105861521 B CN105861521 B CN 105861521B CN 201610314912 A CN201610314912 A CN 201610314912A CN 105861521 B CN105861521 B CN 105861521B
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- streptococcusagalactiae
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- vaccine
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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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- A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
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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
- A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
-
- 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
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a kind of preparation method and applications of Rofe source of fish Streptococcusagalactiae recombination GroEL protein vaccine.The recombination GroEL protein vaccine is prepared by Rofe source of fish Streptococcusagalactiae recombination GroEL albumen.Rofe source of fish Streptococcusagalactiae recombinates the amino acid sequence of GroEL albumen as shown in SEQ ID NO.2.The present invention is cloned into from the Streptococcusagalactiae genome of the Rofe source of fish using Rofe source of fish Streptococcusagalactiae as research objectGroELGene connect construction of expression vector, In with pET28a (+) carrierE.coliProkaryotic expression and purifying are carried out in BL21 (DE3), are obtained a kind of high specificity, the recombination GroEL protein vaccine that is 68.61 ± 7.39% with immune protection effectiveness, are laid a good foundation for the immune protection of Tilapia mossambica Streptococcusagalactiae disease.
Description
Technical field
The invention belongs to molecular vaccinology technical fields.It is recombinated more particularly, to a kind of Rofe source of fish Streptococcusagalactiae
The preparation method and applications of GroEL protein vaccine.
Background technique
Streptococcusagalactiae (Streptococcus agalactiae) it is that a kind of people and animals fish suffers from gram-positive bacteria altogether, it can draw
Play neonatal meningitis, bovine mastitis and fish meningoencephalitis.Streptococcusagalactiae can infect a variety of seawater, freshwater fish and cause
Death causes heavy economic losses to China or even World Aquaculture.Therefore, the relevant prevention and treatment skill of research Streptococcusagalactiae
Art has critically important realistic meaning.
Currently, having some patents relevant to hammer bacteria vaccine at home.Application number: 200580013779.X, invention
Title: streptococcus agalactiae vaccine disclose a kind of β hemolytic Streptococcus agalactie intact killed cells and the bacterium culture it is dense
The combination-vaccine of contracting extract preparation imitates the immunoprotection of the anti-Streptococcusagalactiae of Tilapia mossambica by injecting and impregnating two ways
Fruit is respectively 80% and 35%.Application number: 200780025577.6, denomination of invention: combination vaccine against streptococcus discloses one kind
Combined vaccine, when hardly possible distinguishes that the ratio (based on cell: cell) of streptococcus and Streptococcus iniae in vaccine preparation is 20:1 or 40:
When 1, anti-difficulty can be obtained and distinguish streptococcic 64% relative protection ratio and 71% relative protection ratio of anti-Streptococcus iniae.Application
Number: 201080047156.5, denomination of invention: streptococcal combi-vaccine discloses a kind of combination-vaccine, raw comprising Streptococcusagalactiae
The combination-vaccine of 1 serotype Ia cell of object type and serotype III cell provides raw for the anti-Streptococcusagalactiae of Tilapia mossambica
The 92% of 88.9% protective rate of 1 serotype Ia of object type and anti-1 serotype III of Tilapia mossambica Streptococcusagalactiae bion
Protective rate.Application number: 201010207289.6, denomination of invention: the preparation method of bivalent inactivated vaccine of tilapia streptococcus,
It discloses a kind of Tilapia mossambica Streptococcusagalactiae and Streptococcus iniae mixes the preparation process of bigeminy vaccine in equal volume.Application number:
201210337267.0, denomination of invention: it is complete to disclose a kind of Streptococcusagalactiae for a kind of vaccine preventing Tilapia mossambica streptococcosis
By impregnating, after injection and oral immunity Tilapia mossambica, highest can reach 60%, 90% and 75% immune guarantor to bacterium inactivated vaccine respectively
Shield rate.Application number: 201410121138.7, apply for a kind of title: oral vaccine of Streptococcusagalactiae and preparation method thereof, building
A kind of recombinant attenuated salmonella for expressing Streptococcusagalactiae sip albumen, highest can get after its spice is fed Tilapia mossambica
63% anti-Streptococcusagalactiae immune protective rate.
But the streptococcus agalactiae vaccine disclosed above 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 only have to the consistent Streptococcusagalactiae of serotype
Effect, does not have effect to Streptococcus iniae.
Summary of the invention
The technical problem to be solved by the present invention is to overcome the defect of the above-mentioned prior art and deficiencies, provide a kind of Rofe source of fish
The recombination GroEL albumen of Streptococcusagalactiae GroEL gene coding, and Tilapia mossambica agalasisa hammer is prevented and treated as recombinant protein vaccine
Bacterium disease can generate the protectiveness of specificity to the anti-Streptococcusagalactiae different serotypes of Tilapia mossambica.The system of the recombinant protein vaccine
Preparation Method is simple, safe preparation process, and vaccine reaches 68.61 ± 7.39% to the immune protective rate of the anti-Streptococcusagalactiae of Tilapia mossambica.
The object of the present invention is to provide a kind of preparation methods of Rofe source of fish Streptococcusagalactiae recombination GroEL protein vaccine.
Another object of the present invention is to provide the application of Rofe source of fish Streptococcusagalactiae recombination GroEL protein vaccine.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
A kind of Rofe source of fish StreptococcusagalactiaegroELGene, nucleotide sequence is as shown in SEQ ID NO.1.
The Rofe source of fish StreptococcusagalactiaegroELGene is to clone to obtain from the Streptococcusagalactiae of the Rofe source of fish.
A kind of Rofe source of fish StreptococcusagalactiaegroELThe recombination GroEL albumen of gene coding, amino acid sequence such as SEQ
Shown in ID NO.2.
The recombination GroEL albumen is by above-mentioned Rofe source of fish StreptococcusagalactiaegroELGene encodes to obtain.
In addition, the preparation method of above-mentioned Rofe source of fish Streptococcusagalactiae recombination GroEL albumen, includes the following steps:
S1. primer pair GroEL-F and GroEL-R are utilized, it willGroELGene cloning is contained into prokaryotic expression system
The engineering bacteria of recombinant plasmid pET28-GroEL；
S2. the engineering bacteria of the pET28-GroEL containing recombinant plasmid is expanded culture, purification and recovery obtains recombination GroEL
Albumen；
Wherein, the sequence of the primer GroEL-F is as shown in SEQ ID NO.3, the sequence of primer GroEL-R such as SEQ ID
Shown in NO.4；It is describedGroELThe sequence of gene is as shown in SEQ ID NO.1.
In addition, it is further preferred that the specific method of step S1 is:
S11. it clonesGroELGene: using Rofe source of fish Streptococcusagalactiae genomic DNA as template, with primer GroEL-F
PCR clone is carried out with GroEL-RGroELGene；
S12. GroEL clone strain is constructed: by clone'sGroELGene is connected to pMD18-T carrier, and convertsE.coliCompetent cell screens to obtain positive GroEL grams containing recombinant plasmid pMD18T-GroEL using ampicillin
Grand bacterial strain；
S13. construction recombination plasmid pET28-GroEL: restriction endonuclease EcoR is utilizedIAnd HindIII,By plasmid pMD18T-
GroEL and expression vector pET28a (+) connection；Connection product is transferred to e. coli bl21 (DE3) competent cell, through card
The screening of that penicillin resistance, picking single colonie carry out PCR identification, filter out positive colony cell, as contain recombinant plasmid
The engineering bacteria of pET28-GroEL.
Wherein, it is highly preferred that PCR reaction system described in step S11 are as follows: the 25 μ l reaction system (bases of template DNA containing 50ng
Because of a group DNA), 2.5 μ 10 × PCR of l Buffer(Mg+), 2 μ l dNTP Mixture (2.5mM), 1 μ l GroEL-F, 1 μ l
GroEL-R, 0.125 μ l rTaq (5U/ μ l), adds sterile purified water to 25 μ l.
It is highly preferred that PCR condition described in step S11 are as follows: 94 DEG C of initial denaturation 1min；Then 94 DEG C of denaturation 30s, 55
DEG C annealing 30s, effect 30 recycle under 70 DEG C of extensions 1min；Last 70 DEG C of extensions 10min；16 DEG C of heat preservations.
It is highly preferred that the system of connection described in step S12 are as follows: 10 μ l systems include 4 μ l PCR products, 1 μ l pMD18-T
Vector, 5 μ l Solution I.
It is highly preferred that the specific method is as follows by step S12: by after the product recovery purifying of PCR described in step S11 with
PMD18-T carrier connects, and connects overnight at 16 DEG C；10 μ l connection products are transferred to 50 μ l Escherichia coli Trans1-T1 impression
Then state cell, ice bath 30min, 42 DEG C of heat shock 60s are added 500 μ l LB fluid nutrient mediums, 37 DEG C of shake culture 2h,
4000rpm is centrifuged 5min, stays 100 μ l piping and druming precipitating bacterium solution, mixed liquor is added and contains 100 μ g/ml ampicillins (Amp+)
LB solid medium on, coating uniformly after in 37 DEG C of overnight incubations；10 single colonies identification on random picking plate, mirror
Determining the single colonie that result is the positive is GroEL clone strain.
It is highly preferred that the specific method is as follows by step S13: by plasmid pMD18T-GroEL and expression vector pET28a (+)
EcoR is used respectivelyIAnd Hind IIICarry out double digestion；Digestion products glue is recycled, is connectedgroELGenetic fragment and pET28a
(+), 16 DEG C of connections are stayed overnight, linked system are as follows: 10 μ l contain 4 μ lgroELGenetic fragment, 1 μ l pET28a (+), 5 μ l
Solution I。
It is highly preferred that the system of double digestion described in step S13 are as follows: 50 μ l total volumes contain 5 10 × QuickCut of μ l
Green Buffer, 1 μ l EcoR I, 1 μ l HindIII, 1 μ g pET28a (+) adds the water without nuclease to 50 μ l.
It is highly preferred that the condition of double digestion described in step S13 are as follows: 30 DEG C of digestions 10min, 37 DEG C of digestion 20min.
It is further preferred that the specific method is as follows by step S2:
S21. the engineering bacteria single colonie containing recombinant plasmid pET28-GroEL is inoculated in LB containing kanamycin
In fluid nutrient medium, 9~10h of shake culture；
S22. bacterium solution is added in LB fluid nutrient medium containing kanamycin, expands 1~3h of culture, until OD600 is
0.5~0.7；
S23. after IPTG Fiber differentiation being added into bacterium solution, using imidazole elution purification and recovery, the recombination that is purified
GroEL albumen, the as described Rofe source of fish Streptococcusagalactiae recombinate GroEL albumen, amino acid sequence such as SEQ ID NO.2 institute
Show.
Wherein, more specifically preferably, the specific method is as follows by step S2:
S21. it will contain recombinant plasmid pET28-GroEL'sE.coliBL21 (DE3) single colonie is inoculated in 5ml and contains
In the LB fluid nutrient medium of 50 μ g/ml kanamycins, 9~10h of 200r/min shake culture at 37 DEG C；
S22. 5ml bacterium solution addition 500ml is contained in the LB fluid nutrient medium of 50 μ g/ml kanamycins, 1:100 expands
Big culture 2h, until OD600 is about 0.6；
S23. the IPTG of final concentration of 0.6mmol/l is added into bacterium solution, 16 DEG C of shake cultures are stayed overnight；It is collected by centrifugation overnight
The thallus of induction, Lysis Buffer are resuspended, and supernatant is collected by centrifugation in ultrasonication 15min after ice bath 30min；
S24. purify the recombination GroEL albumen in supernatant with Ni-NTA, first with the elution containing 20mmol/l imidazoles
Liquid washes away impurity, then the elution recombinant protein with the imidazoles containing 200mmol/l；The recombinant protein of elution is super with 10KDa
Chimney filter ultrafiltration removes the imidazoles in eluent, the recombination GroEL albumen purified, the as described Rofe source of fish agalasisa hammer
Bacterium recombinates GroEL albumen, and amino acid sequence is as shown in SEQ ID NO.2.
In addition, above-mentioned Rofe source of fish Streptococcusagalactiae recombinate GroEL albumen as or preparation Rofe source of fish agalasisa hammer
Bacterium recombinates the application in terms of GroEL protein vaccine, also within protection scope of the present invention.
A kind of Rofe source of fish Streptococcusagalactiae recombinates the preparation method of GroEL protein vaccine, is by Rofe source of fish agalasisa chain
Coccus recombination GroEL albumen is diluted to 1mg/ml with sterilizing PBS, completely or Freund's incomplete adjuvant is uniformly mixed by 1:1, system with Freund
For at subunit vaccine.
Further, the vaccine for taking 1ml to prepare, is centrifuged 5min at 3000 × g, and vaccine then can be used without lamination
In follow-up immunization.
The Rofe source of fish Streptococcusagalactiae recombination GroEL protein vaccine and the Tilapia mossambica that the above method is prepared
Source Streptococcusagalactiae recombinates GroEL protein vaccine answering in terms of the drug or preparation of preparation prevention and treatment Tilapia mossambica Streptococcusagalactiae disease
With also all should be within protection scope of the present invention.
The invention has the following advantages:
The present invention provides a kind of preparation method of Rofe source of fish Streptococcusagalactiae recombination GroEL protein vaccine, gained weights
Group GroEL protein vaccine can be used for preventing and treating Tilapia mossambica Streptococcusagalactiae disease, can be to the anti-Streptococcusagalactiae difference serum of Tilapia mossambica
Type generates the protectiveness of specificity, and vaccine reaches 68.61 ± 7.39% to the immune protective rate of the anti-Streptococcusagalactiae of Tilapia mossambica.
Compared with prior art, recombination GroEL subunit vaccine of the invention has apparent advantage, in the prior art to sieve
The prevention and treatment of non-fish Streptococcusagalactiae still relies primarily on conventional medicament, the novel anti-Streptococcusagalactiae disease vaccine in cultivating tilapia
Exploitation is research hotspot in recent years, but focuses primarily upon full bacterium inactivated vaccine and attenuated live vaccine；Although both vaccines
Preparation method is easy, but use of the inactivated vaccine in aquiculture animal needs large dosage, multiple immunity inoculation, and is attenuated
Storage and the traffic condition requirement of live vaccine are high, and validity period is shorter, and exist and return malicious danger；Therefore both vaccines may
Cause biggish toxicity.Subunit vaccine is i.e. as having a vaccine made by immunocompetent pathogenic mycoprotein, in the present invention
Recombination GroEL subunit vaccine contain only a kind of pathogenic bacteria protein, thus the antibody that many irrelevant antigens are induced can be eliminated,
It reduces the side reaction of vaccine bring and because of other diseases caused by vaccine, safety is reliable.Also, with full bacterium inactivated vaccine and
Attenuated live vaccine is compared, and serotype specificity is not present in recombination GroEL vaccine of the invention, can fight the agalasisa of different serotypes
Streptococcus, the scope of application are wider.Therefore, it has a good application prospect in terms of preventing Tilapia mossambica Streptococcusagalactiae.
Moreover, protein vaccine preparation method of the invention uses prokaryotic expression system, preparation method is simple, and process is succinctly fast
Speed, safe preparation process, expression is at low cost, yield is high (1L culture solution can be purified into 20 mg destination proteins), is suitable for extensive
Production application.
Detailed description of the invention
Fig. 1 is Rofe source of fish Streptococcusagalactiae GroEL gene PCR amplified production；Wherein, swimming lane M: DL2000
Marker ；1: PCR amplified production of swimming lane.
Fig. 2 is protein purification result；In figure, M is albumen Marker；Swimming lane 1 is that induction generates rGroELE. coli
BL21 (DE3) ultrasonication solution；Swimming lane 2 is the protein solution for being not bound with pillar；Swimming lane 3 is that 20 mM imidazole concentrations are washed
Wash the solution of pillar；Swimming lane 4-7 is the protein solution that 100 mM imidazole concentrations elute；Swimming lane 8-10 is 200 mM imidazoles
The protein solution that concentration elutes.
Fig. 3 is that recombination GroEL albumen Elisa detects serum antibody titer；Abscissa is the blood for just exempting from the rear 0th, 2,4 week
Clearly；Antibody titer is indicated with the maximum dilution multiple for generating positive findings.
Fig. 4 is to recombinate after Tilapia mossambica is immunized in GroEL protein vaccine to attack malicious survival curve.
Specific embodiment
The present invention is further illustrated below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention
It limits in any form.Unless stated otherwise, the present invention uses reagent, method and apparatus routinely try for the art
Agent, method and apparatus.
Unless stated otherwise, following embodiment agents useful for same and material are commercially available.
Embodiment 1 prepares Rofe source of fish Streptococcusagalactiae and recombinates GroEL albumen
1, design of primers:
Using Rofe source of fish Streptococcusagalactiae as template, a pair of primer GroEL-F comprising restriction enzyme of design and
GroEL-R, sequence are as follows:
Primer GroEL-F(sequence is as shown in SEQ ID NO.3):
5’- GGAATTCATGGCAAAAGATATTAAATTTTC-3’
Primer GroEL-R((sequence is as shown in SEQ ID NO.4)):
5’- GGGTTCGAAGAAGCCACCCATCATAGATGG-3’
Upstream primer GroEL-F underscore part is EcoRIRestriction enzyme site, downstream primer GroEL-R underscore portion
Dividing is HindIIIRestriction enzyme site.
2、GroELGene cloning
(1) PCR amplificationGroELGene
Rofe source of fish Streptococcusagalactiae genomic DNA is extracted, using genomic DNA as template, with above-mentioned primer pair
GroEL-F and GroEL-R carries out PCR reaction.
PCR reaction system are as follows: 25 μ l reaction system template DNAs containing 50ng (genomic DNA), 2.5 10 × PCR of μ l
Buffer(Mg+), 2 μ l dNTP Mixture (2.5mM), 1 μ l GroEL-F, 1 μ l GroEL-R, 0.125 μ l rTaq (5U/
μ l), add sterile purified water to 25 μ l.
PCR condition are as follows: 94 DEG C of initial denaturation 1min；Then 94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 70 DEG C extend
30 circulations are acted under 1min；Last 70 DEG C of extensions 10min；16 DEG C of heat preservations.
Pcr amplification product electrophoretogram is as shown in Fig. 1.
GroELGene nucleotide series are as shown in SEQ ID NO.1.
(2) GroEL clone strain is constructed
It connect after above-mentioned PCR product recovery purifying with pMD18-T carrier, is connected overnight at 16 DEG C.
Linked system are as follows: 10 μ l systems include 4 μ l PCR products, 1 μ l pMD18-T Vector, 5 μ l Solution
I。
10 μ l connection products are transferred to 50 μ l Escherichia coli Trans1-T1 competent cells, ice bath 30min, 42 DEG C of heat shocks
60s, is then added 500 μ l LB fluid nutrient mediums, and 37 DEG C of shake cultures 2h, 4000rpm are centrifuged 5min, stay 100 μ l piping and druming heavy
Shallow lake bacterium solution mixed liquor is added on the LB solid medium containing 100 μ g/ml ampicillins (Amp+), after coating uniformly
In 37 DEG C of overnight incubations.
10 single colonies identification on random picking plate, qualification result are that positive single colonie is GroEL clone bacterium
Strain.
3, construction recombination plasmid pET28-GroEL
GroEL clone strain plasmid is extracted, plasmid pMD18T-GroEL and expression vector pET28a (+) are used respectively
EcoR IAnd Hind IIICarry out double digestion.
Double digestion system are as follows: 50 μ l total volumes contain 5 μ l 10 × QuickCut Green Buffer, 1 μ l EcoR I, 1 μ
l Hind III, 1 μ g pET28a (+) adds the water without nuclease to 50 μ l.
30 DEG C of digestions 10min, 37 DEG C of digestion 20min.
Digestion products glue is recycled, is connectedgroELGenetic fragment and pET28a (+), 16 DEG C of connections are stayed overnight, linked system
Are as follows: 10 μ l contain 4 μ lgroELGenetic fragment, 1 μ l pET28a (+), 5 μ l Solution I.
Connection product is transferred to e. coli bl21 (DE3) competent cell, the screening of Jing Kana penicillin resistance, picking list
Bacterium colony carries out PCR identification, filters out the engineering bacteria that positive colony cell is the pET28-GroEL containing recombinant plasmid.
4, GroEL albumen --- recombinant plasmid pET28-GroEL exists for purifying recombinationE.coliExpression in BL21 (DE3)
And purifying:
(1) it will contain recombinant plasmid pET28-GroEL'sE.coliBL21 (DE3) single colonie is inoculated in 5ml and contains
In the LB fluid nutrient medium of 50 μ g/ml kanamycins, 9~10h of 200r/min shake culture at 37 DEG C adds 5ml bacterium solution
Enter 500ml to contain in the LB fluid nutrient medium of 50 μ g/ml kanamycins, 1:100 expands culture 2h, until OD600 is about
0.6.The IPTG of final concentration of 0.6mmol/l is added into bacterium solution, 16 DEG C of shake cultures are stayed overnight.The bacterium of overnight induction is collected by centrifugation
Body, Lysis Buffer are resuspended, and supernatant is collected by centrifugation in ultrasonication 15min after ice bath 30min.Purify supernatant with Ni-NTA
Recombination GroEL albumen in liquid first washes away impurity with the eluent containing 20mmol/l imidazoles, then with miaow containing 200mmol/l
The elution recombinant protein of azoles.The 10KDa super filter tube ultrafiltration of the recombinant protein of elution removes the imidazoles in eluent,
The recombination GroEL albumen purified, the as described Rofe source of fish Streptococcusagalactiae recombinate GroEL albumen, amino acid sequence
As shown in SEQ ID NO.2.
(2) protein purification result is as shown in Fig. 2, in figure: M is albumen Marker；Swimming lane 1 is that induction generates rGroEL
'sE. coliBL21 (DE3) ultrasonication solution；Swimming lane 2 is the protein solution for being not bound with pillar；Swimming lane 3 is 20 mM
The solution of imidazole concentration washing pillar；Swimming lane 4-7 is the protein solution that 100 mM imidazole concentrations elute；Swimming lane 8-10 is
The protein solution that 200 mM imidazole concentrations elute.
5, Elisa is detected
2, detection method
(1) it is coated with: diluting recombination GroEL albumen to 200 μ g/ml with coating buffer, be added in 96 hole elisa Plates, 100 μ l/
Hole, 4 DEG C of coatings are overnight.
(2) it washs: discarding the coating buffer in 96 holes, cleaned 5 times with less salt Buffer, 3min × 5 time, every time
250 holes μ l/ after liquid-transfering gun is gently blown and beaten, are mitigated oscillation with the speed of 200rpm, are patted dry.
(3) it closes: 1% BSA is added and is closed, 2h, is closed in 250 holes μ l/ by 22 DEG C.It is washed by above-mentioned condition
It washs, pats dry.
(4) primary antibody reacts: the Tilapia mossambica antiserum of acquisition is diluted (such as extension rate: 2,2 by 2 times with PBS step by step2、
23、24、25、26、27、28Deng), it is added by 100 holes μ l/, 22 DEG C, is incubated for 3h.It is washed, is patted dry by above-mentioned condition.
(5) secondary antibody reacts: mouse anti-fish IgM PBS being diluted 10 times, is added, 22 DEG C by 100 holes μ l/, is incubated for 2h；It washes
It washs, pats dry.
(6) three anti-reflective are answered: the antibody PBS of sheep anti-mouse igg-HRP label being diluted 5000 times, is added by 100 holes μ l/
Enter, 22 DEG C of incubation 2h；Washing, pats dry.
(7) it develops the color: TMB substrate solution being added by 100 holes μ l/, incubation at room temperature；After 10min, reaction terminating liquid is added
50 holes μ l/ terminate reaction.
(8) it reads: this 96 ELISA Plate being placed in microplate reader and is read, measure the light absorption value at 450nm, i.e.,
OD450。
If the reading of test group is greater than or equal to twice of control group, it is denoted as the positive, is otherwise feminine gender.Antibody titer with
The maximum dilution multiple of positive findings indicates.
2, result is as shown in Figure 3.There is different degrees of growth in Post-immunisation serum antibody titer, and reaches peak in 4th week
Value is 1:2048.
Embodiment 2 prepares Rofe source of fish Streptococcusagalactiae and recombinates GroEL protein vaccine
By the recombination GroEL albumen for the purifying that embodiment 1 obtains with sterilizing PBS be diluted to 1mg/ml, with Freund completely and
Freund's incomplete adjuvant is uniformly mixed by 1:1, is prepared into subunit vaccine.
The vaccine that 1ml is prepared is taken, 5min is centrifuged at 3000 × g, vaccine then can be used for subsequent exempt from without lamination
Epidemic disease.
3 Rofe source of fish Streptococcusagalactiae of embodiment recombinates GroEL protein vaccine immunity test
1, immunoprotection experiment
(1) bolti is purchased from Guangzhou City Chengyi Marine Product Science Co., Ltd, and specification is 50 ± 5.0g, temporarily supports before testing
Two weeks.
(2) when immunization experiment, Tilapia mossambica is randomly divided into 3 groups, i.e. PBS group, PBS+ adjuvant group, GroEL vaccine group.Every group
14 tails, every group sets three in parallel.
GroEL group and adjuvant component be not injected intraperitoneally the recombinant antigen GroEL that 100 μ l and Freund's adjuvant mix in equal volume with
And PBS, PBS group inject the sterilizing PBS of 100 μ l.
2, immune programme
Tilapia mossambica is immunized twice, initial immunity uses incomplete Freund's adjuvant, and booster immunization is complete using Freund
Adjuvant subunit vaccine.
When initial immunity, 100 μ l vaccines are injected intraperitoneally in every tail fish.After two weeks, every tail fish be injected intraperitoneally 100 μ l vaccines into
Row booster immunization.
The the 2nd and 4 week after just exempting from, every group is selected 2 tail fishes to take a blood sample, after being placed at room temperature for 2h, 4 DEG C, 10000 × g centrifugation 10min,
Supernatant is collected, -80 DEG C of preservations, the measurement for immune indexes after packing.
3, immune protective rate measures
(1) booster immunization after two weeks, carries out bacterium challenge viral dosage with LD50 dosage, 100ul agalasisa is injected intraperitoneally in every tail fish
Streptococcus bacterium solution (1 × 105CFU/ ml).After attacking poison, the situation of Tilapia mossambica is observed daily, pulls dead fish out in time, and record 14
Death condition in it.It is observed continuously 14 days, stops observation, experiment terminates.
The relative protection ratio (RPS) of Tilapia mossambica is calculated according to following formula:
RPS=(1- immune group cumulative mortality/control group cumulative mortality) × 100%.
(2) Tilapia mossambica death condition is as shown in Figure 4 after attacking poison.
The results show that 4th week after initial immunity, is carried out attacking poison with Streptococcusagalactiae.Tilapia mossambica is the 1st day after attacking poison
Start to occur as soon as death, death is concentrated mainly on first 2 days after attacking poison.
In addition, 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.
Recombinate GroEL albumen as anti-Tilapia mossambica Streptococcusagalactiae disease vaccine for injecting immune can get 68.61 ±
7.39% immune protective rate.
SEQUENCE LISTING
<110>Zhongshan University
<120>preparation method and applications of Rofe source of fish Streptococcusagalactiae recombination GroEL protein vaccine
<130>
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 1623
<212> DNA
<213>Rofe source of fish Streptococcusagalactiae groEL gene order
<400> 1
atggcaaaag atattaaatt ttcagcagat gcccgctcag caatggtgcg tggtgttgat 60
attttagctg atacagtcaa agtaacatta ggtcctaaag gccgtaatgt tgttcttgaa 120
aaagcatttg gttcgccttt aattacaaat gatggtgtga caattgctaa agaaattgag 180
ctagaagatc actttgaaaa tatgggagct aaacttgtgt cagaagtggc ttcaaaaact 240
aatgatattg caggggatgg cactacaact gctactgttt tgacccaagc tattgtacgg 300
gaaggtctta aaaatgtaac tgcaggggcg aatccgattg gtattcgtcg tggtattgaa 360
acagctgttt cagcagcagt tgaagagcta aaagagattg cacaaccagt ttcaggcaaa 420
gaagctattg ctcaagttgc agctgtgtct tcacgttctg aaaaagttgg ggaatatatt 480
tctgaagcta tggagcgcgt gggtaatgat ggtgttatca ctattgaaga atcgcgaggt 540
atggaaacag agcttgaagt tgtagaagga atgcagtttg accgtgggta cttgtcacag 600
tatatggtaa ctgataacga gaaaatggtc tctgaacttg agaatccgta tatccttatt 660
acagataaga aaatttcaaa tatccaagaa attttaccat tattagaaga ggttcttaaa 720
acaaatcgtc cattgctaat catcgctgat gatgttgatg gagaagctct cccaacgctt 780
gttcttaata aaattcgtgg aactttcaat gtcgtagctg ttaaagcgcc tggatttggt 840
gatcgtcgta aagccatgct ggaagatatt gctatcctaa caggaggaac tgtcgttact 900
gaagaccttg gtttagactt aaaagatgct actatgcaag ttttaggaca gtctgctaaa 960
gtaacagtag ataaagattc tactgttatt gtcgaaggtg ccggtgactc atcagcaatt 1020
gctaatcgcg tagctatcat taagtcacag atggaggcta caacttctga ttttgatcgt 1080
gaaaaattac aagaacgact tgccaagtta gccggtggtg tagcagtaat taaagttggt 1140
gcagcgactg aaacagaatt aaaagagatg aaacttcgca tcgaagatgc gttaaatgca 1200
acgcgtgctg cagttgaaga aggtattgtt tcaggtggag gtacggctct tgtgaacgtt 1260
attgaaaaag tagcggcact gaaacttaat ggtgatgagg agactggacg taatattgtt 1320
cttcgtgctc tcgaagagcc tgttcgtcaa attgcttaca atgctggata tgaaggttca 1380
gttattattg aacgtttaaa acagtctgaa attggtacag gatttaatgc ggctaatgga 1440
gaatgggtag atatggttac cacaggtatc attgaccctg tcaaagtaac acgttctgca 1500
cttcaaaatg cggcatctgt agcaagtctt atcttgacta cagaagcagt agtagcaaat 1560
aaacctgaac cagaagctcc tacagctcct gcaatggatc catctatgat gggtggcttc 1620
taa 1623
<210> 2
<211> 540
<212> PRT
<213>Rofe source of fish Streptococcusagalactiae recombinates GroEL protein sequence
<400> 2
Met Ala Lys Asp Ile Lys Phe Ser Ala Asp Ala Arg Ser Ala Met Val
1 5 10 15
Arg Gly Val Asp Ile Leu Ala Asp Thr Val Lys Val Thr Leu Gly Pro
20 25 30
Lys Gly Arg Asn Val Val Leu Glu Lys Ala Phe Gly Ser Pro Leu Ile
35 40 45
Thr Asn Asp Gly Val Thr Ile Ala Lys Glu Ile Glu Leu Glu Asp His
50 55 60
Phe Glu Asn Met Gly Ala Lys Leu Val Ser Glu Val Ala Ser Lys Thr
65 70 75 80
Asn Asp Ile Ala Gly Asp Gly Thr Thr Thr Ala Thr Val Leu Thr Gln
85 90 95
Ala Ile Val Arg Glu Gly Leu Lys Asn Val Thr Ala Gly Ala Asn Pro
100 105 110
Ile Gly Ile Arg Arg Gly Ile Glu Thr Ala Val Ser Ala Ala Val Glu
115 120 125
Glu Leu Lys Glu Ile Ala Gln Pro Val Ser Gly Lys Glu Ala Ile Ala
130 135 140
Gln Val Ala Ala Val Ser Ser Arg Ser Glu Lys Val Gly Glu Tyr Ile
145 150 155 160
Ser Glu Ala Met Glu Arg Val Gly Asn Asp Gly Val Ile Thr Ile Glu
165 170 175
Glu Ser Arg Gly Met Glu Thr Glu Leu Glu Val Val Glu Gly Met Gln
180 185 190
Phe Asp Arg Gly Tyr Leu Ser Gln Tyr Met Val Thr Asp Asn Glu Lys
195 200 205
Met Val Ser Glu Leu Glu Asn Pro Tyr Ile Leu Ile Thr Asp Lys Lys
210 215 220
Ile Ser Asn Ile Gln Glu Ile Leu Pro Leu Leu Glu Glu Val Leu Lys
225 230 235 240
Thr Asn Arg Pro Leu Leu Ile Ile Ala Asp Asp Val Asp Gly Glu Ala
245 250 255
Leu Pro Thr Leu Val Leu Asn Lys Ile Arg Gly Thr Phe Asn Val Val
260 265 270
Ala Val Lys Ala Pro Gly Phe Gly Asp Arg Arg Lys Ala Met Leu Glu
275 280 285
Asp Ile Ala Ile Leu Thr Gly Gly Thr Val Val Thr Glu Asp Leu Gly
290 295 300
Leu Asp Leu Lys Asp Ala Thr Met Gln Val Leu Gly Gln Ser Ala Lys
305 310 315 320
Val Thr Val Asp Lys Asp Ser Thr Val Ile Val Glu Gly Ala Gly Asp
325 330 335
Ser Ser Ala Ile Ala Asn Arg Val Ala Ile Ile Lys Ser Gln Met Glu
340 345 350
Ala Thr Thr Ser Asp Phe Asp Arg Glu Lys Leu Gln Glu Arg Leu Ala
355 360 365
Lys Leu Ala Gly Gly Val Ala Val Ile Lys Val Gly Ala Ala Thr Glu
370 375 380
Thr Glu Leu Lys Glu Met Lys Leu Arg Ile Glu Asp Ala Leu Asn Ala
385 390 395 400
Thr Arg Ala Ala Val Glu Glu Gly Ile Val Ser Gly Gly Gly Thr Ala
405 410 415
Leu Val Asn Val Ile Glu Lys Val Ala Ala Leu Lys Leu Asn Gly Asp
420 425 430
Glu Glu Thr Gly Arg Asn Ile Val Leu Arg Ala Leu Glu Glu Pro Val
435 440 445
Arg Gln Ile Ala Tyr Asn Ala Gly Tyr Glu Gly Ser Val Ile Ile Glu
450 455 460
Arg Leu Lys Gln Ser Glu Ile Gly Thr Gly Phe Asn Ala Ala Asn Gly
465 470 475 480
Glu Trp Val Asp Met Val Thr Thr Gly Ile Ile Asp Pro Val Lys Val
485 490 495
Thr Arg Ser Ala Leu Gln Asn Ala Ala Ser Val Ala Ser Leu Ile Leu
500 505 510
Thr Thr Glu Ala Val Val Ala Asn Lys Pro Glu Pro Glu Ala Pro Thr
515 520 525
Ala Pro Ala Met Asp Pro Ser Met Met Gly Gly Phe
530 535 540
<210> 3
<211> 30
<212> DNA
<213>primer GroEL-F sequence
<400> 3
ggaattcatg gcaaaagata ttaaattttc 30
<210> 4
<211> 30
<212> DNA
<213>primer GroEL-R sequence
<400> 4
gggttcgaag aagccaccca tcatagatgg 30
Claims (3)
1. Rofe source of fish Streptococcusagalactiae, which recombinates GroEL albumen, recombinates GroEL albumen epidemic disease in preparation Rofe source of fish Streptococcusagalactiae
Application in terms of seedling, which is characterized in that the amino acid sequence of recombination GroEL albumen is as shown in SEQ ID NO.2.
2. a kind of preparation method of Rofe source of fish Streptococcusagalactiae recombination GroEL protein vaccine, which is characterized in that be by Tilapia mossambica
Source Streptococcusagalactiae recombination GroEL albumen with sterilizing PBS be diluted to 1mg/ml, with Freund completely or Freund's incomplete adjuvant by 1:1 mix
It closes uniformly, is prepared into subunit vaccine；The amino acid sequence of GroEL albumen is wherein recombinated as shown in SEQ ID NO.2.
3. the Rofe source of fish Streptococcusagalactiae that method is prepared according to claim 2 recombinates GroEL protein vaccine.
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| PL239045B1 (en) * | 2018-01-09 | 2021-11-02 | Inst Immunologii I Terapii Doswiadczalnej Polskiej Akademii Nauk | Diagnostic test for detection of infections and/or carrying of Streptococcus agalactiae, using the selected polypeptide markers |
| CN108287244B (en) * | 2018-03-28 | 2020-03-24 | 深圳市伯劳特生物制品有限公司 | Application of heat shock protein groEL in preparation of helicobacter pylori detection reagent |
| WO2024060118A1 (en) * | 2022-09-22 | 2024-03-28 | Bened Biomedical Co., Ltd. | Method for treating sleeping disorders with groel protein |
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| CN102847146A (en) * | 2011-09-15 | 2013-01-02 | 通威股份有限公司 | Vaccine used for preventing tilapia streptococcal disease |
| CN104403958A (en) * | 2014-09-04 | 2015-03-11 | 中国水产科学研究院珠江水产研究所 | Bacillus cereus NY5 capable of effectively inhibiting tilapia-source streptococcus agalactiae |
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