CN102614504B

CN102614504B - Pseudomonas fluorescens recombinant protein vaccine and preparation method thereof

Info

Publication number: CN102614504B
Application number: CN2012100651677A
Authority: CN
Inventors: 胡永华; 孙黎
Original assignee: Institute of Oceanology of CAS
Current assignee: Institute of Oceanology of CAS
Priority date: 2012-03-13
Filing date: 2012-03-13
Publication date: 2013-12-04
Anticipated expiration: 2032-03-13
Also published as: CN102614504A

Abstract

The invention relates to the field of molecular vaccinology, in particular to a pseudomonas fluorescens recombinant protein vaccine and a preparation method thereof. The antigen of the vaccine is shown as the amino acid sequence in the sequence list SEQ ID No. 1. The preparation method comprises the following specific steps of: taking pseudomonas fluorescens TSS1 as a template, using F1/R1 as a primer to perform polymerase chain reaction (PCR) amplification, connecting a PCR product and a carrier p EASY-E2, converting the connecting liquid into escherichia coli DH 5 alpha, and selecting plasmid p E478, wherein the plasmid p E478 converts a recombinant protein containing the amino acids in the sequence list SEQ ID No. 1 and expressed by escherichia coli BL21 (DE 3). The immune protection efficiency of the vaccine to the pseudomonas fluorescens is up to 81 percent.

Description

A kind of pseudomonas fluorescens recombinant protein vaccine and preparation thereof

Technical field

The present invention relates to molecular vaccine and learn field, a kind of pseudomonas fluorescens recombinant protein vaccine and preparation method thereof specifically.

Background technology

Pseudomonas fluorescens (Pseudomonas fluorescens) is a kind of gram negative bacilli, can infect multiple culturing economic Fish, comprises Cyprinus carpio, tilapia, Paralichthys olivaceus etc.Pseudomonas fluorescens infects the disease that Fish are brought out a kind of being known as " erythroderma ", and its symptom is bleeding of skin and festers, thereby causes serious economic loss to industry.At present, the pseudomonas fluorescens disease control there is no effective vaccine, mainly relies on the use of antibiotics.Therefore, need badly and carry out in China for the vaccine research and development of pseudomonas fluorescens.Recombinant subunit vaccine is a kind of common vaccine form, and its cardinal principle is to utilize an antigen of cause of disease self, applies as vaccine after heterogenous expression, thereby plays the immanoprotection action to cause of disease.We find that in early-stage Study the outer membrane protein P478 of pseudomonas fluorescens has good immunogenicity, has been built into it recombinant subunit vaccine on this basis.

Summary of the invention

The object of the invention is to provide a kind of pseudomonas fluorescens recombinant protein vaccine and preparation thereof.

For achieving the above object, the technical solution used in the present invention is:

A kind of pseudomonas fluorescens recombinant protein vaccine, vaccine antigen is shown in the aminoacid sequence in sequence table SEQ ID No.1.Described recombinant protein vaccine is the eukaryotic expression recombination plasmid pE478 that contains list SEQ ID No.1.Described recombinant protein vaccine is the recombiant protein that the eukaryotic expression recombination plasmid pE478 that contains list SEQ ID No.1 transforms escherichia coli expression.

The preparation method of pseudomonas fluorescens recombinant protein vaccine be take pseudomonas fluorescens TSS1 as template, adopting F1/R1 is that primer carries out pcr amplification, the PCR product is connected with carrier pEASY-E2, connecting fluid is transformed into bacillus coli DH 5 alpha, screening plasmid pE478, plasmid pE478 transforms the amino acid whose recombiant protein in sequence table SEQ ID No.1 that contains of e. coli bl21 (DE3) expression; Described primer is F1:5 '-CATATGAACGACAACACTCTTTATG-3 '; R1:5 '-CTCGAGGAAGTTGGCCTCCAGC-3 '.

The application of pseudomonas fluorescens recombinant protein vaccine, described recombinant protein vaccine has the application in prevention and treatment pseudomonas fluorescens bacterin preparation in preparation

The present invention has following advantage:

1. high protectiveness.Vaccine of the present invention reaches 81% to the immunoprotection efficiency of pseudomonas fluorescens.

2. without the commercialization adjuvant.

The accompanying drawing explanation

The vaccine protein (swimming lane 2) of the purification that Fig. 1 provides for the embodiment of the present invention.Swimming lane 1, molecular weight standard.

The specific embodiment

Below in conjunction with embodiment, the invention will be further described.Embodiment is intended to the description of giving an example to the present invention, but not limits the invention in any form.Involved experimental technique routinely all adopts following method in embodiments of the present invention:

1. plasmid extraction, DNA (PCR) product purification are all used the corresponding reagent box of " TIANGEN Biotech (Beijing) Co., Ltd. ".

2. plasmid, the conversion of DNA connecting fluid enter escherichia coli and all use Hanahan method (Sambrook and Russell:Molecular Cloning:A Laboratory Mannual.Cold Spring Harbor Laboratory Press 2001);

3. all restricted enzyme and ligase are all purchased from " Beijing, Niu Yinglun Bioisystech Co., Ltd ".

Embodiment 1

The pseudomonas fluorescens vaccine antigen is (referring to sequence table 1) shown in the aminoacid sequence in sequence table SEQ ID No.1.

Sequence table 1

MSVRDLHVNDNTLYALLLASGLGGFAPLVLADDAQDVGSVNIAGKQTLGSGHMILEESAKARSTVTKQAMDEMSATANAIDKLKYTPGINVSSDDASGTSGTNFTMRGMSSDQVGVSVDGVPINDSGNYSVYSNQLGDPENLAKVFATQGSSEADGPHIGSSGGNIGMITIRPTKDAGVFAKQIVGANALRKTFVRANTGDLGGLKTWVSASHLEGDKWRGKGTLRSDKIEWSSLFEGDNGNSTLATIKYNKQENYNYNSLSKAQFDTEGRRKDYAQSPVYKAGLLSASYKLNRNPFESVNATLTQRWQLQDNLSLTLTPYYYWANGGSFSGQTASNLGPKSDKAGNYDLSNLQSANYYRPSWTETWRPGFTAKMKWDINEEHSLDYGYWYERARQRQTQPFIGINNEGAPDDVWGDYNSGGQVVDKNGATVQGRHYYTVTPAQKLWVQDNWQATPDLSFVGGVAYQYVERDGNNLGSLYDTPEKRNTRYHQFLPNFSAKYQLDDSNQAFYNVTRNMRTPPNYVLYNKGDSVSLKSELSWNQELGWRYTEDDMALSATLFYISFKDRQVSTTDASGDYMVLNVGTVDNKGLELEWSGLLPHNFNYYASYTYTQSEQKDDLLSKNVLLPTSGKQLANVPENMFNLVFGYDDSRFYGNIAGKYVGSFYGDLTNKEKIEGRTVFDLNAGIYLPVDKKVIKSAALRFSMLNVFDKEYLSSARTVAFNSAPVNGLGASTAYYNVGEERTAMVSLEANF

(a) sequence signature:

● length: 753

● type: aminoacid sequence

● chain: strand

● topological structure: linearity

(b) molecule type: protein

(c) suppose: no

(d) antisense: no

(e) initial source: pseudomonas fluorescens TSS1

(f) specificity title: P478

Architectural feature: this albumen has the Plug functional domain (aminoacid 48-156) of a signal peptide structure (amino acid/11-24) and a TonB dependent form albumen.

Embodiment 2

The construction method of pseudomonas fluorescens vaccine expression vector: the structure of plasmid pE478: take pseudomonas fluorescens TSS1 as template, adopting F1/R1 is that primer carries out pcr amplification, and the PCR condition is: 94 ℃ of 60s denaturation template DNAs, then 94 ℃ of 40s, 50 ℃ of 60s, 72 ℃ of 60s, 5 circulations; Then 94 ℃ of 40s, 58 ℃ of 60s, 72 ℃ of 60s, after 25 circulations again at 72 ℃ of extension 10min.The PCR product is connected 2-4 hour with carrier pEASY-E2 (purchased from " Beijing Quanshijin Biotechnology Co., Ltd ") in room temperature after purifying with a day root DNA product purification test kit, connect after mixed liquor transforms bacillus coli DH 5 and cultivate 18 hours on the LB solid medium that contains 100ug/ml peace card penicillin (Ap), transformant of picking, extract plasmid, be plasmid pE478.

Described LB constituent is by weight percentage: 1.0% peptone, 0.5% yeast powder, 1.0% sodium chloride, 97.5% distilled water; Described bacterial strain TSS1 is stored in the common micro-organisms center C GMCC of China Committee for Culture Collection of Microorganisms, deposit number is: CGMCC No.2329, Classification And Nomenclature is pseudomonas fluorescens (Pseudomonas fluorescens), preservation date on January 9th, 2008.

Described primer is F1:5 '-CATATGAACGACAACACTCTTTATG-3 ' and R1:5 '-CTCGAGGAAGTTGGCCTCCAGC-3 '.

The expression and purity of P478 vaccine protein: above-mentioned plasmid pE478 is transformed to e. coli bl21 (DE3) (purchased from " day root biochemical technology company limited " by conventional method, Beijing), cultivate 18-24 hour on the LB solid medium that contains Ap (100ug/ml), transformant of picking, by its called after BL21/pE478.By BL21/pE478 incubated overnight in the LB fluid medium that contains Ap (100ug/ml); Culture fluid after getting 1ml and spending the night, add in the LB fluid medium that contains Ap (100ug/ml) that 100ml is fresh, in 37 ℃ of lower rotating speed 200rpm wave and culture to OD ₆₀₀Be 0.6, add the IPTG that final concentration is 1mM, 37 ℃ of continuation are with rotating speed 160rpm wave and culture 4-5h, then, with 5000g, 4 ℃ of centrifugal 10min, collect bacterium liquid, add the 5ml lysate, slowly shake 1-2 hour in room temperature on shaking table, until bacteria suspension becomes clarification.By bacterium liquid, with 10000g, 4 ℃ of centrifugal 30min, reclaim supernatant.His Trap HP Columns (being purchased from U.S. GE Healthcare company) for albumen in supernatant is reclaimed to purification, the albumen of purification is through SDS-PAGE electrophoresis detection (electrophoresis 25-30min under 8v/cm voltage, electrophoresis 2-2.5h under 15v/cm voltage subsequently), measure its molecular size range (referring to Fig. 1).The albumen of purification, through mass spectral analysis, is confirmed that it is to the antigen protein P478 with the aminoacid sequence in sequence table SEQ ID No1.The 10mM NaH that described lysate is final concentration ₂PO ₄, 10mM Tris and 8M carbamide, pH 8.0.

Embodiment 3

The application of vaccine

Step 1) preparation of adjuvant and vaccine mixed liquor.

Adjuvant preparation: by 5% (mass ratio) NaOH and 5% (mass ratio) Al ₂(SO ₄) ₃With 2: 5 volume ratios, mix, by mixture with 10,000g centrifugal 5 minutes.Precipitation is suspended in PBS to 0.2mg/ml.

Vaccine mixed liquor preparation: the vaccine protein P478 of above-described embodiment purification is diluted to 200ug/ml in PBS; Vaccine protein after dilution is mixed with the adjuvant equal-volume.Adjuvant contrast liquid preparation: PBS is mixed with the adjuvant equal-volume.Described PBS constituent is by weight percentage: 0.8%NaCl, 0.02%KCl, 0.358%Na ₂HPO ₄.12H ₂O, 0.024%NaH ₂PO ₄, surplus is water.

Step 2) immunity of P478 vaccine application.80 Paralichthys olivaceuss (every heavily about 12.2g) are divided into to 2 groups, 40 every group at random.By these 2 groups difference called after A and B group.By every fish of A group lumbar injection 100ul above-mentioned steps 1 respectively) the vaccine mixed liquor, by every fish of B group lumbar injection 100ul above-mentioned steps 1 respectively) adjuvant contrast liquid.

Step 3) preparation of pseudomonas fluorescens suspension.Cultivate pseudomonas fluorescens TSS1 to OD in the LB culture medium ₆₀₀Be 1, centrifugal (5000g, 4 ℃) 10min then.Collect thalline, by its be suspended in PBS to final concentration be 1x10 ⁷Cfu/ml.

Step 4) P478 vaccine immunity protective effect detects.In step 2) after inoculation the 30th day, by above-mentioned steps 3) pseudomonas fluorescens suspension lumbar injection step 2) 2 groups of fishes, the injection volume of every fish is 100ul.In afterwards 20 days, observe and record the death condition of respectively organizing fish every day.After 20 days, statistics is respectively organized total mortality of fish: A group, 7; The B group, 36.Utilize following formula to calculate relative immunity protection efficiency (RPS):

RPS=100x (the total dead percentage ratio of the total dead percentage ratio of 1-immune group fish/matched group fish)

The immunoprotection efficiency of calculating the P478 vaccine according to this formula is 81%.Therefore, obtained vaccine can be protected Paralichthys olivaceus to resist pseudomonas fluorescens to infect efficiently.

Claims

1. a pseudomonas fluorescens recombinant protein vaccine, it is characterized in that: the aminoacid sequence of vaccine antigen is as shown in SEQ ID No.1.

2. by pseudomonas fluorescens recombinant protein vaccine claimed in claim 1, it is characterized in that: described vaccine antigen is by RT-PCR expression plasmid abduction delivering.

3. by the described pseudomonas fluorescens recombinant protein vaccine of claim 1 or 2, it is characterized in that: described vaccine antigen transforms escherichia coli by the RT-PCR expression plasmid and carries out abduction delivering.

4. the preparation method of a pseudomonas fluorescens recombinant protein vaccine claimed in claim 1, it is characterized in that: take pseudomonas fluorescens TSS1 as template, adopting F1/R1 is that primer carries out pcr amplification, the PCR product is connected with carrier pEASY-E2, obtain recombiant plasmid pE478, connecting fluid transforms bacillus coli DH 5 alpha, with peace card penicillin, screened, the plasmid pE478 that screening is obtained transforms the recombiant protein of e. coli bl21 (DE3) express amino acid sequence as shown in sequence table SEQ ID No.1; Described pseudomonas fluorescens TSS1 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preserving number is CGMCC No.2329; Described primer is

F1：5’-CATATGAACGACAACACTCTTTATG-3’；

R1：5’-CTCGAGGAAGTTGGCCTCCAGC-3’。

5. the application of a pseudomonas fluorescens recombinant protein vaccine claimed in claim 1 is characterized in that: for the preparation of the bacterin preparation of prevention and treatment pseudomonas fluorescens disease.