CN100500849C - Expressing Ompk antigen of outer-membrane protein of Vibrio Harveyi and application as constituent of bacteria - Google Patents
Expressing Ompk antigen of outer-membrane protein of Vibrio Harveyi and application as constituent of bacteria Download PDFInfo
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- CN100500849C CN100500849C CNB200310112479XA CN200310112479A CN100500849C CN 100500849 C CN100500849 C CN 100500849C CN B200310112479X A CNB200310112479X A CN B200310112479XA CN 200310112479 A CN200310112479 A CN 200310112479A CN 100500849 C CN100500849 C CN 100500849C
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Abstract
An expression of the Hawei's vibrio outer membrane protein antigen Ompk in colibacillus and the application of the recombinant outer membrane protein Ompk as the component of vaccine in preventing the vibrosis of sea fish are disclosed. Said recombinant protein is prepared through providing Hawei's vibrio EcGS020802 DNA as template, cloning the coding gene of Ompk by PCR method, recombining the gene in the prokaryotic expression carrier pBV220, efficient expression of the recombinant protein in colibacillus to become inclusion body, washing, modifying and renaturation. Its advantages are high purity and high output.
Description
Invention field
The invention belongs to technical field of bioengineering, be specifically related to a kind of dna fragmentation and expression product protein thereof.This dna fragmentation is recombinant expressed in intestinal bacteria, and this recombinant DNA molecules and expression product thereof can be as the vaccine components of anti-vibriosis.
Technical background
Vibrios is a class Gram-negative, tool polar flagella, can move, asporous rod-short or arc bacterium, be distributed in offshore, river mouth seawater and the marine organism, be one of modal bacterium monoid in the ocean environment, wherein the part kind is halobiontic pathogenic bacteria.As pathogenic bacterium, vibrios can infect multiple cultivated animals, as shrimp, clam, Bao, fish etc., often causes mass mortality, brings enormous economic loss.Because they extensively are present in natural waters and sea farming district, vibriosis is worldwide popular, is common and endanger one of severe diseases in the sea farming.Traditional antibiotic therapy has been brought into play important effect in the bacillary vibrios of control sea farming object, but long-term prescription causes drug-resistance of bacteria, makes the dosage of medication increasing, causes drug residue, have a strong impact on the quality of fishery products, even threaten human health.In recent years; the development of beach vibrio piscium vaccine is very fast; playing an increasingly important role aspect the bacillary vibriosis of control; but mainly be inactivated vaccine; owing to there is complicated microbial metabolites in the inactivated vaccine preparation, side effect may occur, and protective antigen may be destroyed and influence its immune effect at inactivation process; different serotype lacks cross-protection, and these have a strong impact on its application.The genetic engineering subunit vaccine of a new generation then can overcome the above problems; carrying out the problem that genetic engineering subunit vaccine research at first will solve is to seek effective protective antigen gene; outer membrane protein is the peculiar structure of gram-negative bacteria cell wall; be positioned at the bacterium outermost layer; has the chance that extensively contacts with body immune system; therefore; become the focus of bacterium protective antigen research; the Ompk of vibrios is the wide host's of a class a vibrios phage receptor protein; avoid bringing into play important effect (FEMS Microbiology Letters125 (1995) 101-106) aspect the phage invasion and attack in the protection vibrios; but it is not seen relevant report as protective antigen; the present invention carries out the Ompk gene recombinant expressed; discover that its albumen has the better protecting antigenic characteristic, can be used as the invasion and attack that vaccine component is resisted vibrios.
Summary of the invention
The object of the present invention is to provide and a kind ofly can effectively resist the genetic engineering subunit vaccine of beach vibrio piscium disease.
The antigenic expression and purification method of Vibrio harveyi outer-membrane protein Ompk that the present invention proposes is to be template with Vibrio harveyi strain EcGS020802 DNA, gene with PCR method clone antigen Ompk coding, with this gene recombination in expressive plasmid pBV220, the recombinant protein of expressing forms inclusion body, inclusion body is behind the washing purifying, recombinant protein purity is good, the yield height.Produce recombinant protein with this method, easy and simple to handle, with low cost.
Another object of the present invention provides a kind of method for preparing the vibrios genetic engineering subunit vaccine.The preparation method utilizes the gene of polymerase chain reaction clone antigen Ompk coding, and it is recombinated to expressive plasmid pBV220, expresses the back purifying in escherichia coli host, adds that suitable adjuvant can obtain the vibrios genetic engineering subunit vaccine of stabilizer type.
Embodiment
The clone of embodiment 1 Ompk gene
The PCR primer: according to the vibrios kind outer-membrane protein Ompk gene conserved sequence design primer of having delivered, primer 1 is 5 ' ATG CGT AAA TCACTT TTA GCT CTT AGC C3 '; Primer 2 is 5 ' TTA CTG CGA TGT AGT GAC CGA AGC3 ', and is synthetic by Shanghai biotechnology company limited.The preparation of Ompk gene: with Vibrio harveyi EcGS020802 genomic dna is template, obtains goal gene with round pcr.PCR circulating reaction condition is: 94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 90s30 circulations.The clone of PCR product and evaluation: the foreign gene of purifying is connected with pGEM T carrier, transforms host e. coli, containing on the LB solid medium of penbritin by blue hickie method screening positive clone, further identifies with the enzyme blanking method.More than operation is all carried out according to a conventional method.With the ABI377 automatic sequencer positive colony is carried out sequential analysis to identify recombinant plasmid.
The length of pcr amplification product is 801 bases, and agarose electrophoresis result as shown in Figure 1.(M) is the dna molecular amount standard of 200bp among the figure, and (1) is pcr amplification product.
Add EcoRI and BamHI restriction enzyme site respectively at Ompk gene mature peptide two ends, with PCR method gene is transformed, with EcoRI and BamHI difference enzyme Qie Jiyin and carrier pBV220,4 ℃ connect 16h, transformed into escherichia coli DH5 α, fall containing on the LB flat board of penbritin the screening positive bacteria, extract the further enzyme of plasmid and cut evaluation.With the positive colony 30 ℃ of cultivations in liquid LB substratum after identifying, when OD value reached 0.4-0.6,42 ℃ of thermal inductions were immediately induced centrifugal collection bacterium behind the 4h, ultrasonic disruption, collection inclusion body.Inclusion body with the plain cracking of 8M urea, after remaining urea element is removed in the water dialysis, obtains the higher recombinant protein of purity after the plain washing of the urea of 2-4M.As shown in Figure 2.The recombinant protein Ompk of (1) purifying among the figure, (2) for not containing the bacillus coli DH 5 alpha of recombinant plasmid, (3) for containing the bacillus coli DH 5 alpha of recombinant plasmid, (4), (5) are not purified inclusion body, (M) are the low molecular weight protein (LMWP) standard.
Do not send out sick healthy cabrilla 150 tails, body weight 25-30g is divided into three groups at random, every group 50 tail.Ompk recombinant protein immune group adds isopyknic Freund's incomplete adjuvant (FCA) abdominal injection with 50ug/ tail antigen.Full bacterium is organized every endnote and penetrates Vibrio harveyi EcGS020802 vaccine 0.1ml.The every endnote of control group is penetrated physiological saline 0.1ml.After the immunity 28 days, use LD
50Dosage Vibrio harveyi Vibrio harveyi EcGS020802 carries out challenge infection to three experimental group, calculates its immune protective rate respectively.Through one-way analysis of variance, Ompk recombinant protein immune group premunition protection ratio is 100%, and full bacterium group immune protective rate is 100%, all with control group difference extremely remarkable (P＜0.01).
Table 1. recombined outer-membrane protein Ompk is to the immune protective rate of cabrilla Vibrio harveyi disease.
Sequence table
＜110〉China's Pearl River Fishery Research Institute of Aquatic Science Research Institute
＜120〉the antigenic expression of Vibrio harveyi outer-membrane protein Ompk and as the application of vaccine component
<140>200310112479X
<141>2003-12-8
<160>2
<210>1
<211>801
<212>DNA
＜213〉Vibrio harveyi (vibrio harveyi)
<220>
<221>CDS
<222>(1)...(798)
<400>1
<210>2
<211>266
<212>PRT
＜213〉Vibrio harveyi (vibrio harveyi)
<400>2
Claims (2)
1. one kind as sequence＜400〉application of polypeptide in the medicine of preparation control vibrios disease of 2 aminoacid sequence.
2. application according to claim 1 is characterized in that: described medicine is the vaccine of control vibrios disease.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102161974A (en) * | 2010-12-22 | 2011-08-24 | 毛芝娟 | Preparation method and application of Vibro harveyi outer membrane protein (ompK) antigen as well as strain for expressing the same |
| WO2020027381A1 (en) * | 2018-07-30 | 2020-02-06 | (주)애드바이오텍 | Antibody against shrimp early mortality syndrome and white spot virus, and use thereof |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101816784B (en) * | 2009-02-27 | 2012-08-01 | 中国科学院海洋研究所 | Immunoprotective subunit vaccine, preparation method thereof and application method thereof |
| CN101818191B (en) * | 2009-02-27 | 2012-08-22 | 中国科学院海洋研究所 | Vibrio harveyi specific molecular genetic marker and application thereof |
| CN102924580B (en) * | 2012-11-19 | 2014-05-28 | 南京师范大学 | Denature and renaturation method of WSSV envelope protein prokaryotic expression inclusion body |
| CN103408642A (en) * | 2013-06-19 | 2013-11-27 | 中国科学院海洋研究所 | Vibrio harveyi outer membrane protein and application thereof |
| CN106924725A (en) * | 2017-03-16 | 2017-07-07 | 中国水产科学研究院珠江水产研究所 | A kind of vibrios outer membrane protein subunit Adjuvanted vaccines and prepare with scale technology |
| CN107596362A (en) * | 2017-09-06 | 2018-01-19 | 广东海洋大学 | A kind of red snapper DNA vaccination for vibrio harveyi |
-
2003
- 2003-12-08 CN CNB200310112479XA patent/CN100500849C/en not_active Expired - Fee Related
Non-Patent Citations (5)
| Title |
|---|
| . WU,S ETAL.Genbank登录号AY332563. 2003 |
| A 26-KDA OUTER MEMBRANE PROTEIN, OMPK,COMMON TO VIBRIO SPECIES IS THE RECEPTOR FOR ABROAD-HOST-RANGE VIBRIOPHAGE, KVP40. TETSUYOSHI INOUE ETAL.FEMS MICROBIOLOGY,No.125. 1995 |
| A 26-KDA OUTER MEMBRANE PROTEIN, OMPK,COMMON TO VIBRIO SPECIES IS THE RECEPTOR FOR ABROAD-HOST-RANGE VIBRIOPHAGE, KVP40. TETSUYOSHI INOUE ETAL.FEMS MICROBIOLOGY,No.125. 1995 * |
| A filamentous phage associated with recent pandemic Vibrioparahaemolyticus O3:K6 strains.. Nasu H, Iida T, Sugahara T, Yamaichi Y, Park KS, YokoyamaK, Makino K, Shinagawa H, Honda T.J Clin Microbiol.,Vol.38 No.6. 2000 |
| WU,S ETAL.Genbank登录号AY332563. 2003 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102161974A (en) * | 2010-12-22 | 2011-08-24 | 毛芝娟 | Preparation method and application of Vibro harveyi outer membrane protein (ompK) antigen as well as strain for expressing the same |
| WO2020027381A1 (en) * | 2018-07-30 | 2020-02-06 | (주)애드바이오텍 | Antibody against shrimp early mortality syndrome and white spot virus, and use thereof |
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