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Cloning, Expressing And Indentifying Of Thermostable Direct Hemolysin From Vibrio Parahaemolyticus

Posted on:2005-02-22Degree:MasterType:Thesis
Country:ChinaCandidate:Q P LiFull Text:PDF
GTID:2120360125954615Subject:Clinical Veterinary Medicine
Abstract/Summary:PDF Full Text Request
In this paper, the cloning and expressing of thermostable direct hemolysin(tdh) of Vibrio parahaemolyticus(V.p) were discussed. A pair of primers with restriction enzyme cutting sites were designed according to the published nucleotide sequence of a putative tdh of V.p. With the specific primers,a target fragernent about 570bp was amplified from Vibrio parahaemolyticus wVp2 strain. With the cleavage of BamHI and EcoRI, the target fragment was inserted oriently into pGEX-4T-l vector. After enzyme restriction and sequence analysis, the nucleotide data had been further analyzed by Antheprot 5.0 and ClustalW softwares. The analysis results showed that the cloned DNA fragement had a open reading frame(ORF) of 570nt, it predicted to be encoded a 189-aa protein was highly hydrophilic, especially the first 24 amino-acid at N terminal, this region could be functional as signal peptide. The homologious comparison proved the cloned gene had 99.82% homology to the sequence of the tdh gene , and the alignment of the amino acid sequence was 99.47%.The recombinant plasmid was transformed into E.coli BL21(DE). The fusion protein was expressed under the IPTG inducing condition, and exhibited a protein band with 49kDa in size on SDS-PAGE gel, the size of the inducing peptide is almost match to the predicted molecular weight of GST-TDH fusion protein. The quantity of 49kDa protein is about 32 percent in total bacterial protein.With Glutathione Sephrose affinity chromatography, the GST-TDH was purified. Then TDH and wVpa extracelluar product was mixed with Freund's adjuvant and immuned rabbits respectively. Furthermore, the TDH protein was specifically recognized by anti-serum which raised against the extracelluar product. Make all together, it proved that the cloned gene represented the wVp2 tdh gene, and the expressed gene products shared indentical antigenicity with the natural TDH.The studies on preparation and application of TDH of wVp2 provided a new approach to fish vaccinology. The successful cloning and expressing the tdh gene of wVp2 made it possible to describe this gene's function under asingle factor level, and also provided technical support for developing an advanced gene engineering vaccine against vibrio parahaemolyticus.
Keywords/Search Tags:vibrio parahaemolyticus, thermostable direct hemolysin, TDH, tdh, cloning, expressing, purification, indentifying.
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