| BACKGROUNDVibrio vulnificus , first described in 1979, is a halophilic gram-negative marine bacterium , causes infectious diseases in humans, especially in people who are immunocompromised or have underlying conditions such as liver cirrhosis , hemochromatosis or alcoholism. It mainly produces two types of disease: primary sepsis via gastrointestinal infection , and edema are observedwith a mortality rate more than 50%. F.vulnificus cytolysin, produced by most pathogenic strains, is a water-soluble polypeptide with a Mr of 51,000. The purified cytotoxin preparations have been show to be cytotoxic to Chinese hamster ovary(CHO)cells,hemolytic for mammalian erythrocytes. As an only extracellular cytotoxin,the cytoxin plays an important part to contribute to the virulence of V.vulnificus.In this study, The whA gene from a standard strain GTC333 of V.vulnificus was amplified by high fidelity PCR.The nucleotide sequence of the target DNA amplification fragment were sequenced after T-A cloning. The recombinant expression vector pET32a(+)-vvA-BL21DE3 were constructed. The expression of whA fusion protein in E.coli BL21DE3 induced by IPTG at different dosages was examined by SDS-PAGE. The whA fusion protein will act as antigens for V.vulnificus pathogenesis and detection kit.MATERIALS AND MTEHODSl.Culture of Vibrio vulnificusStrain GTC333 of Vibrio vulnificus was inoculated on Columbia agar plate containing 10% of sheep blood .The plates was cultivated at 37 for 24h. The bacterium colonies morphology and so on were examined and identified.2. Preparation of DNA templateGenomic DNA of V.vulnificus strain GT333 was extracted by routing phenol-chloroform method, DNase-free RNase digestion and phenol-chloroform extraction again. The DNA template was solved in TE buffer and its concentration and purity were determined by ultraviolet spectrophotometry.3.Polymerase Chain ReactionOligonucleotide primers were designed to amplify the whole sequence of vvhA gene from V.vulnificus strain GT333 based on the published corresponding genome sequence. The sequence of whA sense primer with an endonuclease site of BamH I:5'-GCCGGATCCATGAAGAAAATGACTCTG-3'.The whA sequence of antisense primer with an endonuclease site of Hind III :5' -GCCAAGCTTCTAGAGTTTGACTTGTTG-3' . The parameters for PCR: 94 5min, XI; 94 30S, 52 30S, 72 90S, X10; 94 30S, 52 30S, 72 100S (10S addition for the each of the following cycles), X25; 72lOmin, X 1. The results of PCR were observed under UV light after electrophoresis in 1.5% agarose pre-stained with ethidium bromide.4.T-A Cloning, sequencing and Construction of Expression VectorThe target amplification DNA fragment of whA gene was cloned into pUCm-T vector (pUCm-T-vvhA ) by using the T-A Cloning Kit according to the manufacturer's instruction. The recombinant plasmid was amplified in E.coli strain DH5 a and then extracted by Sambrook's method. A professional company (BEST) was responsible for nucleotide sequence analysis of the inserted fragments. E.coli DH5 a containing pUCm-T-whA and expression vector pET32a were amplified respectively and the two plasmids were extracted. The plasmids were digested with two endonucleases, respectively. The target fragments of whA and pET32a was recovered and then ligased. The recombinant expression vector pET32a(+)-vvhA was transformed into E.coli BL21DE3. pET32a(+)-vvhA were prepared for sequencing again after amplification in its host cell.The nucleotide sequences of the cloned whA gene inserted in the recombinant plasmid vectors were compared for homologies with the published whA gene sequences (M3460) by using a special molecular biological analysis soft ware.5. Expression and Purification of the Fusion ProteinProkaryotic expression system pET32a(+)-whA-BL21DE3 was rotationally cultured in LB medium at 37 induced by IPTG at different dosages of 1.0, 0.5 and 0.1 mmol/L. The supernatant and precipitate were isolated through centrifugation after the engineering bacterium pallet was ultrasonically broken (300V, 5sec X 3). The molecu...
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