Studies On Cloning And Expressing Of Hemolysin Genes Of Vibrio Parahaemolyticus

Studies on cloning and expressing of hemolysin genes ofvibrio parahaemolyticus (Vp)AbstractVp is a major cause of acute gastroenteritis and is especially associated with seafood consumption and travelers?diarrhea. It may cause other diseases such as reactive archritis and heart disease. It is classified into two groups by the Kanagawa penomenon (KP), which is a hemolytic test on Wagatsuma blood agar. Past studies revealed that most cases of Vp gastroenteritis were caused by KP-positive strains. The molecular biological studies showed that these clinical strains possess tdh gene which encoded a protain called thermostable direct hemolysin(TDH). However,cases caused by KP-negative strains are increasing in recent years and this has aroused researchers?much attentions. It has been clear that KP-negative strains can produce a protein TRH encoded by trh gene while its detailed malgenic mechanisms remain unclear. Our study is to clone trh and tdh genes and to induce the expressing of them. Pure proteins are obtained and this makes a good basement for further study.In the first part of this study,we design a pair of primers(p l,p2) and use PCR technique to amplify the special fragments of trh gene from the genomic DNA, then digest it with the restrition endonucleases EcoRI and BamHI, ligate the digested fragements of trh gene with pGEX-3X digested by the same two restrition endonucleases, treansform the ligated liquid into E. ccli DH5 . Screen and identify the positive recombinant clone by PCR and restriction analysis. The nucleotide sequence is sequenced by Bosia technical company. Then we transform the recombinant plasmid into E. coli DE3. The result of SDS-PAGE shows that the protein is expressed in a highly,soluble and fused form. Fragmentate the fused protain and pure TRH is obtained. Hemolytic tests show that the protein is active. The protein is purified and the rabbits are immunized by it. at the first,the third and the forth week with the dosage at 0.5,0.25 and 0.25 mg/kg body weight. Seven days after the last immunization, the rabbit serum is abstracted through vein and is absorbed by DE3. The hemolytic suppression test indicates that some substance which-5-suppress the hemolyticance of Vp exist in the rabbit serum.In the second part of this study, we design another pair of primers(P3,P4) and build tdhIpGEX-3X plasmid by the same method as before. While TDH is expressed in unsloable form in inclusion bodies.We dissolved the inclusion bodies and purified the fused protein by FPLC in the end.