Cloning And Expression Of Toxin-Coregulated Pilus A Gene Of Vibrio Cholerae And Study On Its Immuneprotection

Vibrio Cholerae is one of the most common and damaging pathogen in aquaculture, which not only leads to serious mucous sloughing disease and bacterial enteritis of fish but also causes human's potent intestinal infectious diseases Cholerae by aquatic products.At present,the primary means to control the disease is to use antibiotics,but the effects of its are limited by the infection of V.cholerae universally and the increase of drug resistant strains continuously,which result in abundant death of aquatic animals suffered from those. So,studying on immunoprophylaxis and immunotherapy of the disease are important. Since the first elementary step of initialing infection is to adhere to the intestinal mucosa by toxin-coregulated pilus(TCP) of V.cholerae,blocking adhesion and colonization of V.cholerae to the intestinal mucosa might prevent V.cholerae infenction.In this study TcpA protein of V.cholerae is taken as a target protein.In order to determine the applied value of recombinant protein TcpA in the developing genetic engineering subunit vaccine of V.cholerae,tcpA gene from V.cholerae strain Y1 was cloned and expressed in prokaryotic by genetic engineering technolongy,and then antigenicity and immuneprotection of the recombinant protein GST-TcpA were detected.Firstly,tcpA gene was amplified from strain Y1 isolated in Anhui by PCR method and cloned into pMD18-T vector,then sequenced and analyzed by BioXM software.The results revealed that tcpA complete gene contained an open reading frame(ORF) about 675 bp in length without interruption,encoding 224 amino acids with a approximately 21 kD molecular weight.There are 71.8%-99.7%nucleotide sequences homology and 3.4%-87.6%amino acid sequences homology between strain Y1 and some referent strains of V.cholerae registered in GenBank.These results laid a foundation for subcloning and prokaryotic expression of tcpA gene.Secondly,tcpA gene was subcloned into expressive plasmid pGEX-4T-1 and expressed in E.coli BL21(DE3).A pair of specific primers containing BamHâ… and Xhoâ… restriction enzyme sites were designed.tcpA gene was amplified by PCR method from pMD-18T-tcpA.The purified PCR product was digested by BamHâ… and Xhoâ… ,and then was inserted into pGEX-4T-1 and transformed into E.coli BL21(DE3).The recombinant protein GST-TcpA with 47 kD molecular weight was hyperexpressed in the form of inclusion bodies after pGEX-4T-1-tcpA/ BL21 was induced by IPTG.A large number of the recombinant protein GST-TcpA was extracted by conventional protein separate technique at same time.These results laid a foundation for the study on antigenicity and immuneprotection of the recombinant protein GST-TcpA.Finally,antigenicity and immuneprotection in vitro and in vivo of the recombinant protein GST-TcpA were investigated by western blot,inhibition of cell adhesion and immuneprotection trial respectively.After the Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE),the expessive recombinant protein GST-TcpA induced from pGEX-4T-1-tcpA/BL21 was shifted to PVDF film and detected by western blot with antipilus of strain Y1 serum.Western blot result showed anti-serum could react specifically with the recombinant protein,which indicates that the recombinant TcpA has the same antigenicity as natural one's.Anti-serum were prepared in experimental mice immunized with V.cholerae,pili protein of V.cholerae,recombinant protein GST-TcpA and GST protein respectively,and their adhesion inhibition effect was determined by Hep-2 cell infection model.The results showed that anti-V.cholerae serum could inhibit bacteria adhering to Hep-2 cells completely,anti-pili serum and anti-GST-TcpA serum could reduce the capacity of bacteria adhering to Hep-2 cells markedly,but the anti-GST serum showed no adhesion inhibition effect.These indicates that anti-GST-TcpA serum has a powerful anti-adhesion effect on V.cholerae in vitro and V.cholerae can expess other adhesins besides TcpA.After being immunized four times with recombinant protein GST-TcpA or GST protein,the immunized mice were challenged of V.cholerae strain Y1 with 10~8 CFU/mL dose.The mice immunized with recombinant protein GST-TcpA displayed 81.25%immuneprotection rate,but the mice immunized with GST protein and unimmunized mice wholly died.These suggested recombinant protein could induce an immmune response to provide better protection against V.cholerae infection.The results showed that recombinant protein TcpA remain antigenicity and immuneprotection in vitro and in vivo on the basis of successfully constructing recombinant expressive plasmids pGEX-4T-tcpA and obtaining recombinant protein GST-TcpA,and thus recombinant protein TcpA has the potential to be used for developing of genetic engineering subunit vaccine of V.cholerae.