The Construction And Expression Of Gene Cdta, Cdtb, CdtC Against C.jejuni

Campylobacter jejuni (C.jejuni) has been recognized as one of the most important causes of acute bacterial gastroenteritis in worldwide, which is also the most frequent infectious agent associated with Guillain-BarréSyndrome (GBS). However, due to the poor understanding of genetic property, virulence factors and pathogenesis of C.jejuni inducing GBS, no effective and feasible measures are now available in prevention of C.jejuni inducing diarrhea and GBS. To solve the problems, in this study, we choose the genes which possess pathogenicity, conservatism and immunogenic coloning to pGEM-T vector with template. We construct and express cytolethal distending toxin cdtA and its peptids of highly antigenicity protein and eukaryotic expression recombinant Vector of cdtA, cdtB, cdtC gene by cloning, sequence analysising and predicting of secondary structure cdtA, cdtB, cdtC gene of Campylobacter jejuni. It would lay a foundation for exploring the specific immune responses and immunoprotection in mice challenged with C.jejuni and developing a new C.jejuni vaccine. The topic will be carried out with four parts as follows:PART I The T-A Coloning of candidating vaccine genes against C.jejuniObjective: To choose twenty candidating vaccine genes against C.jejuni which possess pathogenicity, conservatism and immunogenic colone to pGEM-T vector. To extract the best sequencing plasmids.Methods: (1) Coloning of twenty candidating vaccine genes: They were amplified by PCR with templates of the total DNA of C.jejuni O:19, and the homology of the twenty fragments was detected. The purified PCR products of the twenty fragments were selected as target DNA fragments and cloned into pGEM-T to construct the recombinant plasmids respectively. The recombinant plasmids were identified by restriction enzyme analysis and PCR reaction and confirmed by DNA sequencing. (2) Sequencing and big extracting of recombinant plasmids: We choose three to five recombinant plasmids of each gene to sequence and big extract the best recombinant plasmid.Results: (1) The twenty DNA fragments were confirmed to be highly homologous with C.jejuni NCTC11168 strains. The recombinant plasmids were successfully constructed and confirmed by restriction enzyme analysis, PCR reaction and DNA sequencing. (2) The concentration of the twenty big extracting recombinant plasmids was 1μg/μl.Conclusion: The recombinant plasmids of the twenty candidating vaccine genes were constructed successfully.PART II Clone,Sequence analysis and Prediction of secondary structure and B cell epitope of cdtA, cdtB, cdtC gene of Campylobacter jejuniObjective: To predict the secondary structure and B cell antigenic epitopes of cytolethal distending toxin (CDT) protein of Campylobacter jejuni, analyze their features to predict the possibilities for it to be a potential vaccine.Methods: cdtA, cdtB, cdtC fragment was amplified from Cj O:19 chromosomal DNA by PCR. Its T- A was cloned, sequenced and compared with International standard strains NCTC11168 in the GenBank. The cdtA, cdtB, cdtC gene of Cj was collected to predict the secondary structure by using methods of Chou-Fasman, Garnier-Robson and Karplus-Schulz, the hydrophilicity, surface probability and antigenic index of deduced CDTA, CDTB, CDTC by Kyte-Doolittle, Emini and Jameson -Wolf were analyzed respectively. The B cell antigenic epitopes for deduced CDTA, CDTB, CDTC were predicted by synthesizing these methods.Results: cdtA, cdtB, cdtC fragments were composed of 874, 913, 711 base pairs. And their nucleotide homology with Cj NCTC11168 strains on the GenBank were 100%. The predominant epitopes were probably in the region of 11～44 and 54～90 of cdtA, 5～70,78～112 and 137～187 of cdtB, 119～140 and 145～193 of cdtC.Conclusion: The cdtA, cdtB, cdtC fragment was successfully amplified and their homology is high compared with other species. Prediction of the secondary structure and B cell antigenic epitopes of cdtA, cdtB, cdtC lay the basis for studies of the function of the protein, development of epitope based vaccine and preparation of the monoclonal antibody against CDTA, CDTB, CDTC.PART III Construction and Expression of cytolethal distending toxin cdtA and its peptids of highly antigenicity protein of Campylobacter jejuni in EcoliObjective: To express the cytolethal distending toxin cdtA and its peptids of highly antigenicity protein of Campylobacter jejuni in E.coli.Methods: The encoding gene of cdtA, A1, A2 protein was amp lified from cultured Campylobacter jejuni via PCR and cloned into exp ression vector pGEX-5x after identification its sequence. And they are expressed in BL21 of E.coli.Result: Sequencing confirmed that encoding gene fragment of cdtA, cdtA1, cdtA2 protein was obtained and inserted into pGEX - 5x correctly. The target protein was expressed in E.coli was protected protein.Conclusion: The recombinant plasmid was constructed and expressed in BL21 of E.coli successfully. The results obtained lay the foundation for research on development of Campylobacter jejuni DNA vaccine.PART IV Construction and Expression of Eukaryotic Expression Recombinant Vector of Compylobacter Jejuni cdtA, cdtB, cdtC GeneObjective: The recombinant plasmid containing the cytolethal distending toxin (CDT) gene of Campylobacter jejuni (Cj) was constructed to provide a foundation for future development of C. jejuni DNA vaccine.Methods:Primers were designed by Primer 5. 0 software according to CJ-cdtA, cdtB, cdtC gene sequence provided from GenBank. Polymerase chain reaction (PCR) was used to amplify the cdtA, cdtB, cdtC gene. The fragment was subcloned into appropriate site of pcDNA3. 1 (+) eukaryotic expression vector by restriction enzyme digestion and linkin reactions. The positive recombinant was identified by restriction endonuclease digestion, PCR amplification and sequencing.Result:The 807bp, 798bp, 570bp-cdtA, cdtB, cdtC specific gene fragments were amplified. Restriction enzyme analysis and sequencing showed that the eukaryotic expression recombinant pcDNA3. 1 (+)-cdtA, cdtB, cdtC were successfully constructed.Conclusion:The cdtA, cdtB, cdtC gene fragments were successfully amplified, and the eukaryotic expression recombinant pcDNA3. 1 (+)-cdtA, cdtB, cdtC were successfully constructed.