Construction Of A Dual-promoter Expression Plasmid PEPR-apxIIA With APP ApxIIA Delivered By Salmonella Choleraesuis C500

Salmonella choleraesuis C500 strain can be used as the attenuated vaccine preventpiglet paratyphoid, also can act as live vector of other DNA vaccines, oral immunization,can induce immune response to specific antigens carryed by it. In this research, a strongtranscription terminator rrnbT1T2 was inserted into the multiple clone site'(MCS's) downstream of eukaryotic expression plasmid pEGFP-C1, then the recombinant plasmidpEGFP-rrnBT1T2 was constructed. According to the structure of prokaryotic promoterPtrc, this hybrid promoter was designed and artificially synthesized, then Ptrc promoterwas inserted into the eukaryotic promoter CMVIE down stream of pEGFP-rrnBT1T2, sothe dual-promoter expression vector pEGFPPtrcR was constructed and transformed intoSalmonella choleraesuis C500 using 1×TSS, the engineering strian C500/pEGFPPtrcR wasobtained, and the original growth, biochemistry, and serology characteristics of C500 werekept, the stable passages were at least 20 generations. For checking the validity ofpEGFPPtrcR, the expression of report gene EGFP in prokaryotic cells and eukaryotic cellswas measured. The green fluorescent of the bacteria C500/pEGFPPtrcR was observedusing fluorescence microscope. The expressed protein was 27KD measured by SDS-PAGE,according to the expected molecular weight. Mediated by liposome, the plasmidpEGFPPtrcR was transfected into Vero cell, and after 24h, green fluorescent was alsoobserved. This manifested the construction of dual-promoter expression vectorpEGFPPtrcR which was able of eukaryotic and prokaryotic expression was successful andindicated that the exogenous gene carried by the double-promoter expression vector pEGFPPtrcR was able to express in salmonella strain C500 and also in somatocytes. Theincreased expression amount of antigen can induce stronger immune response, theperspective of the research of new combinant salmonella vaccine was promising.Porcine contagious pleuropneumonia (PCP), is a kind of swine infectious respiratorydisease caused by Actinobacillus pleuropneumoniae (APP), and great loss was caused bythis disease to the pig industry worldwidely. Main measures for prevent and control thisdisease were immunization. The APP apxⅡA gene sequence in GenBank was referred todesign a pair of specific primers (apxⅡA land apxⅡA 2). The whole apxⅡA gene wasamplified using the genome DNA of APP-CYY starin isolated by writer's lab as thetemplate. The result of sequencing showed that the apxⅡA gene was 2871bp, the homologyof this gene to the apxⅡA gene of the other 5 APP strains registered in GenBank was99.5-99.9%. Amino acid sequence analysis showed that this gene encoded 957 aminoacids without signal peptide, typical RTX toxin features was showed. This gene wasinserted into the dual-promoter expression vector pEGFPPtrcR MCS, the newdual-promoter expression vector pEPR-apxⅡA was constructed and transformed into hogcholera salmonella strain C500 by means of 1×TSS, the engineering bacteria C500/pEPR-apxⅡA was obtained, the original growth, biochemistry, and serology characteristicsof C500 were kept, the stable passages were at least 20 generations. The green fluorescentwas observed using fluorescence microscope. The expressed fusion protein of EGFP andapxⅡA in C500 was about 130KD measured by SDS-PAGE. Plasmid pEPR-apxⅡA wastransfected into Vero cell mediated by liposome, and 24h later green fluorescent wasdetected. This proved that the construction of dual-promoter expression plasmidpEPR-apxⅡA was successful, and can express in prokaryotic and eukaryotic cells. Thiswould lay a solid foundation for the construction of high performance APP DNA livevaccine using C500 as vector, and the research of porcine contagious pleuropneumonia andpiglet paratyphoid fever double vaccine.The invA gene is the invasion gene of salmonella, homologous recombination can beused for the deletion mutation of this gene, so that the virulence of Salmonella choleraesuisC500 strain may be attenuated. The two sides' genes invB and invE ofinvA were amplifiedusing the genome of C500 as template, pTER with prokaryotic promoter Ptrc and EGPF was amplified using plasmid pEGFPPtrcR as template. According to the orientating andsequence of invB-invA-invE in the gengme of salmonella, they were linked into vectorpUC18. HindⅢand EcoRI were removed to ensure the usage integrity ofpTER in MCS.Transfer vector p18BE-pTER using invB and invE of the double sides' sequence of C500invA as homologous arm and pTER as target shooting was constructed. The constructionof this plasmid would lay a foundation for the construction of genetic engineeringattenuation of C500.