Construction, Evaluation And Mechanism Of The Prokaryotic-eukaryotic Dual Expression Plasmid Of Somatostatin

The prokaryotic-eukaryotic dual expression system compared to a single prokaryotic or eukaryotic expression system has greater advantages and can also start the prokaryotic and eukaryotic expression of foreign genes. The prokaryotic-eukaryotic dual expression system using attenuated strain of Salmonella choleraesuis as the carrier, could express the same proteins in bacteria and mammalian cells, and also can stimulate the immune response respectively by products of prokaryotic expression and eukaryotic expression.Therefore, in theory, prokaryotic-eukaryotic dual expression plasmid can stimulate the body's ability to improve their immune response.To construct the prokaryotic-eukaryotic dual expression vector pPtrc-S/2SS-M4GFP-asd, gene cloning, cell culture, RT-PCR, SDS-PAGE, Western blot, ELISA and other technologies were applied in this research, and the original promoter Ptrc fragment was cloned into the somatostatin eukaryotic expression plasmid without resistence gene (pGS/2SS-M4GFP-asd), then the vector of pPtrc-S/2SS-M4GFP-asd was separately converted into E.colχs6097 and crp-/asd-double deletion attenuated Salmonella choleraesuis C500, soχ6097(pPtrc-S/2SS-M4GFP-asd) and C500 (pPtrc-S/2SS-M4GFP-asd) engineered strains were obtained. Then we studied the identification of pPtrc-S/2SS-M4GFP-asd plasmid and C500(pPtrc-S/2SS-M4GFP-asd) engineered bacteria on prokaryotic and eukaryotic expression. We used the recombinant bacteria to immune 4 week-age mice, then detected the levels of anti-SS antibody and anti-C500 antibody in plasma, in order to discuss the immune effect of attenuated Salmonella choleraesuis as the carrier of somatostatin prokaryotic-eukaryotic dual expression plasmid, and also to develop efficient and safe gene vaccine on growth for providing theoretical basis and technical support.1. Construction and Screening of Somatostatin prokaryotic-eukaryotic expression plasmid pPtrc-S/2SS-M4GFP-asdThe prokaryotic promoter Ptrc was synthesized and subcloned into the CMV promoter downstream of the eukaryotic expression vector pGS/2SS-M4GFP-asd, the somatostatin prokaryotic-eukaryotic dual expression plasmid pPtrc-S/2SS-M4GFP-asd was built, and also was converted into E.coliχ6097 and crp-/asd-double deletion attenuated Salmonella choleraesuis C500. It was proved that gene insertion site, direction and sequence were completely correct, so x6097(pPtrc-S/2SS-M4GFP-asd) and C500(pPtrc-S/2SS-M4GFP-asd) engineered strains were sucessfully obtained.2. Expression and identification of somatostatin prokaryotic-eukaryotic dual expression plasmid pPtrc-S/2SS-M4GFP-asdThe constructed somatostatin engineered strain C500(pPtrc-S/2SS-M4GFP-asd) were cultured for 3h, which were inducted by isopropyl-β-D-thio-pyran-galactoside (IPTG) and non IPTG, then the S/2SS-M4GFP fusion protein with the size of 55KDa was detected; using the method of Western blot, it was proved that the fusion protein could combined the anti-somatostatin antibody; when the somatostatin prokaryotic-eukaryotic dual expression plasmid pPtrc-S/2SS-M4GFP-asd was transfected into 293FT cells, the green fluorescence was detected after 48h, and also RT-PCR transcription products were tested by S segment (352bp), S/SS fragment (491bp) and S/2SS(795bp), therefore the results showed that the plasmid could express respecticely in prokaryotic and eukaryotic cells.3. Effects of the Prokaryotic-eukaryotic dual expression DNA immunization of somatostatin on growth and immune response in miceThe prokaryotic-eukaryotic dual expression of somatostatin C500 (pPtrc-S/2SS-M4GFP-asd) engineered bacteria was used to immune female kunming mice, by three immunization doses(high dose of 0.2×1010CFU/each, medium dose of 0.2×109CFU/each, low dose of 0.2×108CFU/each) to the immune female mice.Throughout the experimental stage, compared to medium-dose, low-dose and eukaryotic control group(pGS/2SS-M4GFP-asd), high-dose group had weight gain increased respectively by 5.5%,8.2%and 7.1%, without no significant difference between those groups (P>0.05), and also had the lowest feed conversion rate(25.9:1).3w after the first vaccination, high-dose group had a higher IgG antibody titer compared to eukaryotic control group by 6.7%; after strengthen vaccination, IgG antibody titers of high-dose group were higher than the eukaryotic control group by 8.2%.3w after the first vaccination, IgGl antibody titers of high-dose group were increased by the eukaryotic control group by 25%, but IgG1>IgG2a, so it was proved that after vaccination, the body tends to produce Th2 type immune response mainly mediate humoral immune response. Those showed that prokaryotic-eukaryotic dual expression plasmid of somatostatin DNA vaccine pGS/2SS-M4GFP-asd could cause stronger immune response especially humoral immune reaction, in order to enchance the relative growth compared with the somatostatin gene vaccine pGS/2SS-M4GFP-asd.