生长抑素基因疫苗质粒pEGS/2SS的构建及表达

Construction and Expression of Somatostatin DNA Vaccine pEGS/2SS in HeLa

将pcS/2SS中的乙肝表面抗原(HB sA g)S基因及插入S基因中的生长抑素(som atostatin,SS)基因亚克隆到pUC 19中,构建成pU SS/S质粒;PCR扩增pcS/SS中SS基因,调整阅读框,融合到pU SS/S质粒S基因后,构建成pU S/2SSG质粒;最后将S/2SSG融合基因克隆到pEGFPN 1中EGFP基因的5′端,构建S/2SS-EGFP融合表达质粒pEG S/2SS。酶切、测序鉴定后脂质体包裹法将pEG S/2SS转染H eL a细胞,荧光显微镜下检测荧光,EL ISA检测表达产物的SS免疫反应原性。酶切和测序鉴定表明,重组质粒pEG S/2SS构建成功。pEG S/2SS转染24 h后的H eL a细胞检测到绿色荧光,72 h检测到强烈荧光,表明融合基因在H eL a细胞获得高表达;EL ISA证实融合蛋白具有SS的抗原抗体反应原性。质粒pEG S/2SS的成功构建及表达为生长抑素基因免疫的机理及安全性研究奠定了基础。

HBsAg S gene inserting SS of pcS/2SS was subcloned into pUC19,and the resultant recombinant plasmid was pUSS/S. SS gene in pcS/SS was amplified by PCR,adjusted open-reading frame ,then fused to close behind the S gene of plasmid pUSS/S,the plasmid pUS/2SSG; was constructed. Subsequently the fused gene of S/2SS was subcloned into the 5＇ end of EGFP gene of pEGFP-N1,and S/2SS-EGFP recombinant plasmid pEGS/2SS obtained. These plasmid were identified by restriction endonuclease digestion and sequencing,then the pEGS/2SS was used to transfect HeLa cells through liposome embedding. The recombinant protein was detected by ELISA and fluorescence microscope. The SS expression vector with GFP was constructed successfully. Green fluorescence was observed at 24 h after transfection and became most strong at 72 h. It indicated that the recombinant gene has expressed in HeLa cells. The fused protein reacted with the antibodies against somatostatin by ELISA. Successful construction and expression of the plasmid pEGS/2SS provides the basis for study the immune mechanism and safety of the somatostatin gene vaccine.