带有6肽组氨酸的HBV S/S145真核表达载体的构建及应用

Constuction of eukaryotic expression vector carring HBV S/S145 antigen by epitope tagging method and its application

目的构建稳定表达带有6肽组氨酸的HBVS/S145蛋白的真核载体。方法应用PCR技术及体外基因重组技术,将S基因和nt587突变的S基因插入带有his-tag的pxF2RH载体中,扩增6个组氨酸和S/S145基因,最后与经NheI、SalI双酶切的线性载体pCI-neo连接,构建成带有6肽组氨酸的稳定表达S/S145的真核表达载体(简称pCI-his-S Y/S145)。用脂质体转染的方法将质粒转至人宫颈癌细胞,通过酶联免疫吸附试验(ELISA)检测细胞上清中抗原的分泌情况。用West-ern-blot方法检测细胞中外膜蛋白的表达情况。结果真核表达载体pCI-his-SY/S145的测序结果与预期大小一致。ELISA方法可检测到细胞上清中的野生型表面抗原,但检测到145突变抗原表达水平低。应用Western-blot方法可以同时检测到野生型和G145R突变的外膜蛋白表达。结论带有his-tag的重组质粒pCI-his-S/S145可以在Hela细胞中成功表达融合蛋白,且组氨酸可作为检测蛋白的标志。

Objective To construct the eukaryon vector which expressing surface antigen of wide type and S145 mutant with six histidine. Methods First, we inserted surface antigen gene of wide type and S145 mutant into the vector pxF2RH which encoding six histidines. Second, S and histidine genes were amplified by PCR. Third, PCR products and vector pCI-neo were digested by different double restriction endonucleases were purified and connected together at same Eppendorf tube with T4 DNA ligase. The target recombinant plasmid may be gained after preparation and purification. Plasmid was transfected into Hale cells by lipofectamine, and the surface antigen secreted in supernatant was measured by ELISA while surface protein was detected by Western-blot. Results Direct sequencing results showed that the homologies of nucleotides between presumed and the sequence obtained were 100%. Only the wide-type surface antige can be detected by monoclonal antibody based commercial diagnostic kit. Moreover, the mutant protein can be recognized by Western-blot. Conclusion We first successfully constructed the plasmid with epitope tagging, which can be transfected into and stably expressed in mammalian cells.