汉坦病毒G1S0.7嵌合基因真核载体的构建及表达

构建含汉坦病毒嵌合基因G1S0.7的真核重组质粒,并在真核细胞中有效表达.从本室前期构建并经测序的重组质粒pShuttle-G1S0.7上双酶切回收得到片段G1S0.7后,克隆入真核表达载体pVAX,并将其通过脂质体介导转染HEK293细胞,表达产物用ELISA和Western-blot进行鉴定.酶切鉴定结果表明,成功构建了含汉坦病毒嵌合基因G1S0.7的重组质粒pVAX-G1S0.7; ELISA检测结果和Western-blot结果显示,汉坦病毒G1S0.7嵌合基因在HEK293细胞中得到了表达,所表达的融合蛋白分子量约90kD,与预期大小一致,并且表达产物可与抗汉坦病毒NP mAb特异性结合.说明所构建的表达载体可在真核细胞中表达出与汉坦病毒抗体有特异性结合活性的融合蛋白,为下一步基因免疫及进一步筛选HFRS基因疫苗候选组分提供实验依据.

Construction and identification of eukaryotic expression vector containing chimeric gene G1S0.7 of Hantavirus

Aim To construct eukaryotic expression vector containing chimeric gene G1S0.7 of Hantavirus and express it in eukaryocyte. Method Fragment G1S0.7 was obtained by cutting from recombinant plasmid pShuttle-G1S0.7 which was constructed formerly and confirmed by sequencing. Then insert the fragment into the expression vector pVAX to construct the recombinant plasmid pVAX-G1S0.7. After transfecting HEK293 cells by lipofectine-mediated method, ELISA and Western-blot were used to identify the expressed product. Result The eukaryotic expression vector was proved to successfully constructed by restriction enzyme analysis. Western-blot results and ELISA results showed that the recombinant expressed transiently in HEK 293 cells and produced protein about 90KD, corresponding to the estimated molecular size, the protein could bind specifically to the hantavirus nucleoprotein specific mAb. Conclusion The successful construction and expression of the eukaryotic expression vector lays the basis for further research on its immunological characteristics and screening candidate components of genetic vaccine for Hantavirus.