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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NPmAb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。
The eukaryotic recombinant plasmid containing Hantavirus chimeric gene G1S0.7 was constructed and expressed efficiently in eukaryotic cells. The recombinant plasmid pShuttle-G1S0.7 was cloned into the prokaryotic expression vector pShuttle-G1S0.7 and cloned into the eukaryotic expression vector pVAX and transfected into HEK293 cells by liposome. The expression product was identified by ELISA and Western-blot. The results of restriction enzyme digestion indicated that the recombinant plasmid pVAX-G1S0.7 containing Hantavirus chimeric gene G1S0.7 was successfully constructed. The results of ELISA and Western-blot showed that the Hantavirus G1S0.7 chimeric gene was expressed in HEK293 cells The expressed fusion protein has a molecular weight of about 90 kD, is consistent with the expected size, and the expression product can specifically bind to anti-Hantaan virus NPmAb. Which indicated that the constructed expression vector could express the fusion protein with specific binding activity to Hantavirus in eukaryotic cells and provide experimental basis for further gene immunization and further screening of candidate components of HFRS gene vaccine.