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目的 :研究含登革 2型病毒 4 3株 (D2 4 3)NS1基因的重组质粒DNA在幼地鼠肾细胞BHK 2 1的表达。方法 :将含信号肽的NS1基因片段插入到pcDNA3.1的KpnⅠ位点和EcoRⅠ位点之间 ,获得重组表达载体pcDNA NS1。用电穿孔法将其导入BHK 2 1细胞 ,G4 18选择培养。挑取单细胞克隆 ,RT PCR及蛋白质印迹法鉴定NS1基因的稳定表达。结果 :在随机挑取的 5个单细胞克隆中 ,有 4个克隆的RT PCR鉴定为阳性 ,蛋白质印迹结果表明NS1基因获得表达。结论 :构建的pcDNA NS1质粒在BHK 2 1细胞中有稳定表达 ,因此含NS1基因的该重组质粒DNA可用作核酸免疫
Objective: To investigate the expression of BHK 2 1 in juvenile rat kidney cells of recombinant plasmid DNA containing 433 (D2 4 3) Dengue virus type 2 viruses. Methods: The NS1 gene fragment containing the signal peptide was inserted into the pcDNA3.1 between the KpnI site and the EcoRI site to obtain the recombinant expression vector pcDNA NS1. It was introduced into BHK21 cells by electroporation, and G418 was selected for culturing. Single cell clones were picked and stable expression of NS1 gene was identified by RT PCR and Western blotting. RESULTS: Of the 5 single cell clones randomly selected, 4 clones were identified as positive by RT PCR and Western blotting results showed that the NS1 gene was expressed. CONCLUSION: The constructed pcDNA NS1 plasmid is stably expressed in BHK 2 1 cells. Therefore, the recombinant plasmid DNA containing the NS1 gene can be used as nucleic acid immunization