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目的构建HIV-1CN54株gagprotease基因嵌合脊髓灰质炎病毒cDNA的表达质粒,并鉴定、检测基因及其表达。方法用PCR技术获得人免疫缺陷病毒CN54株的gagprotease基因,并使其两端带上合适的酶切位点,将其定向插入到包含脊髓灰质炎病毒cDNA的表达质粒pSVA14中,替代其部分结构基因,构建HIV基因嵌合缺陷性脊髓灰质炎病毒基因组的表达质粒。经筛选、鉴定后用脂质体转染技术将新构建的质粒转入Hela细胞内,用WesternBlot方法检测目的基因在Hela细胞内的表达。结果PCR技术扩增所得的人免疫缺陷病毒CN54株gagprotease基因经琼脂糖凝胶电泳、DNA测序证实成功获得,未引入突变碱基,筛选、鉴定证明gagprotease基因被正确定向插入到脊髓灰质炎病毒的cDNA序列之中,WesternBlot检测到gagprotease基因正确表达了相关蛋白。结论成功构建了表达人免疫缺陷病毒CN54株gagprotease基因的缺陷性脊髓灰质炎病毒基因组嵌合质粒,为利用脊髓灰质炎病毒作人免疫缺陷病毒基因的表达载体奠定了基础,此研究对开发以脊髓灰质炎病毒为艾滋病的疫苗载体有重要意义。
Objective To construct the expression plasmid of chimeric poliovirus chimeric HIV - 1 CN54 strain gagprotease gene, and to identify and detect the gene and its expression. Methods The gagprotease gene of human immunodeficiency virus CN54 strain was obtained by PCR and inserted into the pSVA14 plasmid containing the poliovirus cDNA. The gagprotease gene was inserted into both ends of the gagprotease gene to replace its partial structure Gene, construction of HIV gene chimeric defective poliovirus genome expression plasmid. After screening and identification, the newly constructed plasmids were transfected into Hela cells by lipofection technique, and the expression of the target gene in Hela cells was detected by Western Blot. Results The gagprotease gene of human immunodeficiency virus CN54 strain amplified by PCR was successfully confirmed by DNA sequencing. No mutation was introduced into the gagprotease gene. Screening and identification proved that the gagprotease gene was correctly inserted into the poliovirus Among the cDNA sequences, Western Blot detected that the gagprotease gene correctly expressed the related protein. Conclusion The chimeric plasmids of defective poliovirus expressing gagprotease gene of human immunodeficiency virus CN54 were successfully constructed and laid the foundation for the use of poliovirus as human immunodeficiency virus gene expression vector. The plague virus is a vaccine carrier for AIDS and is of great importance.