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目的 构建趋化因子受体CCR5反义RNA真核表达载体并获取重组假病毒颗粒以用于抗HIV 1研究。方法 用RT PCR法从健康人外周血单个核细胞 (PBMCs)中获得趋化因子受体CCR5翻译起始区的基因片段 ,并以正、反两个方向定向插入到真核表达载体pLXSN上。重组载体用脂质体转染剂 (lipofectAMINE)转染PA317包装细胞 ,抗 G418克隆的细胞上清经逆转录后用荧光定量PCR(FQ PCR)测定假病毒滴度 ,进一步感染NIH 3T3细胞。结果 CCR5正、反义RNA的真核表达载体 ,经PA317细胞包装形成的假病毒颗粒已成功地感染NIH 3T3细胞 ,目的基因在该细胞中得到整合与表达。结论 从PBMCs中获得的趋化因子受体CCR5基因片段通过逆转录病毒载体可转移至真核细胞中并得到表达 ,为进一步研究CCR5反义RNA的抗HIV 1作用奠定了基础
Objective To construct eukaryotic expression vector of chemokine receptor CCR5 antisense RNA and obtain recombinant pseudovirions for anti-HIV 1 study. Methods The gene fragment of translation initiation region of chemokine receptor CCR5 was obtained from healthy human peripheral blood mononuclear cells (PBMCs) by RT - PCR and inserted into the eukaryotic expression vector pLXSN in both forward and reverse directions. The recombinant vector was transfected into PA317 packaging cells by lipofectamine, and the supernatant of the anti-G418 clone was reverse-transcribed. The pseudovirus titer was determined by fluorescence quantitative PCR (FQ PCR) and further infected with NIH 3T3 cells. Results The eukaryotic expression vector of CCR5 positive and negative antisense RNA and the pseudovirus particles packaged by PA317 cells were successfully infected with NIH 3T3 cells. The target gene was integrated and expressed in this cell. Conclusion The CCR5 gene fragment obtained from PBMCs can be transferred into eukaryotic cells by retroviral vector and expressed, which lays a foundation for further study on the anti-HIV 1 effect of CCR5 antisense RNA