摘要
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
Objective To construct recombinant vector expressing antisense RNA to CCR 5 in eukaryotic cells and obtain recombinant pseudovirus,which will be used to block HIV-1 infection.Methods The DNA fragment targeted against the initional part of CCR 5 mRNA translation was amplified by using RT-PCR from peripheral blood mononuclear cells (PBMCs) and cloned into retroviral vector pLXSN,then transfected into packaging cell(PA317)with lipofectAMINE. After 2-3 weeks selecting with G418,the pseudovirion in the survival cell's supernatant was detected with RT-PCR(FQ),then was used to infect NIH/3T3 cell. Results The psuedovirion packed from expression vector of sense/antisense RNA to CCR 5 had infected NIH/3T3 cell successfully. The vector had incorporated into its genome and transcripted into RNA. Conclusion The gene fragment of antisense RNA to CCR 5 could be obtained from PBMCs and transfected into eukaryotic cell with retroviral vector. The results made a great foundation for studying its inhibiting effect on HIV-1 infection.
出处
《中华实验和临床病毒学杂志》
CAS
CSCD
北大核心
2002年第1期52-54,共3页
Chinese Journal of Experimental and Clinical Virology
