HIV-1 vpr基因表达载体pAFVPRA的构建

Construction of expression vector of HIV-1 vpr gene

目的 构建可在肝癌细胞中特异性表达人HIV 1vpr基因的重组表达载体。方法 用PCR的方法从 pBSX CYPYVPR xcTHY(简称vpr质粒 )中扩增HIV 1vpr基因片段 ,克隆到pGEM T载体中 ,构建克隆载体 pGEM T/vpr,切下vpr片段后插入到 pCI质粒中 polyA的上游 ,构建中间载体pCI/vpr ,再切下vprA(vpr+polyA)插入到pAF5 .1中AFP启动子的下游 ,从而把AFP启动子和polyA尾分别克隆到vpr基因的上游和下游 ,构建成 pAFVPRA载体。 结果 成功构建了带有AFP启动子和polyA尾的载体 pAFVPRA。结论 pAFVPRA质粒构建成功后 ,可进一步通过合适的方式把HIV 1vpr基因导入肝细胞 。

Objective To construct hepatocarcinoma specific expression vector of HIV-1 vpr gene. Methods HIV-1 vpr gene segment was amplified from pBSXCYPYVPR-xcTHY with polymerase chain reaction (PCR),and then cloned into pGEM-T vector and the cloning vector pGEM-T/vpr was obtained.Then the vpr gene was recovered and inserted into the upstream of polyA in the pCI vector.Finally The vprA (vpr+polyA) fragment was inserted into the downstream of AFP promoter in the pAF5.1 after it was recovered from the intermedia vector pCI/vpr. Results The hepatocarcinoma specific recombinant expression vector was constructed successfully. Conclusion After the pAFVPRA vector was constructed successfully, the HIV-1 vpr gene can be introduced into the hepatocytes and kill liver cancer cells specifically.