携带双自杀基因且可诱导敲除SV40T的逆转录病毒载体的构建与结构鉴定

Construction and Identification of Novel Retroviral Vector containing Double-Suicide Genes and Inducible SV40T Knockout

目的构建携带双自杀基因且可诱导敲除SV40T的逆转录病毒载体,优化目前的肝细胞永生化。方法去除eGFP终止子taa的pSEB-HUS质粒为逆转录病毒基础质粒。先将SV40T及其启动子hEFH亚克隆至pSEB-HUS,再将LoxP位点插入至pSEB-SV40T质粒的抗性基因Blasticidin上游获得pSEB-LoxP-SV40T质粒。同时将CD自杀基因定向克隆至pSEB-HUS的eGFP下游;然后设计一段HSV-tk自杀基因引物,在上游引物的5′端加入方向一致的LoxP序列。PCR获得TK基因后,定向克隆至CD下游获得pSEB-CD-TK,最后将pSEB-CD-TK上的双自杀基因亚克隆至pSEB-LoxP-SV40T质粒上得到携带双自杀基因及SV40T的质粒。结果PCR及酶切鉴定均证实两个自杀基因及SV40T正确克隆至逆转录病毒质粒中,目的基因序列与GenBank报道一致,两个LoxP位点方向相同。结论成功构建携带双自杀基因且可诱导敲除SV40T的逆转录病毒载体。

Objective To construct a new retroviral vector with double-suicide genes and knockout-inducible SV40T gene, with the aim of optimizing immortalization of hepatocytes. Methods By use of pSEB-HUS plasmid containing TAA-deleted eGFP as the based retroviral vector, SV40T gene and its hEF1 promoter were subcloned into the vector. Loxp site was inserted to the upstream of the Blasticidin resistance gene to obtain pHUS-Loxp-SV40T plasmid. CD suicide gene was directed downstream eGFP in pSEB-HUS vector. Using TK＇s primers with Loxp sequence upstream the suicide HSV-TK gene, TK-gene fragment was amplified by PCR. The TK fragment was cloned downstream CD gene. Eventually, the fragment of the double-suicide CD-TK genes was cut off from pHUS-CD-TK and linked to pHUS-SV40T plasmid, thus achieving the resultant retroviral vector which contains double-suicide genes and knockout-inducible SV40T gene. Results Analyses by PCR, restriction enzyme digestion and gene sequencing confirmed the insertion and correct orientation of the double-suicide genes and SV40T gene with two Loxp sites in the retroviral vector. Conclusion A new retroviral vector with double-suicide genes and knockout-inducible SV40T gene has been successfully constructed, which may be used to improve hepatocyte immortalization.