微创医学

目的构建人类免疫缺陷病毒1型(HIV-1)tat基因分泌型真核表达载体。方法聚合酶链反应(PCR)法从缺失pol基因的重组HIV-1基因组pNL4-3中扩增tat基因;将PCR获得的tat基因片段和pSecTag2B分泌型真核表达载体分别用Hind III酶切;pSecTag2B载体片段经小牛碱性磷酸酶去磷酸化后,用T4 DNA连接酶将tat基因与pSecTag2B载体片段连接过夜并转化感受态大肠杆菌DH5α,提取阳性克隆质粒进行Hind III酶切鉴定,并利用Nco I酶切鉴定克隆的tat基因反向。最后选取正向tat基因克隆质粒送基因测序。结果 PCR扩增产物在1%琼脂糖凝胶上约320bp位置出现条带,与预期的324bp大小一致;经Hind III和Nco I分别酶切鉴定,获得2个正向克隆;正向克隆经测序,克隆的tat基因序列与Genbank中登记的HIV-1 tat基因100%同源。结论本研究方法可以成功地构建HIV-1 tat基因分泌型真核表达载体。

Objective To construct secretory eukaryotic expression vector of tat gene from human immunodeficiency type 1( HIV-1) ． Methods HIV-1 tat gene was amplified from pNL4 － 3 plasmid，which was HIV-1 genome deleted pol gene，with polymerase chain reaction ( PCR) ． Tat DNA fragments and secretory eukaryotic expression vector，pSecTag2B were then digested with Hind III． Digested pSecTag2B fragments were further dephosphorylated with calf intestinal alkaline phosphatase ( CIP) ． Digested tat DNA fragments and pSecTag2B fragments were ligated overnight with T4 DNA ligase and were further transformed to E． coli competent cells DH5α． Furthermore，recombinant plasmids were extracted and determined by digestion with Hind III restricted enzyme． Moreover，the direction of inserted tat gene in recombinant plasmids was detected with Nco I restricted enzyme． Consequently，plasmids from norientation clone were sequenced． Results A band about 320bp as expecting ( 324bp) was visible when PCR products were electrophoresis in 1% agarose． Digested with Hind III and Nco I enzyme respectively，2 positive norientation clone were determined． The sequence of tat gene cloned in norientation plasmid was 100% homology with HIV-1 tat gene registered in GenBank． Conclusion HIV-1 tat gene can be cloned into secretory eukaryotic vector successfully．