人端粒酶逆转录酶启动子调节单纯疱疹病毒胸苷激酶和人白细胞介素12融合基因肿瘤细胞特异性表达载体的构建及意义

Construction and identification of tumor cells specific expression vector of combination gene of HSV-TK and hIL-12 driven by hTERT promotor

目的构建由人端粒酶逆转录酶（hTERT）启动子调节的单纯疱疹病毒胸苷激酶基因（HSV—TK）和人白细胞介素12（hIL-12）融合基因表达载体。方法利用聚合酶链反应（PCR）分别扩增hTERT启动子基因、HSV—TK基因和hIL-12p70基因，并将其分别克隆至pShuttle载体上，合成pShuttle—hTERTp—HSV—TK—linker—IL一-12融合基因表达载体，并对所合成的载体酶切、测序检验。结果各基凶片段成功扩增，大小分别为1084、1173、1557bp，并成功联结至pShuttle载体上。对所构建新载体分别行酶切鉴定，各融合基因片段大小与预计大小相符，DNA序列测定结果显示，与目标序列完全一致，无突变发生。结论成功构建肿瘤特异性启动子调控的融合基因表达载体pShuttle—hTERTp—HSV—TK—linker—IL-12。

Objective To construct the tumor cells specific expression vector of combination gene of herpes simplex virus thymidine kinase （HSV-TK） and human inlerleukin 12 （hIL-12） driven by human telomerase reverse transcriptase （hTERT） promotor for the further research. Methods hTERTp, HSV-TK and IL-12p70 genes were amplified by using PCR separately and cloned into the vector pShuttle to establish the combination geue expression vector as pShuttle-hTERTp-HSV-TK-linker-IL-12. Subsequently, the expression vector was identified by enzyme digestion and DNA sequencing. Results The hTERTp, HSV-TK and IL-12p70 genes, which were 1084, 1173 and 1557 bp respectively, were successfully cloned into the expression vector and identified to be correct by enzyme digestion and sequencing. Conclusion The pShuttle-hTERTp-HSV-TK-linker-IL-12 combination gene expression vector was successfully constructed as prediction.