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.