构建重组质粒检测癌基因STAT3诱导表达活性

目的建立基于报告基因的检测方法,检测环境因子饮用水水源有机提取物对癌基因Stat3的诱导表达活性,发现异常活化癌基因的环境致癌因子,为进一步揭示肿瘤发生发展与环境因素的相互作用关系奠定基础。方法将Stat3特异结合序列CRP-APRE克隆到诱导表达型载体pGL2转录启始位点上游,选择氯霉素乙酰转移酶基因(CAT)作为报告基因克隆在转录启始位点下游,构建重组质粒pTKS3-CAT。转染入表达IL-6受体的人肝癌细菌HepG2中,筛选报告基因CAT表达受IL-6诱导的阳性克隆,同时用无关药物检测模型的特异性。用建立的检测方法——ELISA法检测饮用水源有机提取物对CAT表达活性的影响,检测环境因子对Stat3的诱导表达活性。结果CAT的表达受IL-6诱导并呈现剂量依赖关系,EC50等于0.127nmol.L-1,而无关药物rhEPO不能诱导CAT表达。结论在96孔板上用ELISA法测定CAT基因的诱导表达水平,可在基因水平检测癌基因Stat3的诱导表达活性。

Objective To establish a reporter gene based measurement and use it to test induced expressive activity of cancer related gene Stat3 for entironment factor,organic extracts from source water, in order to discover the relationship between cancer and abnormal activation of entironment factor. Methods A recombinant pTKS3-CAT was constructed by inserting CRP-APRE, a sequence composed of seven copy Star3 response elements upstream of promoter and a chloramphenicol acetyltransferase （CAT） gene downstream of promoter, pTKS3-CAT was transfected into HepG2 cells expressing IL-6 receptor. One stably transfected clone was isolated, and used to screen compounds for activity of stimulating CAT gene expression. Results The expression of CAT was induced by IL-6. A dose-dependent espression of CAT gene with halfmaximal induction at 0.127 nmol·L^-1 was observed. After treating with rhEPO CAT activity couldn＇t be tested. Conclusion We can use it to assay CAT from extracts of cells grown with CAT-ELISA method in microtiter wells and then to test induced expressive activity of cancer related gene Star3.