热诱导调控序列的合成及其热诱导特性的研究

目的 探讨合成热诱导性的HSP70启动子调控序列，并鉴定加热条件下诱导其下游报告基因表达的特性。方法 利用PCR方法，以肝癌细胞株HepG2基因组为模板，体外合成热休克蛋白70（HSP70）启动子序列。利用双酶切法构建HSP70启动子调控的含EGFP报告基因的真核表达载体pcDNA-HSP70-EGFP。以脂质体为载体，体外转染肝癌细胞株HepG2，在不同的温度下（37℃、39℃、41℃、43℃、45℃）加热不同时间（0、15、30、45、60min）后，利用流式细胞术检测报告基因EGFP表达强度，从而判定温度及时间对HSP70启动子热诱导活性的影响。结果 合成的HSP70启动子序列测序结果与GeneBank 中AL671762所提供序列完全一致。低温加热后EGFP在HepG2细胞中的表达强度不同程度增强，在43℃/30 min 时最为明显，为未加热组的3.3倍。结论 成功合成可热诱导的HSP70启动子序列；HSP70启动子在低温加热下能明显增强其下游外源基因的表达，为进一步研究肿瘤热疗-基因治疗提供了实验基础。

Study on synthesis of heat induced regulative sequence and its heat induced feature

Objective To investigate the synthesis of regulative sequence of heat induced HSP70 promoters and identify the feature of induced downstream reporter gene expression in the heating condition. Methods Using PCR method and liver cancer cell strain HepG2 gene group served as mould plate, the heat shock protein 70（HSP70） promoter sequences was synthesized in vitro. Using double-enzyme cutting method,the HSP70 promoter regulative contained EGFP（Enhanced Green Fluorescence Protein）reporter gene eucaryotic expression carrier pcDNA-HSP70-EGFP was constructed. The liposome was used as carrier. The liver cancer cell strain HepG2 was transfected in vitro. Under different temperature and after heating different time,using flow-cytometry to detect the expression intensity of reporter gene EGFP,the temperature and time to influence on HSP70 promoter heat induced activity was judged. Results It was agreed with completely on the synthesized HSP70 promoter sequence detected results and Gen Bank AL671762 providing sequence.After low-temperature heating, EGFP in HepG2 cells expression intensity had enhancement in different degree.The obvious enhancement was in 43℃/30 minutes, and it was 3.3 times on the unheating group.Conclusion Heating induced HSP70 promoter sequence was synthesized successfully. Under low-temperature heating, the HSP70 promoter would significantly enhance the downstream exogenous gene expression and it provides experimental basis for further studying tumor heat-therapy-gene therapy.