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靶向Mcl-1的shRNA重组质粒的构建及其对HepG2细胞增殖和凋亡的影响

期刊名称：华中科技大学学报(医学版)
时间：0
页码：158-161
语言：中文
分类：R512.6[医药卫生—临床医学;医药卫生—内科学]
作者机构：[1]华中科技大学同济医学院附属同济医院肝病研究所,湖北武汉430030
相关基金：基金项目：国家自然科学基金（30872237）
相关项目：一种干预乙肝相关性肝癌发生的新策略：通过调控两种门控蛋白启动的DNA DSB修复途径遏制HBV的整合

关键词：乙型肝炎病毒, 缺失, LA蛋白, 稳定性, Hepatitis B virus, Deletion, La protein, Stability

中文摘要：

目的观察乙型肝炎病毒e抗原mRNA（HBeAg mRNA）上La蛋白结合位点的缺失对其细胞内稳定性的影响,从而进一步研究这个位点在乙肝病毒生命周期中的作用。方法针对HBeAg mRNA上La蛋白结合位点,在HBV DNA水平上利用PCR定点突变技术,构建突变型HBV载体,使其转录出来的HBeAg mRNA缺失La蛋白结合位点,将其命名为pHBV-mLa;应用脂质体将突变型HBV载体瞬时转染HepG2细胞,同时将转染了野生型HBV载体的细胞作为对照组;在各组中,用半定量逆转录PCR（RT-PCR）检测HBeAg mRNA,同时酶联免疫吸附试验（ELISA）检测细胞培养上清中HBeAg。结果酶切鉴定和碱基测序证明,成功构建了La蛋白结合相关位点部分碱基缺失的突变型HBV载体—pHBV-mLa。转染后半定量RT-PCR检测发现,突变后HBeAg mRNA水平明显低于未突变的对照组,ELISA检测发现突变组中HBeAg表达下调。结论HBV-DNA上引入的突变可以破坏HBeAg mR-NA上La蛋白结合位点,影响HBeAg mRNA在细胞内的稳定性。HBeAg mRNA上La蛋白结合位点对乙肝病毒生命周期至关重要。

英文摘要：

Objective To investigate the stability of HBeAg mRNA in cells with deletion of the La protein binding site in HBeAg mRNA and to further study the role of the La protein binding site in hepatitis B virus life cycle.Methods The HBV vector with mutation that resulted in the deletion of La protein binding site in HBeAg mRNA was constructed by PCR-based site-directed mutagenensis,termed pHBV-mLa.The mutant vector was transfected into HepG2 cell using liposome, and the wild-type HBV vector served as the control.The HBeAg mRNA level was detected by semi-quantitative RT-PCR,and HBeAg secreted into culture media was detected by ELISA in each group.Results The identification of restriction analysis and sequencing supported that the mutant HBV vector was successfully constructed.Semi-quantitative RT-PCR and ELISA showed that the HBeAg mRNA level in the mutant vector group was significantly lower than that in the control group,and the expression of HBeAg was reduced in the mutant vector group.Conclusion The HBeAg mRNA stability in cells decreased due to the deletion of the La protein binding site in HBeAg mRNA that was caused by the mutation in HBV-DNA.The La protein binding site in HBeAg mRNA plays an important role in hepatitis B virus life cycle.

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