靶向整合于MHCC97-H细胞的HBx基因shRNA表达载体的构建和鉴定

Construction and Identification of shRNA Eukaryotic Expression Vectors Specific to HBx Gene Integrated in Human Hepatocellular Carcinoma Cell Line MHCC97-H

目的构建靶向整合于人肝癌MHCC97-H细胞乙型肝炎病毒x基因(HBxgene)基因的shRNA真核载体。方法依据从人肝癌细胞株MHCC97-H中克隆的HBx基因序列,设计合成X169、X220及MOCK(无关对照)3对寡核酸,经退火成互补双链并克隆至pGenesil-Ⅰ载体;经酶切、PCR和测序鉴定;将鉴定的shRNA质粒脂质体转染MHCC97-H细胞,流式细胞分析转染效率。结果酶切、PCR及测序证实pshRNA-X169/X220/MOCK质粒构建符合设计,转染48h后MHCC97-H细胞可激发绿色荧光,pshRNA-X220转染效率最低。结论成功构建靶向整合于人肝癌细胞HBVx基因特异性shRNA表达载体,并成功转染人肝癌细胞株MHCC97-H细胞,为沉默HBx基因实验性肝癌基因治疗奠定基础。

Objective To construct and identify shRNA eukaryotic expression vectors specific to HBx gene integrated in human hepatocellular carcinoma cell line. Method shRNA sequences, two selected from HBx gene integrated in the genome of human hepatocellular carcinoma cell hne MHCC97-H cells named X169 and X220 and another control sequence named MOCK,were synthesized and cloned into pGenesil-Ⅰ vector. After confirmed by restriction enzyme reaction,PCR assay and sequencing,the shRNA vectors were used to transfect MHCC97-H cell line by lipofectamine 2000. The transfection efficiency was assayed by FACS. Results The shRNA vectors were constructed successfully by confinning procedures. After transfection, EGFPs expressed by transfected vectors gave out green light in MHCC97-H cells. The transfection efficiency of pshRNA-X220 was the lowest. Demise MHCC97-H cells after pshRNA-X220 transfection were more than those of the other groups. Conclusion The shRNA vectors can be constructed and expressed in MHCC97-H cells successfully, which will facili- tate the further study on the role of RNAi on HBx gene expression in gene therapy for HCC.