摘要
目的构建靶向乙型肝炎病毒X基因(HBX)的shRNA表达载体pSilencer3.1-shHBX,观察其体外抑制HBX在HepG2细胞中表达的作用,为应用RNA干扰技术进一步研究HBX基因的功能奠定基础。方法设计并构建靶向HBX的shRNA表达载体pSilencer3.1-shHBX,脂质体转染法将HBX表达载体pcDNA3.1-HBX与pSilencer3.1-shHBX共转染人肝癌HepG2细胞,培养72 h后以RT-PCR检测HBX基因表达情况,以Western blot检测HBX蛋白的表达量。结果经酶切和测序鉴定,构建的重组质粒pSilencer3.1-shHBX与设计一致。该质粒使HBX基因mRNA表达量降低47.1%,使HBx蛋白表达量降低58.9%,而阴性对照质粒无此作用。结论成功构建靶向HBX shRNA表达载体pSilencer3.1-shHBX,该质粒可体外抑制HBX在HepG2细胞中的表达。
Objective To construct the shRNA expression vector targeting the hepatitis B virus X gene （HBX） and to observe the inhibitory effect of this vector on HBX protein expression in vitro. Methods A recombinant pSilencer3.1-shHBX plasmid, which can transcribe the shRNA targeting HBX, was constructed by cloning the annealed synthesized sequences into linearized pSilencer3.1-H1 vector and then named pSilencer3.1-shHBX. Then, pcDNA3.1-HBX and psilencer3.1-shHBX were cotransfected into HepG2 cells by the liposome transferring method. Cells were cultured for 72 hours after transfection and RT-PCR and Western blot were performed to evaluate the inhibitory effect of pSilencer3.1-shHBX against the expression of the HBX gene and HBx protein, respectively. Results The recombinant pSilencer3.1-shHBX plasmid identified by restriction enzyme digestion and sequencing, was consistent with the design. As opposed to the negative control, the recombinant pSilencer3.1-shHBX plasmid efficiently inhibited the expression of HBX mRNA and HBx protein. The inhibitory rates were 47. 1% and 58.9%, respectively. Conclusion The shRNA expression vector targeting HBX was successfully constructed and was able to inhibit the expression of the HBx protein.
出处
《中国病原生物学杂志》
CSCD
2009年第5期337-339,358,共4页
Journal of Pathogen Biology
基金
江苏省教育厅自然科学基金项目(No.07KJD310220)
