设计特异性结合HBV X基因启动子的人工锌指蛋白以抑制HepG2.2.15细胞中HBV的转录

Inhibition of HBV transcription in HepG2.2.15 cells by artificial Zinc finger protein designed specifically to target HBX gene promoter

摘要:

目的：设计人工锌指蛋白（zinc finger protein，ZFP）特异性结合于乙型肝炎病毒X蛋白（hepatitis B virus X protein，HBX）基因启动子，观察ZFP在体外对乙型肝炎病毒（hepatitis B virus，HBV）转录的抑制。方法：将pcDNA3.1-ZFP转染入HepG2.2.15细胞24 h后，用Western blot检测HBX蛋白的含量；用ELISA和real-time PCR检测上清液中乙型肝炎核心相关抗原（hepatitis B e antigen，HBeAg）和HBV DNA含量，用RT-PCR检测胞内HBV mRNA水平。结果：ZFP可抑制HepG2.2.15细胞中HBX的表达；相比空质粒组，ZFP组细胞培养上清液中HBV DNA 拷贝量和HBeAg 含量分别下降了51.7%（t=23.079，P=0.000，９５％CI=44.98%～58.52%）、33.8%（t=3.887，P=0.003，９５％CI=12.12%～55.48%）；细胞内HBV mRNA也明显减少（t=3.616，P=0.022）。结论：人工设计的可特异性结合于HBX的ZFP可于HepG2.2.15细胞中有效抑制HBV转录。

Abstract:

Objective：To observe the inhibition of hepatitis B virus（HBV） transcription in vitro by Zinc finger protein（ZFP） which was designed specifically to target X gene promoter of HBV（HBX）. Methods：Recombinant plasmid pcDNA3.1-ZFP was transfected into HepG2.2.15 cells. Expression of HBX protein was detected by Western blot at 24 h after being transfected. Hepatitis B e antigen（HBeAg） and HBV DNA levels in supernatant of HepG2.2.15cells at 24 h after being transfected with pcDNA3.1-ZFP were detected by ELISA and real-time PCR. HBV mRNA was tested by RT-PCR. Results：ZFP could inhibit the expression of HBX in HepG2.2.15 cells. In the presence of ZFP，HBV DNA level and HBeAg was considerably reduced by 51.7%（t=23.079，P=0.000，９５％CI=44.98%-58.52%） and 33.8%（t=3.887，P=0.003，９５％CI=12.12%-55.48%） respectively，while HBV mRNA was sharply decreased（t=3.616，P=0.022）. Conclusion：ZFP protein designed to target X gene promoter of HBV can inhibit the transcription of HBV in HepG2.2.15 cells effectively.