设计特异性结合HBVX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

作　　者：祝艺耘 付愚[1] 李中堂[1] 刘锐[1] 魏续福[1] 吴忠均[1] 

机构地区：[1]重庆医科大学附属第一医院肝胆外科,重庆400016

出　　处：《重庆医科大学学报》2014年第7期929-932,共4页Journal of Chongqing Medical University

基　　金：国家自然科学基金资助项目(编号:81171562)

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24h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 eantigen，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HBVmRNA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。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 supematant 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=-O.O00,95%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.

关 键 词：乙型肝炎病毒 X基因启动子 锌指蛋白 HEPG2 2 15细胞 

分 类 号：R373.2[医药卫生—病原生物学]