摘要
目的了解泛素特异性蛋白酶18（USP18）对乙型肝炎病毒（HBV）复制的影响及其潜在机制。方法 1）USP18和Pol质粒构建：采用常规基因克隆方法,将人USP18编码序列及HBV多聚酶（Pol）编码序列分别构建到pcDNA3.1-3tag真核细胞表达载体内。2）研究Pol对USP18的影响：高表达转染Pol质粒至Hep AD38细胞内,抽提细胞内总RNA,逆转录成c DNA,用实时荧光定量PCR（qRT-PCR）检测Pol对USP18的影响。3）研究USP18对HBV的影响：高表达转染USP18质粒至Hep AD38细胞内,抽提细胞内总RNA和总DNA,qRT-PCR检测USP18对HBV复制的影响。4）研究Pol与USP18之间的相互作用：将Pol质粒按一定比例转染到铺有293T细胞的10cm培养皿中,采用免疫共沉淀的方法检测Pol与USP18之间是否存在相互作用。结果成功构建USP18和Pol表达质粒,并在HepAD38细胞中高表达;过表达Pol蛋白促进USP18表达;过表达USP18刺激HBV复制;Pol与USP18存在相互作用。结论初步证明USP18可能通过与Pol相互作用促进HBV复制。
Objective To investigate the impact of Ubiquitin Specific Protease 18（USP18） on the replication of Hepatitis B virus（HBV） and its potential mechanisms. Methods USP18 and HBV Polymerase（Pol） plasmids were constructed via conventional methodsbased on the pc DNA3. 1-3 tag eukaryotic cell expression vector. The effect of Pol over-expression on USP18 in HepAD38 cells was evaluated byReal-time Quantitative polymerase chain reaction（qPCR）.Similarly,the effect of USP18 over-expression on HBV was also evaluatedby q PCR. The interaction between Pol and USP18 was confirmed by Co-Immunoprecipitation（CO-IP）.Results The USP18 or Pol plasmids were constructed successfully and further confirmed to exist in the HepAD38 cells by Western Blot（WB）. The Over-expression of Pol promoted USP18 expression while theover-expression of USP18 promoted the replication of HBV. Certain interactions between Pol and USP18 were mapped out by CO-IP.Conclusion USP18 stimulates the replication of HBV,which may be achieved via the interaction between Pol and USP18. However,thepotential mechanismof this stimulation process remains to be further explored.
作者
姚敏
李玉佳
叶海艳
廖鑫忠
赵航
李世林
陈利民
YAO Min;LI Yufia;YE Haiyan;LIAO Xinzhong;ZHAO Hang;LI Shilin;CHEN Limin.(Institute of Blood Transfusion, Chinese Academy of Medical Sciences, Peking Union Medical College, Provincial Key Laboratory for Transfusion-transmitted Diseases of Sichuan Province, Chengdu, 610052 China)
出处
《中国输血杂志》
CAS
2018年第4期361-365,共5页
Chinese Journal of Blood Transfusion
基金
四川省科技厅国际合作项目(2017HH0024)
中国医学科学院医学与健康科技创新工程(CAMS-2016-I2M-3-025)
