HBV C编码链抗HBV潜在药物靶标的设计、筛选及鉴定  

Design,screening and identification of HBV C coding chain as anti-HBV potential drug targets

作　　者：许桂丹[1] 肖树荣 韦武均 彭彬 农顺强 邓益斌[1,3,4] XU Guidan;XIAO Shurong;WEI Wujun;PENG Bin;NONG Shunqiang;DENG Yibin(Department of Laboratory Center,Affiliated Hospital of Youjiang Medical University for Nationalities,Baise,533000;Department of Laboratory Center,Yangjiang People’s Hospital,Yangjiang,529500;Guangxi Clinic Medicine Research Center of Hepatobiliary Disease,Baise,533000;Department of Infectious Diseases Center,Affiliated Hospital of Youjiang Medical University for Nationalities,Baise,533000,China)

机构地区：[1]右江民族医学院附属医院医学检验科,广西百色533000 [2]广东省阳江市人民医院检验科,广东阳江529500 [3]广西肝胆疾病临床医学研究中心,广西百色533000 [4]右江民族医学院附属医院感染性疾病科,广西百色533000

出　　处：《右江医学》2020年第1期12-16,共5页Chinese Youjiang Medical Journal

基　　金：国家自然科学基金(81460123);广西自然科学基金(2018GXNSFAA281187);广西肝胆疾病临床医学研究中心(桂科AD17129025-04);广西卫生和计划生育委员会自筹经费科研课题(Z20180219);百色市科学研究与技术开发计划项目(百科计20182513);博导培育计划人选项目(右医附院院字[2018]32号);博点学科建设项目(右医附院院字[2018]29号)

摘　　要：目的针对HBV C编码链设计合成反基因锁核酸(locked nucleic acid,LNA)片段,以HepG2.2.15细胞为研究对象,筛选并鉴定出能特异性阻断HBV复制和表达的有效治疗靶点药物。方法利用RNAstructure 5.0软件针对HBV C区编码链1914~1928 nt、1969~1983 nt、2002~2016 nt、2053~2067 nt、2069~2073 nt、2162~2176 nt和2404~2418 nt位点设计合成7条反基因LNA片段(分别以SQ1~SQ7表示),以阳离子脂质体lipofectamine 3000(lipo3000)介导转染HepG2.2.15细胞,隔3天对应给药,连续给药3次,分别于给药后第3、6、9天收集培养上清液,采用酶联免疫吸附试验(ELISA)检测HBeAg水平;实时荧光定量聚合酶链反应技术(FQ-PCR)测定HBV-DNA浓度。结果针对HBV C区编码链2162~2176 nt(SQ6)和2404~2418 nt(SQ7)位点的反基因LNA片段对HBV-DNA复制和HBeAg表达的抑制效果最明显,用药后第3、6、9天,SQ6对HBeAg的平均抑制率分别为43.91%、59.95%和59.07%,HBV-DNA则分别为42.91%、50.09%和51.14%;SQ7对HBeAg的平均抑制率分别为44.18%、60.81%和60.02%,HBV-DNA则分别为47.28%、54.16%和52.22%。结论针对HBV C区编码链2162~2176 nt和2404~2418 nt位点的反基因LNA片段体外能有效抑制HBV的复制和表达,为抗HBV治疗提供一定的理论依据和实验依据。Objective Anti-gene locked nucleic acid(LNA) fragment were designed and synthesized for the HBV C coding chain.HepG2.2.15 cells were used as the research object to screen and identify effective therapeutic target drugs that could specifically block HBV replication and expression.Methods Using RNAstructure 5.0 software to design and synthesize 7 HBV C region coding strands 1914~1928 nt,1969~1983 nt,2002~2016 nt,2053~2067 nt,2069~2073 nt,2162~2176 nt and 2404~2418 nt loci.Antigenic LNA fragments(represented by SQ1 to SQ7,respectively) were transfected into HepG2.2.15 cells with cationic liposome lipofectamine 3000(lipo3000),and the corresponding administration was performed every 3 days,the administration was continued 3 times respectively.Culture supernatants were collected on days 3,6,and 9 after administration.HBeAg levels were measured with an enzyme-linked immunosorbent assay(ELISA);HBV-DNA concentration was measured by FQ-PCR.Results Anti-gene LNA fragments targeting the 2162 to 2176 nt(SQ6) and 2404 to 2418 nt(SQ7) loci of the HBV C region coding chain have the most significant inhibitory effects on HBV-DNA replication and HBeAg expression.The average inhibition rates of SQ6 on HBeAg were 43.91%,59.95%,and 59.07%;the average inhibiton rates on HBV-DNA were 42.91%,50.09%,and 51.14%;the average inhibition rates of SQ7 on HBeAg were 44.18%,60.81%,and 60.02%;the average inhibition rates on HBV-DNA was 47.28%,54.16% and 52.22%,respectively.Conclusion Antigenic LNA fragments targeting the 2162~2176 nt and 2404~2418 nt sites of the HBV C region coding chain can effectively inhibit HBV replication and expression in vitro,and provide theoretical and experimental support for anti-HBV therapy.

关 键 词：乙型肝炎 乙型肝炎病毒 锁核酸 基因治疗 

分 类 号：R512.62[医药卫生—内科学]