乙型肝炎病毒核心蛋白小干扰RNA载体的构建与验证  

Construction and identification of small hairpin RNA vector targeting HBV core protein in HepG2.2.15 cells

作　　者：赵培庆[1,2] 柴巨川 盖潇潇[1] 高立芬[1] 马春红[1] 梁晓红[1] 

机构地区：[1]山东大学医学院免疫学研究所,济南250012 [2]淄博市中心医院中心实验室 [3]山东大学医学院检验科

出　　处：《中华实验和临床病毒学杂志》2014年第6期479-481,共3页Chinese Journal of Experimental and Clinical Virology

基　　金：本课题受国家自然基金青年基金资助（基金编号30700973）;国家自然基金资助（基金编号81171642）

摘　　要：目的 构建针对乙型肝炎病毒核心蛋白的小干扰RNA真核表达载体,并验证其干扰效果.方法 设计针对HBV基因组（ayw亚型）HBc基因的小干扰RNA序列（位于2147 bp ～2165 bp序列）,目的片段与载体成功连接后转入大肠埃希菌DH5α,酶切及测序验证其正确性.最后以脂质体转入HepG2.2.15细胞中,检测其干扰效果.结果 成功构建针对乙型肝炎病毒核心蛋白的小干扰RNA真核表达载体,并命名为pshRNA-HBc..酶切及测序验证正确,pshRNA-HBc对HepG2.2.15细胞中HBc表达具有特异性干扰效果.结论 构建针对乙型肝炎病毒核心蛋白的小干扰RNA真核表达载体,为后续研究HBc的生物学功能奠定良好基础.Objective To construct eukaryotic expression vector of small hairpin RNA vector targeting hepatitis B virus core protein in HepG2.2.15 cells,and to verify the interfering effect.Methods To design and synthesize small interfering RNA sequence against HBc gene （ayw subtype） which locates between 2147 bp to 2165 bp in the HBV genome.The siRNA fragment were inserted into pSilencer3.1-H1 neo vector.The recombinants were transformed into E.coli DHSα successfully.The recombinant plasmids were identified by Eco RI enzyme digestion and sequenced.Finally,The HepG2.2.15 cells were transfected with the vector by using lipofectamine method to identify its interfering effect.Results The eukaryotic expression vector containing small hairpin RNA targeting HBV core protein was constructed successfully and named as pshRNA-HBc.The recombinant plasmids were identified by SalI or EcoRI enzyme digestion and sequenced.The silencing effect was detected by using revere transcription polymerase chain reaction （RTPCR） and the secretion of HBsAg and HBeAg in the supernatant of HepG2.2.15 cells.The result showed that the interfering effect was specific and efficient.Conclusion The small interfering RNA targeting HBV core protein in HepG2.2.15 cells was constructed successfully,which may be useful for studying the biological functions of HBc in the future.

关 键 词：肝炎病毒 乙型 病毒核心蛋白质类 

分 类 号：R373[医药卫生—病原生物学] R392[医药卫生—基础医学]