HBV全基因组克隆转染细胞系的建立.docVIP

HBV全基因组克隆转染细胞系的建立 HBV全基因组克隆转染细胞系的建立 　　 作者：赵艳芳，闫永平，苏海霞，王安辉，张磊，张景霞，门可，徐德忠? 【关键词】? 肝炎病毒,乙型；基因组,病毒；克隆,分子；转染；细胞培养；基因表达 ??? 【Abstract】 AIM: To construct a new cell culture system for producing HBV in : Fulllength HBV DNA was cloned into pcDNA3 vector (named pcDNA33HBV). HepG2 cells were transfected with pcDNA33HBV and screened with antibiotic G418. HBsAg and HBeAg were identified by ELISA and HBcAg was identified by immunocytochemical staining. S gene mRNA expression was tested by RTPCR and HBV DNA in the supernatant of transfected cells was tested by PCR. RESULTS: The plasmid pcDNA33HBV was constructed successfully. After stable transfection, HBsAg and HBeAg could be detected in the supernatant of transfected cells. HBcAg positive staining was located mainly in the cytoplasm. S gene mRNA expression was verified. S gene and preS gene could be detected in the supernatant by RTPCR. The copies of HBV genome can reach 1×108 copies/L detected by realtime quantitative PCR. CONCLUSION: Rebinant plasmid pcDNA33HBV could be expressed, transcribed and replicated in HepG2 cells. This transfectionbased cell culture system could produce high copies of HBV genome. ??? 【Keywords】 hepatitis B virus; genome, viral; cloning, moleculor; transfection; cell culture;? gene expression ??? 【摘要】 目的： 建立HBV体外细胞培养体系. 方法： 构建HBV全基因克隆质粒pcDNA33HBV，稳定转染HepG2细胞，G418筛选. ELISA检测细胞上清液HBsAg，HBeAg的表达，免疫组化检测细胞内HBcAg的表达，RTPCR检测细胞S基因mRNA的表达，PCR检测细胞上清液DNA. 结果： 成功构建了HBV全基因克隆质粒pcDNA33HBV，稳定转染HepG2后，培养上清HBsAg，HBeAg阳性，细胞内HBcAg呈核浆型分布，且以浆型分布为主. RTPCR证实有HBV S基因 mRNA 的表达，上清中HBV S及前S基因阳性，荧光定量PCR检测显示培养上清中HBV 滴度达1×108拷贝/L. 结论： 重组质粒pcDNA33HBV能在HepG2细胞中表达、转录、复制，该细胞培养体系能产生较高水平的HBV. ??? 【关键词】 肝炎病毒，乙型；基因组，病毒；克隆，分子；转染；细 者双酶切，琼脂糖凝胶电泳鉴定后送北京三博远志生物技术有限公司进行序列测定. ??? 脂质体介导下pcDNA33HBV基因转染转染前一日将处于对数生长期的HepG2细胞用胰酶消化并换无抗生素的DMEM培养基，将细胞接种于24孔板，密度达90%时进行转染. 转染方法按说明书进行. 24 h后传代，48 h换含600? mg/L G418培养基进行筛选. 设未转染的HepG2细胞为对照. 待对照组细胞全部死亡后，用含300 mg/L G418的培养基维持. 筛选过程不含其他抗生素. ??? 测抗原表达取转染细胞的上清液用ELISA法测HBsAg，HBeAg. 操作及结果判定按ELISA试剂盒说明书进行，终止反