HBV全基因C区突变株在永生化B淋巴母细胞系中的稳定表达作者.doc

HBV全基因C区突变株在永生化B淋巴母细胞系中的稳定表达 作者：张明霞；周福元；刁志宏；何海棠；侯金林；骆抗先　　(南方医科大学南方医院感染内科，广东??广州??510515) 摘要：目的 构建携带V60、G87和L97突变位点的HBV 全基因组真核表达载体，转染慢性HBV感染者永生化B淋巴母细胞系(LCLs)。方法 用定点突变技术将HBVp3.8Ⅱ质粒C基因区3个氨基酸V60、G87、L97进行突变(p3.8Ⅱ- V60，G87，L97)，转化E.coli. XL1-Blue感受态细胞扩增筛选，用限制性内切酶SacⅠ和KpnⅠ分别将野生型和突变型p3.8Ⅱ质粒进行双酶切， 插入EBV-plpp真核细胞表达载体，转染LCLs，潮霉素稳定筛选后，鉴定目的基因在转染细胞能够稳定表达。结果与结论DNA序列分析表明，野型HBV DNA核心区第60、87、97位氨基酸发生了预期的突变，Western Blotting和微粒子免疫荧光法证明转染的LCLs能稳定表达HBV 抗原，证实成功构建了预期细胞模型。 关键词： 乙型肝炎病毒；变异；真核细胞表达载体；永生化B淋巴母细胞 中图分类号：R512.6??文献标识码：A??文章编号：1673-4254(2006)06-0725-05 Stable expression of HBV C gene mutants in immortalized human B-cell linesZHANG Ming-xia； ZHOU Fu-yuan；DIAO Zhi-hong； HE Hai-tang； HOU Jin-Lin LUO Kang-xianDepartment of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou 510515, ChinaAbstract: Objective To provide an cell model of immortalized lymphoblstoid B-cell lines for studying the biological characteristics of full-length hepatitis B virus (HBV) genome carrying the hot-spot mutations V60, G87, and L97. Methods V60, G87, and L97 mutation points were introduced into HBV p3.8Ⅱ plasmid containing 1.2 copy of HBV genome by means of site-directed mutagenesis. The HBV genome was amplified by PCR from p3.8Ⅱ and p3.8Ⅱ- V60, G87, L97 plasmid, and the PCR product was inserted into EBO-plpp eukaryotic expression vector. The recombinant vectors and the EBO-plpp vector were transfected into immortalized human lymphoblasts with lipofectamine 2000 and selected with hygromycin. Steady expression of the target genes was determined by RT-PCR, Western blotting and microparticle enzyme immunoassay. Results DNA sequence analysis indicated that the desired mutation was introduced into wild-type HBV DNA. HBsAg, HBeAg and HBcAg could be detected in EBO-HBV- transfected cell lysate or culture supernatant. Conclusion Transfectants that stably express HBV mutant antigen may provide a cell model to study the biological characteristics of HBV carrying hot-spot mutation in v