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

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Sac I和Kpn I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。

Stable expression of HBV C gene mutants in immortalized human B-cell lines

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 p3.8 II plasmid containing 1.2 copy of HBV genome by means of site-directed mutagenesis. The HBV genome was amplified by PCR from p3.8 II and p3.8 II-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 vitro.