[1]张明霞,周福元,刁志宏,等.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[J].南方医科大学学报,2006,(06):725-729.
 ZHANG Ming-xia,ZHOU Fu-yuan,DIAO Zhi-hong,et al.Stable expression of HBV C gene mutants in immortalized human B-cell lines[J].Journal of Southern Medical University,2006,(06):725-729.
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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()
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《南方医科大学学报》[ISSN:1673-4254/CN:44-1627/R]

卷:
期数:
2006年06期
页码:
725-729
栏目:
论著·基础研究
出版日期:
2000-01-01

文章信息/Info

Title:
Stable expression of HBV C gene mutants in immortalized human B-cell lines
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; 南方医科大学南方医院感染内科 广东广州510515; 广东广州510515;
Author(s):
ZHANG Ming-xia ZHOU Fu-yuan DIAO Zhi-hong HE Hai-tang HOU Jin-Lin LUO Kang-xian Department of Infectious Diseases Nanfang Hospital Southern Medical University Guangzhou 510515 China
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
Keywords:
hepatitis B virus mutation eukaryotic expression vector immortalized B-lymphoblasts
分类号:
R392
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HBVDNA核心区第60、87、97位氨基酸发生了预期的突变,WesternBlotting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。?
Abstract:
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 vitro.?

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备注/Memo

备注/Memo:
全军医学科学技术研究“十五”计划重点课题(01Z046)
更新日期/Last Update: 1900-01-01