南方医科大学学报

南方医科大学学报 ›› 2006, Vol. 26 ›› Issue (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

张明霞； 周福元； 刁志宏； 何海棠； 侯金林； 骆抗先；   

  1. 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；
  • 出版日期:2006-06-20 发布日期:2006-06-20
  • 基金资助:
    全军医学科学技术研究“十五”计划重点课题(01Z046)

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

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   

  1. 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；
  • Online:2006-06-20 Published:2006-06-20

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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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