干扰素作用下乙型肝炎病毒的变异机制  

Mutation of hepatitis B virus with treatment of interferon

作　　者：王卫峰[1] 周晓东[2] 

机构地区：[1]浙江医院消化内科,浙江省杭州市310013 [2]中山大学附属第二医院医学研究中心,广东省广州市510120

出　　处：《世界华人消化杂志》2005年第6期724-728,共5页World Chinese Journal of Digestology

基　　金：国家科技部973计划项目;No;G1999054105~~

摘　　要：目的:体外研究干扰素-α2b(IFN-α2b)作用下乙肝病毒(HBV)突变机制. 方法:以不同浓度IFN-α2b反复作用于HBV分泌型HepG2.2.15细胞,Western blot方法检测培养细胞上清中AFP,提取培养细胞上清中HBV基因组DNA,以聚合酶联反应扩增HBV C基因,克隆入pGEM-T easy 载体,鉴定后行测序分析. 结果:Western blot方法检测各组培养细胞上清中AFP, 实验组AFP未见明显减少;序列分析表明,对照组前C/ C基因克隆有两种类型,同源性93%,有10个氨基酸的差别,其中6个氨基酸相比,氨基酸的性质相同; 实验组同样具有这两种克隆,其中优势株所占比例分别为12/15、14/14和9/15,高剂量组与对照组相比优势株所占比例差异有统计学意义(P<0.05);实验组高剂量IFN 组出现了四个插入/缺失突变克隆,造成前C/C基因移框突变,其中C31-2插入突变发生在前C区,另外3个突变均发生在C区,与优势株相比平均同源性97%以上. 结论:在rhIFN-α高剂量条件选择下,能检测到HepG2. 2.15HBV前C/C基因插入或缺失突变株,突变株与优势株相比基因序列同源性平均在97%以上.AIM: To investigate the mutation of hepatitis B virus (HBV) with the treated with interferon (IFN)-α2b. METHODS: Different doses of IFN-α2b were added to the HepG2.2.15 cell culture. The α-fetoprotein (AFP) in the supernatant was assayed by Western blot. HBV DNA was isolated from the culture medium. HBV C gene amplified by PCR was cloned by T-A cloning into pGEM-T easy vector. The recombinatant was sequenced with T7/SP6 primer, and analyzed by DNA software. RESULTS: AFP in the culture medium was detected, which was not reduced after IFN-α treatment. There were two subtypes of preC/C gene in the control group. The homogeneity was 93%, with 10 amino acids different, 6 of which were of identical characteristics. The same subtypes were found in the experiment group. The proportion of the dominant strain gene was altered in the high dose IFN group. There were 4 insertion/deletion mutations in the high dose IFN experiment group, which resulted in reading frame shift. The mean homogeneity was more than 97%. CONCLUSION: HBV pre-C/C gene insertion/deletion mu- tants exist in HepG2.2.15 cells treated with high dose IFN-α. Compared with the dominant strain, the mean homogeneity of the mutant strain was more than 97%.

关 键 词：乙型肝炎病毒 变异机制 HEPG2.2.15细胞 Western 乙肝病毒(HBV) rhIFN-α 细胞上清 基因组DNA HBVC基因 聚合酶联反应 前C/C基因 blot 干扰素-α 序列同源性 AFP 实验组 氨基酸 高剂量 不同浓度 突变机制 体外研究 测序分析 

分 类 号：R373.21[医药卫生—病原生物学]