乙型肝炎病毒大S蛋白基因纵向研究方法及应用  

Variations in the large surface protein gene of hepatitis B virus in vivo:a longitudinal study and application

作　　者：余南[1] 崔进[1] 周国宝[1] 张云娇[1] 陈晶砺[1] 

机构地区：[1]广州开发区医院生物中心实验室广州开发区医院传染科,广东省广州市510730

出　　处：《世界华人消化杂志》2009年第27期2840-2845,共6页World Chinese Journal of Digestology

基　　金：广州市医药卫生科技基金资助项目;No.2006-YB-196~~

摘　　要：目的:建立乙型肝炎病毒(HBV)大S蛋白基因纵向研究方法,并将其应用于疾病进程中HBV的准种动态研究.方法:以2例HBV感染者(对象A,男,38岁;对象B,女,22岁.对象A研究期间未经任何抗病毒治疗,B则1年持续接受ADV抗病毒药物治疗)体内不同时期的血清样本核酸提取物为模板,通过特异性扩增获取病毒大S蛋白基因序列并克隆测序,依据生物信息学方法对发生于序列结构不同区域的变异进行分析,利用比对数据对不同样本HBV准种予以描述与评价.结果:所得24条HBV大S蛋白基因序列中2条源于对象B样本的基因序列PreS2编码区存在30nt的缺失,其余22条序列大小均为1203bp;24条序列的HBV基因型均为C型.对象A大S蛋白基因序列分散值DA、DB、DC分别为:0.29、0.16、0.23,对象B则为0.80、2.30、1.82.来自对象A的全部10条HBV大S蛋白基因序列内部的"a"决定簇与参考基因完全一致;对象B的14条序列期初有2条(29%)期终有6条(86%)存在G145R突变,期终序列中有1条序列"a"决定簇内发现4个位点的突变.结论:人体内感染HBV以准种形式存在且可以用分散值DA、DB、DC对其进行描述,并应用于HBV准种动态的纵向研究.而抗病毒药物ADV的1年期治疗可使本例HBV准种分散性增高,可能增加本例耐药性突变出现的风险,同时增加本例"a"决定簇的变异出现.AIM：To develop a method for longitudinal study of variations in the large surface protein gene of hepatitis B virus （HBV） in vivo and apply it to the investigation of the dynamics of HBV quasispecies. METHODS：Two HBV carriers were included in the study. Patient 1 was a 38-year-old male who received no anti-virus therapy before and during the longitudinal study,while patient 2 was a 22-year-old female who received continuous adefovir dipivoxil （ADV） therapy during the oneyear longitudinal study. Serum samples were collected from the two patients over the longitudinal period. Nucleic acids were extracted from the serum samples and used as templates to amplify the HBV large surface protein gene. The resulting PCR products were cloned into the pMD18-T vector and sequenced. Variation analysis was performed using sequence alignment tools to characterize and evaluate the dynamic changes of HBV quasispecies. RESULTS：Of 24 large surface protein gene sequences （10 from patient 1 and 14 from patient 2） analyzed,two from patient 2 contained a 30-nt deletion in PreS2 coding region. Online HBV genotype analysis showed that all the 24 sequences belonged to genotype C. The divergence values （DA,DB and DC） of HBV large surface protein gene from patient 1 were 0.29,0.16 and 0.23,respectively,while those from patient 2 were 0.80,2.30 and 1.82,respectively. The ＇a＇ determinant region was perfectly identical among the 10 sequences from patient 1. In patient 2,G145R mutation was found in two sequences （29%） at the beginning and in six sequences （86%） at the end of the study. Four point mutations also were noted in the ＇a＇ determinant region of one sequence from patient 2 at the end of the study. CONCLUSION：Our results support the quasispecies distribution of HBV inside human hosts. It is feasible to use divergence values DA,DB and DC to characterize and evaluate the dynamic changes of HBV quasispecies. ADV therapy may increase the diversity of HBV quasispecies and thereby raise the risk of devel

关 键 词：乙型肝炎病毒 大S蛋白 变异 “a”决定簇 准种 

分 类 号：R512.62[医药卫生—内科学] R450[医药卫生—临床医学]