[来自重型乙型肝炎患者血清样本的完整乙肝病毒脱氧核糖核酸克隆及序列]
[Complete HBV DNA clone and sequence from serum samples of severe hepatitis B patients].
作者信息
Wu Wei, Li Lan-juan, Chen Yu, Li Jun, Qian Xiang, Cheng Dong-qing
机构信息
Department of Infectious Diseases, First Hospital of Zhejiang University, Hangzhou 310003, China.
出版信息
Zhonghua Gan Zang Bing Za Zhi. 2005 Oct;13(10):734-7.
OBJECTIVE
To study the association between hepatitis B virus (HBV) mutants and the pathogenesis of severe hepatitis B by full-length HBV genome.
METHODS
Serum samples from 10 severe hepatitis B patients were collected in our hospital. Serum HBV DNAs were extracted using DNA mini Kit, and amplified by LA Taq DNA polymerase to yield full-length HBV DNA. PCR products were isolated and cloned into vector pUCm-T, then transfected into DH-5 alpha cells. Positive clones were selected and checked by digestion, and full-length HBV DNAs were sequenced.
RESULTS
4 cases were cloned into vector pUCm-T successfully and completed the full-length sequencing. Among them, 3 cases had a G to A mutation at nucleotide 1896 in pre-C region and 1 had a double mutation of T1762-A1764 in the core promoter region. Some amino acid changes occurred within the known CTL, B or T cell epitopes of the PrS2 and C regions.
CONCLUSIONS
This method could serve to study the relationship between HBV genome and the pathogenesis of severe hepatitis B.
目的
通过乙肝病毒（HBV）全基因组研究HBV突变体与重型乙型肝炎发病机制之间的关联。
方法
收集我院10例重型乙型肝炎患者的血清样本。使用DNA小提试剂盒提取血清HBV DNA，并用LA Taq DNA聚合酶进行扩增以产生全长HBV DNA。分离PCR产物并克隆到载体pUCm-T中，然后转染到DH-5α细胞中。挑选阳性克隆并通过酶切进行鉴定，对全长HBV DNA进行测序。
结果
4例成功克隆到载体pUCm-T中并完成全长测序。其中，3例在前C区核苷酸1896处发生G到A的突变，1例在核心启动子区发生T1762-A1764的双重突变。在PreS2和C区已知的CTL、B或T细胞表位内发生了一些氨基酸变化。
结论
该方法可用于研究HBV基因组与重型乙型肝炎发病机制之间的关系。
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