启东肝癌高发区乙肝病毒流行株全基因分析

目的：通过对江苏启东地区乙型肝炎病毒（HBV）全基因序列的分析，探讨该地区肝癌高发的分子病毒学病因。方法：以蛋白酶K消化后，酚／氯仿抽提7例肝炎和7例肝癌患者血清中DNA。应用聚合酶链反应（PCR），扩增血清中HBV基因全长，克隆至T载体后行全自动测序。以PHYLIP软件构建的系统进化树判断HBV的基因型；以Clastal W软件对序列进行突变分析。结果：14例标本中有12例为C基因型，2例为B基因型。肝癌和肝炎组中HBV基因型类别分布无差异。有5例HBV发生了PreS2的缺失突变，其中肝癌标本4例（57．1％），肝炎标本仅1例（14．3％）。HBV基因组中常见的点突变为PreS1区的nt．3116及核心启动子区的nt．1762／1764，发生率高达78．6％（11／14）。与肝炎组相比，肝癌中点突变发生率显著增高的位点为前C区nt．1899G→A的突变（P=0．01）及核心启动子区nt．1653C→T的突变（P=0．05）。结论：启东地区HBV以C基因型为主；启东HBV基因组中可能存在着与肝癌相关的点突变和缺失突变。

Full-genome sequence analysis of hepatitis B virus isolated from hepatocellular carcinoma in high incidence area Qidong

Objective:To investigate the genotype of hepatitis B virus(HBV)in Qidong area and further characterize the se- quence properties of HBV isolated from hepatocellular carcinoma(HCC)and chronic hepatitis(CH)patients.Methods:DNA was ex- tracted from serum samples of 7 HCC and 7 CH patients by conventional proteinase K digestion/phenol extraction method.The full-ge- nome of HBV was amplified by polymerase chain reaction(PCR)using serum DNA as the template.PCR product was cloned into T- vector and DNA sequence was obtained by ABI auto-sequencing.The genotypes of HBV were determined by the Phylogenetic tree using PHYLIP software based on the entire HBV sequences and mutation analysis was carried out by the Clastal W software.Results:Among the total 14 HBV strains,12 belonged to genotype C and 2 belonged to genotype B.No significant difference of genotype distribution ex- isted between HBV and HCC groups.Deletion in PreS2 region was observed in 4(57.1%)HCC patients and 1(14.3%)CH patient. The mutational hotspots,which incidence was as high as 78.6%(11/14),were located at nt.3116(PreS1 region)and nt.1762/1764 (core promoter/X region).Compared with HBV isolated from of CH patients,two point mutations,i.e.nt.1899A in PreC region(P= 0.01)and nt.1653T in core promoter/X region(P=0.05),occurred more frequently in HCC patients.Conclusion:The prevalent HBV genotype in Qidong area is genotype C.HCC-related point mutation and deletion of HBV may exist in Qidong genomes.