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为从全基因组水平研究乙型肝炎病毒 (HBV)的核苷酸结构及复制和抗原表达特性 ,分别从 2例HBsAg和HBeAg阳性、HBVDNA滴度为 10 14 和 10 13 拷贝 /ml的慢性乙型肝炎患者 (编号为 5 6和 2 18)血清中扩增、克隆了HBV全基因组 ,并测定了核苷酸序列。两株病毒基因组转染HepG2细胞的培养上清中HBsAg表达水平基本相同 ,但 # 5 6毒株表达的HBeAg约为 # 2 18株的 3倍 ,Southern印迹及核酸杂交显示 ,# 5 6HBV株复制效率显著高于 # 2 18株 ,两株HBVDNA全长均为 32 15个核苷酸 ,同源性为 98 7% ,均为B基因型 (adw亚型 )。两者相差的42个核苷酸分布在C、Pre S1、Pre S2、P及X基因编码区 ,其中有位于SPⅠ、SPⅡ、Xp和ENHⅠ基因调控序列区中。 # 5 6患者感染的毒株基因组中Pre S2起始密码ATG变异为GTG ,但由Pre S1起始翻译形成的大蛋白中包括Pre S2的部分氨基酸 ,故 # 5 6血清和 # 5 6株转染细胞的培养上清仍可与Pre S2抗体出现阳性反应。
In order to study the nucleotide structure, replication and antigen expression of hepatitis B virus (HBV) from genome-wide level, we analyzed the nucleotide and amino acid sequences of two hepatitis B virus (HBsAg) positive and HBeAg positive HBVDNA titer of 10 14 and 10 13 copies / Hepatitis patients (numbered 56 and 218) were amplified in serum and the entire genome of HBV was cloned, and the nucleotide sequence was determined. The expression levels of HBsAg in the supernatant of HepG2 cells transfected by the two virus genomes were basically the same, but the # 5 6 strains expressed about 3 times as much HBeAg as the # 2 18 strain. Southern blotting and nucleic acid hybridization showed that the # 5 6HBV strain replicated The efficiency of HBVDNA was 32 15 nucleotides in length, with a homology of 98.7%. All of them were genotype B (adw subtype). The difference of 42 nucleotides is located in the C, Pre S1, Pre S2, P and X gene coding regions, which are located in the SP Ⅰ, SP Ⅱ, Xp and ENH Ⅰ gene regulatory region. # 5 6 The PreS2 start codon in the genome of a patient infected with the ATG variant is GTG, but the partial protein formed by the initial translation of Pre S1 includes a part of the amino acids of Pre S2, so # 5 6 serum and # 5 6 strains Cell culture supernatants were still positive with Pre S2 antibody.