两株乙型肝炎病毒基因组结构分析及其复制和抗原表达特性的比较

Functional Comparison of Two Full-length Genomes of Hepatitis B Virus Strains Isolated from Two Chronic Hepatitis B Patients

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。

For studying the characterisitcs of gemomic structures, replication and antigen expression of hepatitis B virus (HBV) strains, the full-length HBV genomes from sera of two chronic hepatitis B patients with high level of serum HBV DNA (10 14 and 10 13 copies/ml respectively) were separately cloned. The full-length genomes of the two HBV strains were sequenced and used to transfect HepG2 cells separately. The HBsAg expressed in the supernatant of transfected HepG2 cells was similar between the two strains, but the level of HBeAg of strain #56 was three times higher than that of strain #2-18 Replication efficiency of HBV strain #56 was significantly higher than that of strain #2-18 detected by Southern blot and hybridization anlaysis of intracelluar viral particles and extracellular replicative intermediates. Both genomes were 3215 nucleotides in length, belonged to genotype B (adw subtype), and were of 98.7% homology. The 42 diverse nucleotides distributed in the C, Pre-S1, Pre-S2, P and X genes and in Sp I, SpII, Xp and ENH I regulatory regions. Since HBV strain #56 had a point mutation at Pre-S2 initial codon (from ATG to GTG), the major protein translated from Pre-S1 contained partial amino acids of Pre-S2, therefore serum sample #56 and the supernatant of transfected HepG2 cells reacted positively with the Pre-S2 antibodies.