慢性乙肝病毒B亚型感染儿童不同HBeAg状态下CP基因变异测定

Detection of mutation in core promoter in children at different status of serum HBeAg with chronic hepatitis B

目的了解HBV基因型B/adw2型的慢性HBV感染儿童不同血清HBeAg状态下HBVC区启动子基因的变异。方法选择HBV基因型为B型/血清型adw2的慢性HBV感染儿童64例,按血清HBeAg不同状态分为血清e抗体阳性组(n=34)和e抗原阳性组(n=30)2组,以HBVDNA为模板行PCR反应扩增S区序列(确定HBV基因型)及C区启动子(CP)基因片断,CP基因片断经胶回收纯化后,应用T-A克隆技术构建重组质粒pGEM-CP,经蓝白斑筛选,阳性克隆以凝胶电泳、双酶切及PCR验证后将鉴定正确的克隆测序,将测序结果与Entrezdata-base中血清亚型及基因型相同的HBV序列相比较并进行分析。结果S区基因测序结果证实64例儿童感染的HBV基因型均为B型/血清型adw2。CP基因测序结果证实2组均有CP基因变异,血清e抗体阳性组CP变异率(29.41%)高于e抗原阳性组(6.67%)(P<0.05)。e抗体阳性组有4个变异热点,而e抗原阳性组无变异热点。结论慢性乙肝病毒B亚型感染儿童存在CP基因变异,且可能与e抗原血清转换有关。

Aim ： To investigate the relationship between the different status of serum HBeAg and mutation in core promoter in children with chronic hepatitis B, who had same genotype B/adw2 of HBV. Methods： A total of 64 children infected with hepatitis B virus （genotype B/adw2 ） were divided into two groups according to status of serum HBeAg. Core promoter was amplified by PCR and cloned into pGEM-T vector with T-A cloning technique. The recombinant plasmid pGEM-CP was confirmed by digestion with restriction enzyme Apa Ⅰ and Sac Ⅰ . The correct clones were selected to be sequenced from each patient. Results： In each group the genotypes of HBV were B/adw2. There was significant difference in mutations of core promoter between the two groups with different serum status of HBeAg（ 29.41% vs 6.67% , P 〈 0.05 ）. There were four hot mutational positions in the HBe antibody positive group. Conclusion ： The mutation of HBV CP is related to HBe seroconversion .