摘要
目的 探讨乙型肝炎病毒前C信号肽酶裂解位点变异后乙型肝炎病毒e抗原(HBeAg)表达的改变。 方法 以聚合酶链反应结合限制性片段长度多态性 (PCR RFLP)方法筛检前C信号肽酶裂解位点变异的病例 ,经PCR扩增出编码HBeAg的前C/C区基因并克隆于EB病毒真核表达载体 (EBO) ,以脂质体介导方法将变异株与野株分别转染HepG2细胞 ,利用SEAP报告系统监测转染效率 ,用酶联免疫吸附方法 (ELISA)和免疫印迹 (Westernblot)方法检测转染不同病毒株的细胞外和细胞内HBeAg的量。 结果 转染野株的细胞培养上清经ELISA方法可检测到HBeAg阳性 ;而转染变异株的细胞培养上清中HBeAg为阴性。培养细胞内部以Westernblot方法检测发现野株可表达三种蛋白 ,即HBeAg(分子量 170 0 0 )和两种HBeAg前体 (2 2 0 0 0和 2 5 0 0 0 ) ,而变异株只能检测到两种HBeAg前体 ,且变异株的HBeAg前体密度带明显比野株强。 结论 乙型肝炎病毒前C信号肽酶裂解位点变异后可能影响HBeAg的翻译后加工 ,导致大量的HBeAg前体在细胞中蓄积 ,这可能导致HBV感染者血清HBeAg阴性。
Objective To study HBeAg change in patients infected hepatitis B virus(HBV) with pre-C signal enzyme cleavage site mutation. Methods Mutation in pre-C signal enzyme cleavage site was detected by PCR-RFLP. The PreC/C gene with mutation was amplified by PCR and was cloned to EB viral eukarotic expression vector. Then transfect the vector with wild type or mutant PreC/C gene to HepG2 cell. SEAP reporter system was used to monitor the efficiency of transfection. HBeAg and its precursor in the supernatant and HepG2 cell were detected by ELISA and Western blot. Results HBeAg was positive in the supernatant of wild type and negative control in T1862 vaniant by ELISA. In HepG2 cell transfected with wild type, three proteins were detected by Western blot, they were HBeAg(17 000) and two HBeAg precursor(22 000 and 25 000). And in HepG2 cell transfected T1862 vaniant, only two HBeAg precursor was detected. The precursor in cells transfected withT1862 vaniant were significantly stronger than cells transfected with wild type. Conclusion Mutation in pre-C signal enzyme cleavage site may affect the decoration of HBeAg, which may cause great of HBeAg precursor locating in cells and lead to HBeAg negative in serum of patients infected with HBV.
出处
《中华检验医学杂志》
CAS
CSCD
北大核心
2004年第1期17-19,共3页
Chinese Journal of Laboratory Medicine
基金
国家自然科学基金资助项目 (3 963 0 2 0 80 )
军队医药卫生重点课题资助项目 (96Z0 2 4)
