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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 investigate the change of hepatitis B virus e antigen (HBeAg) expression after hepatitis B virus pre-C signal peptide cleavage site mutation. Methods The cases of pre-C signal peptide cleavage site mutation were screened by polymerase chain reaction and restriction fragment length polymorphism (PCR RFLP). The pre-C / C region gene encoding HBeAg was amplified by PCR and cloned EBV eukaryotic expression vector (EBO). The mutant and wild strain were transfected into HepG2 cells by liposome-mediated method. The transfection efficiency was monitored by SEAP reporter system. The expression of EBV was detected by enzyme-linked immunosorbent assay (ELISA) Westernblot) method was used to detect the extracellular and intracellular HBeAg transfection of different virus strains. Results The HBeAg positive cells were detected by ELISA in the supernatant of the transfected wild-type cells, but negative in the cell culture supernatant of the transfected mutant. The results of Western blot showed that the wild type strain could express three proteins, HBeAg (170 000) and two HBeAg precursors (2200 and 25000), while the mutants could only detect To the two HBeAg precursors, and mutant strains of HBeAg precursor density band was significantly stronger than wild plants. Conclusion The variation of pre-C signal peptide cleavage site of hepatitis B virus may affect the post-translational processing of HBeAg, resulting in the accumulation of a large number of HBeAg precursors in the cells, which may result in the serum HBeAg-negative in HBV-infected patients.