乙型肝炎病毒C基因变异株的体外构建及其生物学意义

Construction of mutant gene of HBV and investigating its biological significance

目的 了解我国乙型肝炎病毒 (HBV)C区变异的生物学意义。方法 采用酶切位点消除法定点突变构建HBVC基因变异株 (V6 0、G87、L97)真核细胞表达载体 ;转染HepG2 细胞用ELISA法检测各毒株宿主细胞上清液中HBeAg。 结果 与野生毒株相比 ,构建的变异株前C/C区碱基除目的突变点外 ,其他碱基均同源。在重组质粒转染的宿主细胞中检测到目的DNA片段。L97变异株宿主细胞上清液HBeAg的A值在不同时间点均高于野生株和其他变异株 (P <0 .0 0 1) ;G87宿主细胞上清液中HBeAg的A值均为阴性 ,但浓缩 10倍后阳性 ;V6 0宿主细胞上清液中HBeAg的A值与野生株差异无显著性。结论 HBV前C/C区V6 0。

Objective To study the biological significance of the common mutant preC/C gene in clinical HBV in China. Methods Site directed mutagenesis based on the unique enzyme site elimination was used to construct eukaryocyte expression vector with mutant HBV/C gene(V60、G87、L97). Expression vectors with wild and mutant preC/C gene were transferred into HepG2 cell. Culture supernatant was detected by ELISA for HBeAg. Results Result of DNA sequencing showed that the constructed mutant HBV preC/C gene had only one specific site variation compared with the wild type sequence. Goal DNA fragment was detected positive in the HepG2 cells transferred with wild and mutant preC/C gene. A value of HBeAg in the supernatant of the cells harboring L97 variant was higher than that of the wild and other variant strains( P <0.001). However, A value of HBeAg in G87 was negative which resumed positive in the 10 times concentrated supernatant. While in V60, A value had no significant difference from that of the wild strain. Conclusion The variant strains of V60, G87 and L97 in HBV preC/C region might have different HBeAg secretion.