S基因显性阴性突变体干扰乙肝病毒颗粒组装的研究

The study of interfering on HBV particle assembly in S gene dominant negative mutant

目的 探讨S基因突变对HBV病毒颗粒组装的影响。方法 构建野生型S基因、S基因突变的真核表达载体pcDNA3 S和pcDNA3 mS及S基因突变的全长HBV基因组表达载体pHBV mS ,用pHBV mS与pcDNA3 S共转染HepG2细胞 ,pHBV mS与pcDNA3共转染HepG2细胞 ,以adwR9与pcDNA3共转染为对照 ;pcDNA3 mS和adwR9共转染HepG2细胞 ,以adwR9和pcDNA3共转染为对照 ,用荧光定量PCR检测胞内和上清中病毒量 ,用ELISA方法检测上清中S抗原。结果 pHBV mS与pcDNA3共转染组上清中病毒量较对照组低而胞内病毒量二者相同。pHBV mS与pcDNA3 S共转染组上清、胞内病毒量与对照组相同。pcDNA3 mS和adwR9共转染HepG2细胞其上清中病毒量较对照组低而胞内病毒量两者相同。上清中S抗原实验组较对照组低。结论 S突变体干扰HBV病毒颗粒的组装 ,导致的HBV病毒颗粒分泌下降。

Objective To investigate the effect on assembly of HBV particle in S gene dominant negative mutant. Methods The S mutation vectors were constructed through the molecular clone and PCR based on site directed mutagenesis in vitro, and then transfected transiently the cell HepG2. Western blot assay and ELISA quantitatively evaluated the express of S protein. HBV DNA was quantitatively evaluated by PCR. Results Full length of mutant HBV gene, which expressed S mutated protein, was transfected in HepG2 cell lines. Its virus load was lower than that of wild HBV control group. Virus load of both groups was same S protein of single S gene mutant cotransfected with wild HBV was less than that of control group. Virus load of single S gene mutant cotransfected with wild HBV was lower than that of wild HBV in the culture medium. Virus load of both groups was same in the cell. Conclusion S mutant could interfere the assembly of HBV particle and cause reduce secretion of HBV particle.