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目的 探讨包膜蛋白突变对HBV包装的影响。方法 构建包膜蛋白突变的全长HBV基因组表达载体:前S1突变HBV表达载体pHBV mS1、Sloop突变HBV表达载体pHBV mS和前S1、S共突变HBV表达载体pHBV mS1S。分别转染HepG2细胞,以野生型HBV质粒 (adwR9 )转染HepG2细胞为对照;pHBV mS1S和pcDNA3分别与adwR9共转染HepG2细胞。ELISA方法检测上清液中和胞内S抗原;荧光定量PCR检测胞内和上清液中病毒量。结果 突变体pHBV mS1、pHBV mS和pHBV mS1S与对照组adwR9中S蛋白表达量和分泌量无明显差别;单独转染的突变体胞内病毒量较adwR9高,尤其以pHBV mS1S明显,而突变体上清中病毒量较对照组adwR9低,尤其以pHBV mS1S明显;pHBV mS1S与adwR9共转染组上清液中病毒量较pcDNA3与adwR9共转染组低。结论包膜蛋白突变对S蛋白表达量和分泌量无影响,但影响病毒包装,使病毒分泌量下降。
Objective To investigate the effect of envelope protein mutation on HBV packaging. Methods The full - length HBV genome expression vector with envelope protein mutation was constructed. The former S1 mutant HBV expression vector pHBV mS1, the Sloop mutant HBV expression vector pHBV mS and the former S1 and S common mutation HBV expression vector pHBV mS1S. HepG2 cells were transfected with HepG2 cells, respectively, and HepG2 cells were transfected with wild-type HBV plasmid (adwR9). HepG2 cells were co-transfected with pcDNA3 and pHBV mS1S respectively. ELISA method to detect the supernatant and intracellular S antigen; fluorescence quantitative PCR detection of intracellular and supernatant virus. Results There was no significant difference in S protein expression and secretion between the mutants pHBV mS1, pHBV mS and pHBV mS1S and the control group adwR9. The intracellular virus levels of mutants transfected alone were higher than those of adwR9, especially at pHBV mS1S, The amount of virus in supernatant was lower than that of adwR9, especially at pHBV mS1S. The amount of virus in supernatant of co-transfection group of pHBV mS1S and adwR9 was lower than that of pcDNA3 and adwR9. Conclusion The mutation of envelope protein has no effect on the expression and secretion of S protein, but affects the viral packaging and decreases the secretion of virus.