HBx基因缺失突变对HBV复制和转录的影响

Effect of absence of HBx expression on HBV replication and transcription

目的构建HBx基因缺失突变质粒pUC-HBV1.0.X7,研究HBx缺失对HBV复制和转录的影响。方法以包含HBV全长基因组的野生型质粒pUC-HBV1.0为模板,用定点突变技术构建HBx基因缺失突变质粒pUC-HBV1.0.X7,并酶切和DNA测序鉴定。用SapⅠ单酶切这两种质粒,获得线性的野生型和HBx缺失突变型HBV基因组,经转染试剂PolyJetTMReagent介导分别瞬时转染到HepG2细胞中。在转染后各时间点Western blot检测HBx蛋白的表达,Southern blot和Real-time PCR同时检测细胞质DNA复制和细胞核cccDNA的合成,Real-time PCR分析定量HBV前基因组RNA(pgRNA)的转录。结果在野生组中可检测到HBx蛋白的表达,而在HBx突变组中HBx蛋白的表达低于Western blot的检测范围。两组中各时间点合成的cccDNA水平无统计学差异(P>0.05)。而在转染后96 hHBx突变组中细胞质DNA的复制和pgRNA的转录都分别比野生组减少50%～70%(P<0.05)。结论 HBx缺失虽然不影响细胞核HBV cccDNA的合成,但是它能下调细胞质HBV DNA的复制和pgRNA的转录。

Objective To construct the HBx-defective mutant plasmid pUC-HBV1.0. X7, and investigate the effect of the absence of HBx on HBV replication and transcription. Methods The wild-type plasmid pUC-HBV1.0 which containing full-length HBV genome was used as a template, and the plasmid pUC- HBV1.0. ）（7 with a stop codon at amino acid 7 of HBx was constructed by site-directed mutagenesis, which was then identified by Hind Ⅲ and Sac I restriction digestion and DNA sequencing. Then the plasmids pUC- HBV1.0 and pUC-HBV1.0. X7 were both digested by Sap I enzyme to obtain linear HBV DNA, and the two kinds of linear HBV DNA were separately transfected into HepG2 cells with PolyJetTM Reagent. The expression of HBx was detected by Western blotting. Cytoplasmic HBV DNA and cccDNA were both detected by Southern blotting and real-time PCR. The transcription of HBV pgRNA was quantified by real-time PCR at different time points after transfection. Results The HBx-deficient plasmid pUC-HBV1.0. ）（7 was verified to be constructed successfully by double digestion and DNA sequence analysis. The expression of HBx was detected in the group of wild-type, while it was below the detection limit by Western blotting in the HBx mutant. The levels of cccDNA formation were not significantly different in the wide type HBx and HBx mutant （ P 〉 0.05 ）. The levels of cytoplasmic HBV DNA and pgRNA in the HBx mutant were reduced by 50% to 70% respectively compared to those in the wild type HBx at 96 h （P 〈 0.05）. Conclusion Although the absence of HBx does not affect cccDNA formation, it can downregulate HBV replication and transcription.