E645R变异MXA蛋白抗HBV复制体外研究

目的研究E645R变异MXA蛋白抑制乙型肝炎病毒(hepatitis B virus,HBV)复制活性。方法对pcDNA3.1-MXA重组质粒采用定点突变构建E645R变异MXA蛋白表达重组质粒pcDNA3.1-MXA-E645R。将pcDNA3.1-MXA(MXA组),pcDNA3.1-MXA-E645R(E645R组)和pcDNA3.1空质粒(对照组)分别与PU19-1.24HBV重组质粒按2∶1比例瞬时共转染HepG2细胞,转染3d后检测MXA蛋白表达和各组细胞上清HBsAg和HBeAg的量以及上清和细胞内HBVDNA水平。结果酶切和测序表明定点突变成功构建pcDNA3.1-MXA-E645R重组质粒;MXA、E645R转染组有较好的MXA蛋白表达。E645R组与MXA组HBsAg较对照组分别下降23%和20%(P>0.05),E645R组和MXA组HBeAg较对照组分别下降61%和66%(P<0.05)。E645R组与MXA组上清HBVDNA水平较对照组分别下降1.9个和2.2个log值,细胞内HBVDNA水平均下降1.7个log值。结论E645R变异MXA蛋白具有较好的抗HBV活性,E645R变异不影响MXA蛋白抑制HBV复制。

In vitro investigation on the influence of interferon-inducible mutant E645R on the inhibitory effect of MXA protein upon the replication of hepatitis B virus

To investigate on the influence of interferon-inducible mutant E645R on the inhibitory effect of MXA protein upon the replication of Hepatitis B virus(HBV),the expression vector pcDNA3 1-MXA-E645R was constructed by site-mutagenesis.The plasmid pcDNA3 1-MXA(MXA group),pcDNA3 1-MXA-E645R(E645R group)and pcDNA3 1 empty plasmid(control group)were co-transfected with PU19-1,24 HBV in a proportion of 2∶1 to the HepG2 cells. Three days after transfection,the expression of the MXA protein was detected by Western blotting and lasar confocal microscopy;the amounts of HBsAg and HBeAg in the cell culture and supernatants were measured by Abbott analysis and HBV DNA levels in the supernatants and cells were determined by real-time PCR. It was demonstrated that the pcDNA-3 1-MAX-E645R recombinant plasmid was successively constructed by site mutagenesis as revealed by enzyme digestion and sequencing. As demonstrated by Western blotting and confocal microscopy,the MXA protein was expressed robustly in HepG2 cells in MXA and E645R groups. Compared with the control group,the amount of HBsAg in supernatants of E645R and MXA groups was decreased by 23% and 20% respectively, while those of HBeAg were decreased by 61% and 66% respectively,in which this showed a marked difference with that of the control group.Furthermore,in comparison with the control group,the extracellular HBV DNA in MAX and E645R groups were decreased by 1.9 logs and 2.2 logs respectively;while the intracellular HBV DNA were decreased by 1.7 logs in both groups. From these observations,it is clear that the MXA protein of the mutant E645R shows an anti-viral activity against HBV,but the E645R mutation does not influence the interferon-induced MXA protein to inhibit the replication of HBV.