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目的研究乙型肝炎病毒(HBV):RNA上La蛋白结合位点的缺失对S基因mRNA在细胞内稳定性的影响,评价HBV RNA上这个位点对HBV生命周期的重要性,从而寻找新的抗HBV靶点。方法构建HBV突变载体, 使其转录出来的HBV RNA缺失La蛋白结合位点;计算机预测突变后这个位点所在的HBV RNA片段的二级结构;将未突变的HBV载体和HBV突变载体分别瞬时转染HepG2细胞;半定量逆转录聚合酶链反应法检测HBV S基因的 mRNA,酶联免疫吸附法检测乙型肝炎表面抗原。结果酶切鉴定和测序证明,成功构建了La蛋白结合相关位点部分碱基缺失的HBV突变载体——pHBV—mLa;突变后的HBV RNA二级结构同突变前的比较,完全改变;转染细胞后半定量逆转录聚合酶链反应法检测发现,突变后HBV S基因的mRNA水平显著降低(t’=12．703,P<0．05);酶联免疫吸附法检测发现突变引起乙型肝炎表面抗原表达明显下调(f=4_4．648,P<0．01)。结论HBV RNA上La蛋白的结合位点以及局部特殊二级结构的形成,影响着自身转录后的稳定性,对于HBV的生命周期至关重要。
Objective To study the effect of hepatitis B virus (HBV) deficiency of La protein binding site on the intracellular stability of S gene mRNA in order to evaluate the importance of this site on HBV life cycle in HBV RNA, Anti-HBV target. Methods The HBV mutated vector was constructed so that the HBV RNA transcribed from it lacked the binding site of La protein. The secondary structure of the HBV RNA fragment at the site of this mutation was predicted by computer. The unmutated HBV vector and the HBV mutated vector were transiently transfected HepG2 cells were infected with HepG2 cells. The mRNA of HBV S gene was detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), and HBsAg was detected by enzyme-linked immunosorbent assay. Results The results of restriction enzyme digestion and DNA sequencing showed that the HBV mutated vector -pHBV-mLa with partial base deletion at the site of La protein binding was constructed successfully. The secondary structure of the mutated HBV RNA was completely changed compared with that before mutation, Semi-quantitative reverse transcriptase-polymerase chain reaction assay showed that the mRNA level of HBV S gene was significantly decreased (t ’= 12.703, P <0.05) after mutation, and the mutation was found to be induced by enzyme-linked immunosorbent assay Hepatitis surface antigen expression was significantly down-regulated (f = 4_4.648, P <0.01). Conclusion The binding site of La protein and the formation of special secondary structure in HBV RNA affect the post-transcriptional stability of HBV RNA and are crucial for the life cycle of HBV.