【作者】 林园园；

【导师】 林菊生；

【作者基本信息】 华中科技大学 ， 内科学， 2006， 硕士

【摘要】 狼疮自身抗原蛋白(the lupus autoantigen protein,La protein,SSB)是一种细胞核浆中的磷蛋白,最初是在系统性红斑狼疮和干燥综合症患者的血清中发现的。它与多种RNA有关系,包括细胞RNA和病毒RNA,涉及到它们的代谢。近年来发现它与HBV RNA也有着密切的关系。【目的】研究HBV RNA上La protein结合位点的缺失对HBV RNA在宿主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】应用PCR,分子克隆和定点突变技术,针对HBV RNA上La protein结合的相关位点,构建HBV突变载体,命名为pHBV-mLa,其转录出来的HBV RNA缺失La protein结合位点;通过计算机预测,突变后这段序列所在的HBV RNA片段的二级结构;应用脂质体将未突变的HBV载体和HBV突变载体分别瞬时转染入HepG2细胞株中;半定量RT-PCR检测各组转染细胞中HBsAg-mRNA和HBeAg-mRNA的水平,ELISA检测各组转染细胞HBsAg和HBeAg表达情况。【结果】酶切鉴定和碱基测序证明,成功构建了La protein结合相关位点部分碱基缺失的HBV突变载体——pHBV-mLa;突变后,在La protein结合位点处的HBV RNA二级结构同突变前比较,完全改变。转染细胞后,半定量RT-PCR检测发现:突变的HBV载体转染组细胞中HBsAg-mRNA和HBeAg-mRNA水平都明显低于未突变HBV载体转染组(t’=12.703,P<0.05,和t’=11.085,P<0.05)。ELISA检测发现:同未突变的HBV载体转染组比较,突变的HBV载体转染组细胞培养液上清中,HBsAg和HBeAg的表达均明显下调(t=44.648,P<0.01和t=42.048,P<0.01)。【结论】由于HBV DNA上的人工突变,所引起的HBV RNA上La protein结合位点相应的改变,使得HBV RNA特殊的二级结构改变,导致La protein与HBV RNA结合松弛、解离,无法保护HBV RNA(HBsAg-mRNA和HBeAg-mRNA),RNA酶就可以引起HBV RNA的降解,从而使HBV RNA水平降低,影响HBV各种蛋白的表达。本实验表明,在HBV DNA上,相应碱基序列的存在和保守使HBV RNA上La protein结合位点可以形成特殊的二级结构,这个结构影响着HBV RNA在宿主细胞内的稳定性,所以HBV RNA上这个位点对HBV生命周期至关重要。更多还原

【Abstract】 The lupus autoantigen protein (La protein) is a nuclear phosphoprotein. It was first identified as an autoantigen in serum of patients suffering from systemic lupus erythematosus and Sjogren’s syndrome. La protein interacts with a large variety of cellular and viral RNAs, and involves in their metabolism. In recent years, some evidences for the association between La protein and HBV RNA were found.【Objective】To investigate the effect of the deletion of La protein binding site in HBV RNA on the stability of HBV RNA in host cell for further study on the significance of the site in HBV life cycle, and searching a new anti-HBV target.【Methods】The HBV expression vectors with mutation related with the La protein binding site were constructed by a molecular clone and PCR based site directed mutagenensis in vitro, named pHBV-mLa. The HBV RNA secondary structure of the site was calculated with the program RNAdraw V1.1b2 by computer. The normal HBV vectors and mutant vectors were respectively transfected into HepG2 cells by Lipofectamineam2000. In two groups, the HBV RNA level were analyzed by semi-quantitative RT-PCR, and HBV antigens secretion into culture media were tested by ELISA.【Results】The HBV vectors with mutation related with the La protein binding site were successfully constructed, it was confirmed by the identification of restriction analysis and sequencing. In La protein binding site, The HBV RNA secondary structure of the mutant vectors was completely different to the stem-loop structure of the normal HBV vectors. After transfection into HepG2 cells, Semi-quantitative RT-PCR and ELISA showed that, both the level of HBsAg-mRNA and HBeAg-mRNA in the mutant vectors group were significantly lower than that in the normal HBV vectors group(t’=12.703, P<0.05, and t’=11.085, P<0.05), and both the expression efficacy of HBsAg and HBeAg in mutant group were reduced(t=44.648,P<0.01 t=42.048,P<0.01), compared with the nomal group.【Conclusion】The change of La protein binding site in HBV RNA due to the mutation in HBV DNA disorganizes the predicated stem-loop structure in the site shared by all HBV RNAs. As a result of the structural change, La protein cannot bind the site and stabilize HBV RNAs, including in HBsAg-mRNA and HBeAg-mRNA, which are exposed the cleavage site in the upstream of the stem-loop structure to endoribonuclease. It contributes to HBV RNA decay and effects expression of HBV gene. This study shows, it is necessary for the formation of the stem-loop structure in La protein binding site that the sequence of this site in HBV DNA is conserved; the disorganization of the stem-loop structure affects the HBV RNA posttranscriptional stability in host cell. La protein binding site in HBV RNA as well as the special secondary structure in the site is crucial to Hepatitis B Virus life cycle.更多还原