β2糖蛋白I与乙型肝炎表面抗原亲合力的研究

发布时间：2018-04-22 01:00

  本文选题：β2糖蛋白I + 乙型肝炎病毒　； 参考：《吉林大学》2009年硕士论文

【摘要】： 乙型肝炎病毒(HBV)是一种嗜肝脱氧核糖核酸病毒,目前全球约有4亿人群感染HBV。多年来学者对HBV感染肝细胞的早期过程进行研究,提出了多种观点并对可能的靶细胞受体进行鉴定,但仍无定论。β2糖蛋白I(β2-GPI)又称为载脂蛋白H,是血浆中一种含量较丰富的糖蛋白。近年研究认为,β2-GPI是抗磷脂抗体综合征(APS)的主要抗原之一。APS是与抗磷脂抗体(aPL)密切相关,是以血栓形成、习惯性流产和血小板减少等症状为特点的一组综合征。β2-GPI与aPL结合后能识别带负电荷的磷脂复合物,并使β2-GPI的表面结构发生变化,使机体易形成血栓。随着研究的拓展,Mehdi发现重组乙型肝炎表面抗原(rHBsAg)可与正常人肝细胞膜通过β2-GPI在膜外结合,表明β2-GPI可能参与HBV感染肝细胞。本研究组深入研究后提出,β2-GPI可能作为中介分子,与HBsAg形成复合物,再与肝细胞膜表面的特异性蛋白结合,从而介导HBV入肝过程。研究组已从肝癌细胞株SMMC-7721表面经鉴定出结合蛋白为膜联蛋白Ⅱ。在本研究中,我们将着重探讨β2-GPI与HBsAg特异性结合的亲和力及二者相互作用的机制,为上述观点提供更有力的实验依据,为今后的研究奠定基础。本研究利用大肠杆菌M15(pQE30-hβ2-GPI)高效表达目的蛋白,通过亲和层析纯化带有融合6个组氨酸标签的β2-GPI包涵体,并进行复性。原核系统表达的目的蛋白具有无糖基化修饰的特点,故这种rβ2-GPI可用于研究分子构象对蛋白结合作用的影响。我们首次利用放射性免疫法(radioimmunoassay,RIA)测定人β2GPI与rHBsAg结合的亲和常数(Ka),以此推断血浆中β2GPI与HBsAg的亲和力,并通过亲和常数比较与原核系统表达的rβ2-GPI免疫活性的差异。梯度浓度的两组来源不同的β2GPI分别与125I-rHBsAg结合,利用标准曲线Adrion法分别测得两组蛋白的Ka。采用嵌套实验设计进行数据分析,血浆中提取的β2-GPI组与原核系统表达的rβ2-GPI组的Ka无明显统计学差异(P0.05)。最终求得血浆人β2-GPI组:Ka1=(2.795±1.846)×10~8 L/ mol。结果表明血浆β2-GPI与HBsAg结合的亲和力较强,且血浆中提取的β2-GPI与原核系统表达的rβ2-GPI与rHBsAg的结合力相似。我们得出结论,血浆中β2-GPI与HBsAg亲和力强,可能在血浆中二者较易形成复合物,且二者的结合与β2GPI的糖基化结构无关,即糖基化与否不影响β2-GPI的免疫活性。因此,β2-GPI无论发生何种构象改变,均不影响其与HBsAg的结合。这种结合可能参与介导HBV嗜肝过程,甚至参与肝细胞癌的发生发展。
[Abstract]:Hepatitis B virus (HBV) is a hepatophilic deoxyribonucleic acid virus. At present, about 400 million people worldwide are infected with HBV. Over the years, researchers have studied the early process of HBV infection in hepatocytes, put forward a variety of viewpoints and identified possible target cell receptors. 尾 2 glycoprotein I (尾 2 GPI), also known as apolipoprotein H, is a rich glycoprotein in plasma. In recent years, it has been considered that 尾 2-GPI is one of the major antigens of anti-phospholipid antibody syndrome (APSs). APS is closely related to anti-phospholipid antibody (2-GPI). A group of syndromes characterized by habitual abortion and thrombocytopenia. 尾 2-GPI combined with aPL can recognize the phospholipid complex with negative charge and change the surface structure of 尾 2-GPI and make the body easily form thrombosis. With the development of the study, Mehdi found that recombinant hepatitis B surface antigen (HBs) could bind to normal human hepatocytes through 尾 2-GPI, suggesting that 尾 2-GPI may be involved in the infection of hepatocytes with HBV. This study suggests that 尾 2-GPI may act as a mediator, form a complex with HBsAg, and then bind to specific proteins on the surface of the hepatocyte membrane, thus mediating the process of HBV entering the liver. The binding protein was identified as integrin 鈪，

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