一种乙肝表面抗原结合蛋白（SBP）的原核分泌型表达、纯化及其作用机理的初步研究

发布时间：2018-03-01 15:03

本文关键词： 乙肝表面抗原结合蛋白乙肝病毒 Fc受体分泌型表达　出处：《复旦大学》2010年硕士论文　论文类型：学位论文

【摘要】：乙型肝炎(hepatitis B, HB)是世界范围内流行的一种严重危害人类健康的疾病,由乙型肝炎病毒(HBV, hepatitis B virus)引起.我国是乙肝高流行区,乙肝疫苗是预防感染乙型肝炎和降低乙肝病毒携带率的最有效措施。但是,某些个体在按标准程序接种乙肝疫苗后不能产生足够的抗体以抵抗乙肝病毒的感染. HBsAg-BP (Hepatitis B surface antigen binding protein, SBP)是本室以血清来源的乙肝表面抗原(HBsAg)为探针,通过印迹免疫技术从人肝cDNA噬菌体表达库中筛选获得,其特征是可以与乙肝表面抗原HBsAg特异性的相互作用. 本文分两部分,第一部分,将SBP表达框克隆到大肠杆菌分泌型表达载体pET22b中,获得能够分泌表达SBP的BL21转化菌株；对培养工艺进行了优化,发现SBP表达菌株在IPTG浓度为0.5mM的LB培养基中低温诱导12小时,达到最佳表达水平；在最优条件下诱导表达SBP并破菌得到周质蛋白,通过亲和层析的方法纯化获得了一定量的重组SBP蛋白,ELISA法证实重组SBP具有与HBsAg特异性结合的能力,计算了二者间亲和常数。第二部分,探究SBP细胞表面受体以及SBP促进乙肝表面抗原与细胞结合的作用机理,并构建了红色荧光示踪的DsRed-SBP融合蛋白为今后SBP作用机理的研究提供有利的条件。通过ELISA法证实DsRed-SBP具有与HBsAg特异性结合的能力,荧光定量检测证明发光强度与蛋白含量的线性关系。通过流式细胞试验和Western-blot试验,结果显示SBP的结合受体为CD64,CD32,FcRn等受体,其中FcRn是较为主要的SBP细胞表面受体。通过荧光共聚焦试验和流式细胞仪检测,说明SBP和HBsAg共同结合之后可能形成共作用体发挥作用。ELISA试验证实,封闭CD64,CD32,FcRn等Fc受体后,可以阻断肝细胞对乙肝病毒的吸附作用。
[Abstract]:Hepatitis B virus (hepatitis B HB) is a worldwide epidemic of a serious hazard to human health disease, hepatitis B virus (HBV hepatitis, B virus). China is a high prevalence of hepatitis B, hepatitis B vaccine to prevent infection of hepatitis B and hepatitis B virus carrying the most effective measures to reduce the rate of strip but. Some individuals, according to the standard procedures in inoculation of hepatitis B vaccine cannot produce enough antibodies against hepatitis B virus infection.
HBsAg-BP (Hepatitis B surface antigen binding protein, SBP) is the room with the hepatitis B surface antigen (HBsAg) serum derived probe by immune blot from human liver cDNA phage expression library was screened, which is characterized by interacting with hepatitis B surface antigen specific HBsAg.
This paper is divided into two parts, the first part, the SBP expression box was cloned into Escherichia coli expression vector pET22b, get the expression and secretion of SBP BL21 strain; on training process was optimized, showed that the expression of SBP strain in IPTG concentration was 0.5mM LB medium low temperature induced 12 hours, to achieve the best expression under the optimal conditions; induced expression of SBP and break the bacterial periplasmic protein, purified by affinity chromatography to obtain a certain amount of recombinant SBP protein, ELISA assay confirmed that the recombinant SBP binding ability and specificity of HBsAg, among the two affinity constants were calculated.
The second part, the mechanism of SBP cell surface receptors and SBP promote the integration of hepatitis B surface antigen and cell, and constructed to provide favorable conditions to study the red fluorescent labeled DsRed-SBP fusion protein in SBP mechanism. By the method of ELISA DsRed-SBP confirmed the binding ability with HBsAg specifically, proof of the luminous intensity and fluorescent quantitative detection the protein content of the linear relationship. By flow cytometry test and Western-blot test, the results showed that SBP receptor CD64, CD32, FcRn receptor, wherein FcRn is SBP cell surface receptors are mainly through fluorescence confocal assay and flow cytometry, indicating that SBP and HBsAg together with the total effect may be formed after the body play a role in.ELISA tests, CD64 CD32, FcRn closed, Fc receptor, can block the liver cells of hepatitis B virus adsorption.

【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392.1

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