本文關(guān)鍵詞： 乙肝表面抗原結(jié)合蛋白 乙肝病毒 Fc受體 分泌型表達　出處：《復(fù)旦大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】：乙型肝炎(hepatitis B, HB)是世界范圍內(nèi)流行的一種嚴(yán)重危害人類健康的疾病,由乙型肝炎病毒(HBV, hepatitis B virus)引起.我國是乙肝高流行區(qū),乙肝疫苗是預(yù)防感染乙型肝炎和降低乙肝病毒攜帶率的最有效措施。但是,某些個體在按標(biāo)準(zhǔn)程序接種乙肝疫苗后不能產(chǎn)生足夠的抗體以抵抗乙肝病毒的感染. HBsAg-BP (Hepatitis B surface antigen binding protein, SBP)是本室以血清來源的乙肝表面抗原(HBsAg)為探針,通過印跡免疫技術(shù)從人肝cDNA噬菌體表達庫中篩選獲得,其特征是可以與乙肝表面抗原HBsAg特異性的相互作用. 本文分兩部分,第一部分,將SBP表達框克隆到大腸桿菌分泌型表達載體pET22b中,獲得能夠分泌表達SBP的BL21轉(zhuǎn)化菌株；對培養(yǎng)工藝進行了優(yōu)化,發(fā)現(xiàn)SBP表達菌株在IPTG濃度為0.5mM的LB培養(yǎng)基中低溫誘導(dǎo)12小時,達到最佳表達水平；在最優(yōu)條件下誘導(dǎo)表達SBP并破菌得到周質(zhì)蛋白,通過親和層析的方法純化獲得了一定量的重組SBP蛋白,ELISA法證實重組SBP具有與HBsAg特異性結(jié)合的能力,計算了二者間親和常數(shù)。 第二部分,探究SBP細(xì)胞表面受體以及SBP促進乙肝表面抗原與細(xì)胞結(jié)合的作用機理,并構(gòu)建了紅色熒光示蹤的DsRed-SBP融合蛋白為今后SBP作用機理的研究提供有利的條件。通過ELISA法證實DsRed-SBP具有與HBsAg特異性結(jié)合的能力,熒光定量檢測證明發(fā)光強度與蛋白含量的線性關(guān)系。通過流式細(xì)胞試驗和Western-blot試驗,結(jié)果顯示SBP的結(jié)合受體為CD64,CD32,FcRn等受體,其中FcRn是較為主要的SBP細(xì)胞表面受體。通過熒光共聚焦試驗和流式細(xì)胞儀檢測,說明SBP和HBsAg共同結(jié)合之后可能形成共作用體發(fā)揮作用。ELISA試驗證實,封閉CD64,CD32,FcRn等Fc受體后,可以阻斷肝細(xì)胞對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.

【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392.1

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相關(guān)期刊論文 前1條

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本文編號：1552419