HCV核心蛋白单克隆抗体制备及其与载脂蛋白A1、转位蛋白相互作用

发布时间：2018-05-22 11:40

本文选题：丙型肝炎病毒 + 核心蛋白　；参考：《安徽理工大学》2008年硕士论文

【摘要】： 目的:本实验构建丙型肝炎核心蛋白(HCV core)原核表达载体,在大肠埃希菌中进行表达、并纯化融合蛋白;制备抗core蛋白单克隆抗体并进行鉴定。研究HCVcore在肝源性细胞系中与载脂蛋白AⅠ(apoAⅠ)、转位蛋白(Translin)相互作用,为丙型肝炎病毒引起的脂肪肝和肝癌的分子机制提供理论基础。方法:构建原核表达载体pET-32a(+)HCV-core,转化大肠埃希菌BL21中,以IPTG诱导获得core融合蛋白。利用Ni~+亲和柱对表达蛋白进行纯化及上柱复性。用纯化蛋白免疫BALB/c小鼠,取小鼠脾细胞与骨髓瘤细胞融合,获得抗HCV-core蛋白的单克隆抗体。利用Western blot、ELISA法和免疫组化对单克隆抗体进行特异性和灵敏度分析及鉴定。构建真核表达质粒:pDNA3.1-myc-his-apoAⅠ、pDNA3.1-myc-his-Translin、pDNA3.1(-)core、pact-apoAⅠ、pact-Translin和pBIND-core,共转染到HepG2细胞中。用免疫共沉淀(CoIP)和哺乳动物双杂交的方法,Westren blot分析免疫共沉淀杂交带,用Dual Luciferase Reporter AssaySystem检测荧光素酶强度。结果:1:HCV核心蛋白基因PCR产物及连接到pET32a(+)中,经双酶切和DNA测序鉴定,成功构建pET-32a(+)-core,HCV-core融合蛋白表达成功。通过Ni~+亲和柱对表达蛋白进行纯化,获得core蛋白(42KD)。用纯化蛋白常规免疫BALB/c小鼠,并鼠尾静脉取血。用ELISA检测免疫后小鼠血清出现抗core抗体,并且血清抗体滴度成逐渐增高趋势。Western blot证明血清抗体能检测到HCV肝硬化患者肝组织中core蛋白,进一步证明我们制备的core融合蛋白与天然存在的core蛋白结构基本一致。用脾细胞与骨髓瘤细胞融合,细胞融合率59.2%。用ELISA和Westren blot筛选单克隆抗体株阳性率6.3%。有限稀释再次筛选共获得稳定分泌抗体的杂交瘤细胞株。用ELISA测定其效价,上清效价最高的细胞株是4E10。以4E10细胞上清作为一抗,Western blot检测纯化抗原可检测到8ng的抗原;免疫组织化学发现:在HCV肝癌组织中,HCVcore主要位于肝细胞胞浆中,呈灶性或弥漫性分布。 2:共转染pDNA3.1-myc-his apoAⅠ和pDNA3.1(-)HCVcore进入HepG2细胞中,经免疫共沉后Western blot结果显示:单独core蛋白的杂交带相对分子量大概19kDa,共沉淀带相对分子量大约50kDa。所构建的真核载体能在HepG2细胞中表达,并且HCVcore和apoA1二种蛋白能相互结合。而且也用pDNA3.1-myc-hisapoAⅠ和pDNA3.1(-)HCVcore单独转染入HepG2细胞中,每种质粒都能在细胞中过表达,且不与HepG2细胞内其它蛋白结合。pACT-apoAⅠ和pBIND-core共转染时,相对荧光素酶活性值较pACT和pBIND空载体转染组以及pACT-apoA1和pBIND空载体、pACT空载体和pBIND-core转染组明显升高(12～16倍)。表明HCVcore和apoAⅠ在体内能相互结合,且单独转染后没有自身激活作用。 3:共转染pDNA3.1-myc-his Translin和pDNA3.1(-)HCVcore进入HepG2细胞中,经免疫共沉后Western blot结果显示:单独core蛋白的杂交带相对分子量大概19kDa,共沉淀带只出现Translin蛋白杂交带相对分子量大约21.8kDa。pACT-Translin和pBIND-core共转染时,相对荧光素酶活性值较其他组明显升高(6～8倍)。表明HCVcore和Translin在体内能相互结合。结论:成功表达、纯化HCV-core基因融合蛋白,获得高特异性、高效价鼠抗HCV-core单克隆抗体,为研究core基因的生物学功能提供了新的手段。HCV-core可与apoAⅠ、Translin在体内相互作用,为慢性丙型肝炎致肝脂肪变和肝癌的机制的研究提供理论依据。
[Abstract]:Objective: to construct the prokaryotic expression vector of hepatitis C core protein (HCV core), to express in Escherichia coli and to purify the fusion protein; to prepare monoclonal antibodies against core protein and to identify it. The interaction of HCVcore in the hepatogenic cell line with apolipoprotein A I (apoA I) and transposition protein (Translin) was used as a hepatitis C virus. The molecular mechanism of virus induced fatty liver and liver cancer provides a theoretical basis.
Methods: the prokaryotic expression vector, pET-32a (+) HCV-core, was transformed into the Escherichia coli BL21, and the core fusion protein was induced by IPTG. The expression protein was purified by the Ni~+ affinity column. The purified protein was used to immunize the BALB/c mice and the murine splenocytes were fused with the myeloma cells to obtain the monoclonal antibody against the HCV-core protein. The specificity and sensitivity of the monoclonal antibodies were analyzed and identified by Western blot, ELISA and immunohistochemistry. The eukaryotic expression plasmids were constructed: pDNA3.1-myc-his-apoA I, pDNA3.1-myc-his-Translin, pDNA3.1 (-) core, pact-apoA I, pact-Translin and pBIND-core, and were transferred to HepG2 cells. Immunoprecipitation (CoIP) and mammal double were used. The method of hybridization was used to analyze the co immunoprecipitation hybrids with Westren blot, and Dual Luciferase Reporter AssaySystem was used to detect the intensity of luciferase.
Results: the PCR product of 1:HCV core protein gene and its connection to pET32a (+) were identified by double enzyme cutting and DNA sequencing. The pET-32a (+) -core was successfully constructed and the expression of HCV-core fusion protein was successfully expressed. The expression protein was purified by Ni~+ affinity column and core protein (42KD) was obtained. The purified protein was routinely immune to BALB/c mice and the rat tail vein was taken blood. After immunization, the serum anti core antibody was detected and the serum antibody titer increased gradually in.Western blot. The serum antibody showed that the serum antibody could detect the core protein in the liver tissues of the patients with HCV liver cirrhosis, and further demonstrated that the core fusion protein prepared by us was basically consistent with the natural existing core protein structure. The fusion of splenocyte and myeloma cells was the same. The cell fusion rate 59.2%. was selected by ELISA and Westren blot to screen the hybridoma cell line of the monoclonal antibody positive rate 6.3%. to obtain the stable secretory antibody again. The titer was measured by ELISA. The highest titer of the supernatant was 4E10. with the 4E10 cell supernatant as one anti, Western blot detection of the purified antigen could detect 8ng. Immunohistochemistry revealed that in HCV hepatoma tissues, HCVcore was mainly located in the cytoplasm of hepatocytes, with a focal or diffuse distribution.
2: co transfected pDNA3.1-myc-his apoA I and pDNA3.1 (-) HCVcore into HepG2 cells. The results of Western blot after immunization showed that the relative molecular weight of the single core protein hybrid band was about 19kDa, and the eukaryotic carrying capacity of the coprecipitation zone was expressed in the HepG2 cells by the relative molecular weight of approximately 50kDa.. PDNA3.1-myc-hisapoA I and pDNA3.1 (-) HCVcore were also transfected into HepG2 cells separately, each plasmid could be overexpressed in the cell, and the relative luciferase activity value was compared with pACT and pBIND no-load transfection groups and pACT-apoA1 and pBI when the other proteins in the HepG2 cells were co transfected with.PACT-apoA I and pBIND-core. ND no-load, pACT no-load and pBIND-core transfection group were significantly increased (12~16 times). It showed that HCVcore and apoA I could be combined in the body, and there was no self activation after transfection.
3: cotransfected pDNA3.1-myc-his Translin and pDNA3.1 (-) HCVcore into HepG2 cells. The result of Western blot after immunization showed that the relative molecular weight of the single core protein hybrid band was about 19kDa, and the relative molecular weight of the Translin protein hybridization band was only about 21.8kDa.pACT-Translin and co transfection. The activity of enzyme was significantly higher than that of other groups (6~8 times), indicating that HCVcore and Translin could bind to each other in vivo.
Conclusion: successfully expressed, purified HCV-core gene fusion protein and obtained high specificity and high efficiency mouse anti HCV-core monoclonal antibody. It provides a new means to study the biological function of core gene,.HCV-core can interact with apoA I, Translin in the body, and provides a theory for the study of the mechanism of hepatic steatosis and liver cancer induced by chronic hepatitis C inflammation. Basis.
【学位授予单位】：安徽理工大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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