HCV“截短型”核心蛋白（HCV Core125）的表达纯化及抗体的制备

发布时间：2018-06-19 16:10

本文选题：HCV + 核心蛋白　；参考：《昆明理工大学》2012年硕士论文

【摘要】：丙型肝炎病毒(Hepatitis C virus, HCV)是一种RNA病毒,主要为血液传播,可造成急、慢性病毒性肝炎,并导致慢性进行性肝炎、肝硬化(Hepatocellular cirrhosis, HC和肝细胞癌(Hepatocellular carcinoma, HCC)。全球约有1.7-2亿人感染HCV,中国有近4千万人感染HCV,属于HCV流行比较严重的国家之一。由于尚无预防性疫苗和有效治愈的药物,对HCV进行早期诊断,对控制其传播就显得尤为重要。目前HCV检测技术主要包括抗体检测、核酸检测、核心抗原检测。HCV抗体检测一般存在7-10周的窗口期,且病毒有6种主要的基因型和超过50种亚型易造成漏检。HCV核酸检测虽然能有效的缩短窗口期,但因RT-PCR操作复杂,容易污染且价格昂贵,使之不能广泛应用。HCV核心抗原感染后2-3天就能检测到,有效的缩短了窗口期,但核心抗原在血液里的含量极其低,高灵敏度检测天然HCV core抗原的匹配抗体是建立HCV core抗原诊断试剂的关键和难点。鉴于此,本研究的目的在于表达并纯化HCV core蛋白,并制备出高效价的抗体,以期望能够用于建立高灵敏度HCV core抗原诊断试剂盒。我们通过对HCV core蛋白基因序列的分析,发现HCV Core蛋白N端的前120个左右氨基酸构成了主要的亲水结构域,这段也几乎包括了HCV Core蛋白的所有保守性B细胞表位,而C端富含疏水性氨基酸不利于表达和纯化。因此,研究选择HCV core蛋白125aa (HCV core125)作为目的蛋白,以期获得高的表达量并能够产生高效价的抗体。首先设计1b基因型特异性引物,对HCV核心蛋白前375bp (125aa)的基因片段进行扩增,将Core125基因片段克隆到PET28a表达质粒,成功地构建了重组表达载体PET28a-Core125。重组原核表达菌在1.Omm IPTG进行诱导下,Core125蛋白(21kD左右)得到了大量的表达,主要以包涵体的形式存在。Ni2+-NTA亲和层析柱纯化的包涵体Core125蛋白,分子量大小约为21kD,与预期结果一致,但存在分子量约为30kD的杂蛋白,再经过Strep-Tactin系统亲和层析纯化后,得到高纯度目的蛋白。以感染不同基因型HCV的病人血浆为结合抗体,对纯化后的Core125蛋白经过Western blot检测,结果显示纯化的Core125蛋白能够与HCV几种基因型和亚型(la,lb,3a,3b,6a,6n,6u)病人血清反应,说明此蛋白具有良好的抗原性及基因型间检测通用性。用Core125蛋白免疫新西兰大白兔,以间接ELISA法检测产生的多克隆抗体血清效价达到1：1024000。多克隆抗体血清经过Western blot鉴定,显示了其具有很好的特异性。用Core125蛋白免疫BALB/C小鼠后,以间接ELISA检测血清效价达到1：51200,显示获得了很好的免疫效果,可作为筛选获得阳性杂交瘤细胞的B细胞供体。但由于单克隆抗体的制备中细胞融合率较低,未能成功制备单抗。本论文成功表达了截短型的HCV核心蛋白(Core125),获得了高纯度的目的蛋白,纯化的Core125蛋白能够与不同HCV基因型病人血清反应,显示了此蛋白具有良好的抗原性及基因型间检测通用性；制备出的Core125蛋白多克隆抗体具有高的效价,为HCV抗原检测提高其灵敏度的研究奠定了基础；另外,本研究获得Core125蛋白的多克隆抗体,并初步进行小鼠免疫以获得阳性杂交瘤细胞,为制备Core125蛋白的单克隆抗体做了准备工作,为HCV核心抗原检测试剂的开发奠定了前期工作基础。
[Abstract]:Hepatitis C virus (Hepatitis C virus, HCV) is a RNA virus, mainly for blood transmission, can cause acute, chronic viral hepatitis, and lead to chronic progressive hepatitis, liver cirrhosis (Hepatocellular cirrhosis, HC and hepatocellular carcinoma (Hepatocellular carcinoma, HCC). There are about 40 million people in the world, and nearly 40 million people in China are infected, " HCV is one of the most popular countries. Because there is no preventive vaccine and effective cure, the early diagnosis of HCV is very important to control its transmission. At present, HCV detection technology mainly includes antibody detection, nucleic acid detection, and core antigen detection of.HCV antibody detection generally has 7-10 weeks window period, and the virus has 6 The main genotypes and more than 50 subtypes can easily lead to a leak detection.HCV nucleic acid detection, although it can effectively shorten the window period, but because of the complexity of the RT-PCR operation, easy pollution and high price, it can not be detected at 2-3 days after the widespread application of.HCV core antigen infection, effectively shortening the window period, but the core antigen in the blood content extremely It is the key and difficult point to detect the matched antibody of natural HCV core antigen with low and high sensitivity. In view of this, the purpose of this study is to express and purify the HCV core protein and prepare the high efficient antibody, so as to be expected to be used for the establishment of a high sensitivity HCV core antigen diagnostic kit.
By analyzing the sequence of the HCV core protein gene, we found that the first 120 amino acids of the N terminal of the Core protein form the main hydrophilic domain. This segment also almost includes all the conserved B cell epitopes of the HCV Core protein, and the C terminal rich in hydrophobic amino acids is not beneficial to the epitope and purification of the B. Therefore, the HCV core protein 125A is studied. A (HCV core125) is used as the target protein to obtain high expression and produce high effective antibody. First, the specific primers of 1B genotype were designed to amplify the gene fragment of 375bp (125aa) before the core protein of HCV, and the Core125 gene fragment was cloned into the PET28a expression plasmid, and the recombinant expression vector PET28a-Core125. was successfully constructed. The Recombinant Prokaryotic Expression bacteria were induced by 1.Omm IPTG, and the Core125 protein (21kD) was expressed in a large amount. The inclusion body Core125 protein was purified mainly in the form of inclusion body. The molecular weight was about 21kD, which was consistent with the expected result, but there was a protein with a molecular weight of about 30kD, and then through Strep-Tactin. High purity target protein was obtained after purification by system affinity chromatography. The plasma of infected patients with different genotype HCV was combined with antibody, and the purified Core125 protein was detected by Western blot. The results showed that the purified Core125 protein could react with the sera of several HCV genotypes and subtypes of HCV (LA, LB, 3a, 3b, 6a, 6N, and 6N). Good antigenicity and genotypes are used to detect generality.
Core125 protein was used to immunize New Zealand white rabbits. The serum titer of polyclonal antibody was detected by indirect ELISA method. The serum titer of 1:1024000. polyclonal antibody was identified by Western blot. It showed that the serum titer was good specificity. After immunizing BALB/C mice with Core125 protein, the serum titer of indirect ELISA was reached to 1:51200. Good immune effect can be used as a B cell donor for screening positive hybridoma cells. However, the McAb has not been successfully prepared because of low cell fusion rate in the preparation of monoclonal antibodies.
In this paper, a truncated HCV core protein (Core125) was successfully expressed and a highly purified target protein was obtained. The purified Core125 protein could react with the sera of different HCV genotypes, showing that the protein has good antigenicity and generality between genotypes, and the prepared Core125 protein polyclonal antibody has high potency. The study laid the foundation for the study of HCV antigen detection to improve its sensitivity. In addition, this study obtained the polyclonal antibody of Core125 protein and preliminarily immunized the mice to obtain positive hybridoma cells, prepared the monoclonal antibody for the preparation of Core125 protein, and laid a preliminary working basis for the development of the HCV core antigen detection reagent. Foundation.
【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R373.21

【共引文献】