SARS病毒S1基因的表达及其ELISA检测方法的建立

发布时间：2018-05-21 03:35

  本文选题：SARS + S1　； 参考：《河北师范大学》2006年硕士论文

【摘要】：SARS冠状病毒主要蛋白质包括RNA聚合酶蛋白、S蛋白、E蛋白、N蛋白等。其中S蛋白是负责病毒侵染的主要部分,S蛋白的差异可导致病毒宿主的更迭,并可使宿主产生感冒、腹膜炎、肠胃炎等多种疾病。S蛋白同时也是影响冠状病毒侵染宿主程度的主要成分。本文主要研究了SARS冠状病毒S1基因的原核表达,并对表达蛋白进行初步纯化,利用纯化的重组蛋白建立了检测SARS抗体的ELISA方法。另外还构建了S1基因的真核表达载体。为今后核酸疫苗的研制奠定了基础。 首先以含有S1基因的质粒为模板,对S1基因进行PCR扩增,将扩增的基因片段克隆到pGEM T-Easy载体,经双酶切后进行序列测定和同源性分析。在此基础上构建了S1基因的重组表达质粒pGEX-4T1-S1,转化宿主菌BL21(DE3)。表达的S1蛋白经SDS-PAGE分析,在56KD处有一特异表达条带。激光密度扫描结果表明,融合蛋白占细菌总蛋白的34.36%。经Western blot检测,表达的蛋白能够被SARS阳性血清所识别,为SARS病毒感染诊断和SARS抗体检测方法的建立奠定了基础。 将表达的S1蛋白初步纯化后作为抗原,建立了检测SARS病毒抗体的间接ELISA方法。对ELISA的反应条件进行了研究,结果表明蛋白抗原的最适包被浓度为5.0μg/ml,最适包被条件选37℃孵育一小时然后4℃过夜,HRP-山羊抗人IgG的工作浓度为1:400。其特异性较好,患者血清未与正常血清发生交叉反应。建立的间接ELISA为我国进行SARS病毒的诊断和血清学检测提供了一种技术手段,并为SARS检测试剂盒的研制与开发奠定了前期工作基础。 最后以含有S1基因质粒为模板,对S1基因进行PCR扩增,序列分析正确后构建了真核表达质粒pVAX-S1,转染Hela细胞后的免疫荧光检测结果表明重组质粒在体外细胞中得到表达,为SARS DNA疫苗的研究奠定了一定的基础。
[Abstract]:The main proteins of SARS coronavirus include RNA polymerase protein, S protein, protein E protein, protein N, etc. Among them, S protein is the main part responsible for virus infection. The difference of S protein can lead to the change of virus host, and can make the host produce colds and peritonitis. Many diseases, such as gastroenteritis, are also the main components of coronavirus infection. In this paper, the prokaryotic expression of S1 gene of SARS coronavirus was studied, and the expressed protein was preliminarily purified. A ELISA method for detection of SARS antibody was established by using the purified recombinant protein. In addition, the eukaryotic expression vector of S1 gene was constructed. It lays a foundation for the development of nucleic acid vaccine in the future. Firstly, the S1 gene was amplified by PCR using the plasmid containing S1 gene as the template. The amplified gene fragment was cloned into the pGEM T-Easy vector and sequenced and homologous analyzed by double enzyme digestion. On this basis, the recombinant expression plasmid pGEX-4T1-S1 of S1 gene was constructed, and the host strain BL21DE3 was transformed. The expressed S1 protein was analyzed by SDS-PAGE and there was a specific expression band in 56KD. The results of laser density scanning showed that the fusion protein accounted for 34.36% of the total bacterial protein. The expressed protein could be recognized by SARS positive serum by Western blot detection, which laid a foundation for the diagnosis of SARS virus infection and the establishment of SARS antibody detection method. The expressed S1 protein was initially purified as antigen, and an indirect ELISA method for detection of SARS virus antibody was established. The reaction conditions of ELISA were studied. The results showed that the optimal coating concentration of protein antigen was 5.0 渭 g / ml, and the optimal coating conditions were 37 鈩，

本文编号：1917584