SARS冠状病毒S蛋白表位的筛

发布时间：2018-01-29 09:34

  本文关键词： SARS-CoV S蛋白 基因表达 融合蛋白 合成肽 免疫活性　出处：《南华大学》2005年硕士论文　论文类型：学位论文

【摘要】：目的:分析筛选SARS冠状病毒(SARS associated coronavirus,SARS-CoV)S蛋白的抗原表位,分别采用人工合成抗原肽和基因表达方法获取抗原蛋白并对其免疫原性和免疫反应性进行分析,为诊断SARS-CoV的早期感染、排查疑似病例和大规模流行病学调查的检测试剂盒的制备奠定实验基础。 方法:通过生物信息学方法分析SARS-Cov主要结构蛋白,用多参数预测其B细胞表位,从S蛋白中筛选出保守、特异的表位,在S1和S2蛋白部分各合成一段均为28aa的多肽(peptide1和peptide2);同时合成S1部分一段801bp(304aa～571aa)的基因片段克隆到pUCm-T载体,转化大肠杆菌JM109,蓝白筛选挑取阳性重组子pUCm-T/SARS-S1进行限制性内切酶酶切、PCR及测序鉴定,并对测序结果用Blast软件进行分析。pUCm-T/SARS-S1经限制性内切酶BamH Ⅰ、Sal Ⅰ双酶切,回收酶切目的片段并定向克隆至原核细胞表达载体pET-28b(+),PCR、双酶切和通用引物测序鉴定。重组子pET-28b/SARS-S1转化E.coli ril后,以1mMIPTG诱导表达,并用SDS-PAGE和Western blot鉴定表达产物(rSARS-S1),经三种方法洗涤包涵体后,采用Ni-NTA Spin kit对沉淀中的融合蛋白在变性条件下进行纯化,用急性期或恢复期SARS-CoV患者血清检测产物的免疫反应性。将获得的抗原免疫新西兰兔制备其相应多克隆抗体并纯化,用ELISA和Western blot鉴定其识别peptide1、SARS-S1表达蛋白、SARS-CoV及其裂解产物的特异性。
[Abstract]:Objective: to analyze and screen the epitopes of SARS-CoV S protein of SARS coronavirus. The antigen protein was obtained by synthetic peptide and gene expression, and its immunogenicity and immunoreactivity were analyzed in order to diagnose the early infection of SARS-CoV. The preparation of test kit for screening suspected cases and large-scale epidemiological investigation laid the experimental foundation. Methods: the main structural proteins of SARS-Cov were analyzed by bioinformatics, and their B cell epitopes were predicted by multiple parameters. Conservative and specific epitopes were screened from S protein. One peptide, peptide1 and peptide2, were synthesized in S1 and S2 proteins respectively. At the same time, the gene fragment of S1 was cloned into pUCm-T vector and transformed into Escherichia coli JM109. The positive recombinant pUCm-T/SARS-S1 was selected by blue and white screening for restriction endonuclease restriction endonuclease digestion and sequencing. The sequencing results were analyzed by Blast software. PUCm-T / SARS-S1 was digested by restriction endonuclease BamH 鈪，

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