發(fā)布時(shí)間：2018-01-29 09:34

  本文關(guān)鍵詞： SARS-CoV S蛋白 基因表達(dá) 融合蛋白 合成肽 免疫活性　出處：《南華大學(xué)》2005年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:分析篩選SARS冠狀病毒(SARS associated coronavirus,SARS-CoV)S蛋白的抗原表位,分別采用人工合成抗原肽和基因表達(dá)方法獲取抗原蛋白并對其免疫原性和免疫反應(yīng)性進(jìn)行分析,為診斷SARS-CoV的早期感染、排查疑似病例和大規(guī)模流行病學(xué)調(diào)查的檢測試劑盒的制備奠定實(shí)驗(yàn)基礎(chǔ)。 方法:通過生物信息學(xué)方法分析SARS-Cov主要結(jié)構(gòu)蛋白,用多參數(shù)預(yù)測其B細(xì)胞表位,從S蛋白中篩選出保守、特異的表位,在S1和S2蛋白部分各合成一段均為28aa的多肽(peptide1和peptide2);同時(shí)合成S1部分一段801bp(304aa～571aa)的基因片段克隆到pUCm-T載體,轉(zhuǎn)化大腸桿菌JM109,藍(lán)白篩選挑取陽性重組子pUCm-T/SARS-S1進(jìn)行限制性內(nèi)切酶酶切、PCR及測序鑒定,并對測序結(jié)果用Blast軟件進(jìn)行分析。pUCm-T/SARS-S1經(jīng)限制性內(nèi)切酶BamH Ⅰ、Sal Ⅰ雙酶切,回收酶切目的片段并定向克隆至原核細(xì)胞表達(dá)載體pET-28b(+),PCR、雙酶切和通用引物測序鑒定。重組子pET-28b/SARS-S1轉(zhuǎn)化E.coli ril后,以1mMIPTG誘導(dǎo)表達(dá),并用SDS-PAGE和Western blot鑒定表達(dá)產(chǎn)物(rSARS-S1),經(jīng)三種方法洗滌包涵體后,采用Ni-NTA Spin kit對沉淀中的融合蛋白在變性條件下進(jìn)行純化,用急性期或恢復(fù)期SARS-CoV患者血清檢測產(chǎn)物的免疫反應(yīng)性。將獲得的抗原免疫新西蘭兔制備其相應(yīng)多克隆抗體并純化,用ELISA和Western blot鑒定其識別peptide1、SARS-S1表達(dá)蛋白、SARS-CoV及其裂解產(chǎn)物的特異性。
[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，

本文編號：1473095