本文關(guān)鍵詞：快速診斷和分型診斷漢坦病毒基因工程重組抗原研究　出處：《青島大學》2005年碩士論文　論文類型：學位論文

  更多相關(guān)文章： 漢坦病毒 腎綜合征出血熱 重組核蛋白 免疫印跡法

【摘要】：①目的:在大腸桿菌中克隆和表達漢灘病毒S基因的截短片段,型特異性抗原漢灘型和漢城型,表達的重組蛋白可用于漢坦病毒的快速診斷和分型診斷。②方法:從一名急性期HFRS病人外周血中提取漢坦病毒的RNA為模板,以特異性的引物逆轉(zhuǎn)錄獲得相應的cDNA,經(jīng)套式PCR擴增出漢坦病毒S片段部分編碼基因,插入T-A載體后進行核苷酸測序,將獲得的序列使用NCBI Blast軟件對排分析證實為漢灘型,申請GenBank號:AY425612。對該序列所編碼的部分核衣殼蛋白進行抗原性分析,證實其具有組、型特異性抗原決定簇,這些決定簇均為非構(gòu)象依賴性。人工合成漢灘型S基因733-936bp片段,以漢灘病毒截短核蛋白的重組質(zhì)粒為模板,PCR擴增得到漢灘病毒S基因1-249bp片段,PCR連接兩片段,得漢灘型特異性抗原基因,同時人工合成漢城型739-951bpS基因片段。將上述基因分別克隆到原核表達載體pGEX4T-2中,重組質(zhì)粒轉(zhuǎn)化宿主菌DH5α,經(jīng)IPTG誘導表達目的融合蛋白,蛋白純化后用SDS-PAGE與Western blotting鑒定其抗原性和特異性。③結(jié)果:獲得表達漢坦病毒,漢灘病毒和漢城病毒抗原的三種重組菌,Western blotting鑒定表明重組蛋白有良好的抗原性和特異性,可用于漢坦病毒快速診斷和分型診斷。④結(jié)論:在原核表達系統(tǒng)中成功重組表達了漢坦病毒,漢灘病毒和漢城病毒抗原,三種蛋白在漢坦病毒的診斷中具有一定的價值。
[Abstract]:Objective: to clone and express the truncated fragment of Hantaan virus S gene, type specific antigen Hantan and Seoul type in Escherichia coli. The expressed recombinant protein can be used for rapid diagnosis and typing diagnosis of Hantavirus. Methods: RNA of Hantavirus was extracted from peripheral blood of an acute HFRS patient as template. The corresponding cDNA was obtained by reverse transcription with specific primers. The partial coding gene of Hantavirus S fragment was amplified by nested PCR and inserted into T-A vector for nucleotide sequencing. The sequence was confirmed as Hantan type by NCBI Blast software. Application GenBank: AY425612. the antigenicity analysis of some nucleocapsid proteins encoded by this sequence confirmed that the nucleocapsid protein had a group, type specific antigen determinant. These determinants were conformation-independent. A 733-936bp fragment of Hantaan S gene was synthesized and the recombinant plasmid of truncated nucleoprotein of Hantaan virus was used as template. The 1-249bp fragment of Hantaan virus S gene was amplified by PCR and ligated into two fragments, and the Hantaan type specific antigen gene was obtained. The above genes were cloned into prokaryotic expression vector pGEX4T-2, and the recombinant plasmid was transformed into host strain DH5 偽. The target fusion protein was induced by IPTG. After purification, the antigenicity and specificity of Hantavirus were identified by SDS-PAGE and Western blotting. The identification of three recombinant strains of Hantaan virus and Seoul virus by Western blotting showed that the recombinant protein had good antigenicity and specificity. Conclusion: Hantavirus, Hantavirus and Seoul virus antigens were successfully expressed in prokaryotic expression system. The three proteins are valuable in the diagnosis of Hantavirus.
【學位授予單位】：青島大學
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：R392

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