快速诊断和分型诊断汉坦病毒基因工程重组抗原研究

发布时间：2018-01-01 11:28

本文关键词：快速诊断和分型诊断汉坦病毒基因工程重组抗原研究　出处：《青岛大学》2005年硕士论文　论文类型：学位论文

【摘要】：①目的:在大肠杆菌中克隆和表达汉滩病毒S基因的截短片段,型特异性抗原汉滩型和汉城型,表达的重组蛋白可用于汉坦病毒的快速诊断和分型诊断。②方法:从一名急性期HFRS病人外周血中提取汉坦病毒的RNA为模板,以特异性的引物逆转录获得相应的cDNA,经套式PCR扩增出汉坦病毒S片段部分编码基因,插入T-A载体后进行核苷酸测序,将获得的序列使用NCBI Blast软件对排分析证实为汉滩型,申请GenBank号:AY425612。对该序列所编码的部分核衣壳蛋白进行抗原性分析,证实其具有组、型特异性抗原决定簇,这些决定簇均为非构象依赖性。人工合成汉滩型S基因733-936bp片段,以汉滩病毒截短核蛋白的重组质粒为模板,PCR扩增得到汉滩病毒S基因1-249bp片段,PCR连接两片段,得汉滩型特异性抗原基因,同时人工合成汉城型739-951bpS基因片段。将上述基因分别克隆到原核表达载体pGEX4T-2中,重组质粒转化宿主菌DH5α,经IPTG诱导表达目的融合蛋白,蛋白纯化后用SDS-PAGE与Western blotting鉴定其抗原性和特异性。③结果:获得表达汉坦病毒,汉滩病毒和汉城病毒抗原的三种重组菌,Western blotting鉴定表明重组蛋白有良好的抗原性和特异性,可用于汉坦病毒快速诊断和分型诊断。④结论:在原核表达系统中成功重组表达了汉坦病毒,汉滩病毒和汉城病毒抗原,三种蛋白在汉坦病毒的诊断中具有一定的价值。
[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 伪. 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.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392

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