SARS冠状病毒分离鉴定、疫苗构建及免疫血清制备研究

发布时间：2017-12-31 02:37

本文关键词：SARS冠状病毒分离鉴定、疫苗构建及免疫血清制备研究　出处：《吉林大学》2006年博士论文　论文类型：学位论文

【摘要】：本研究进行了吉林省部分SARS病人及与SARS病人密切接触人员血清和漱口液的病毒收集、病毒分离;采用荧光定量RT-PCR和ELISA方法检测了大量临床样品;从2例SARS疑似病例漱口液中分离得到了SARS病毒。人工合成了编码SARS冠状病毒(SARS-CoV)结构蛋白的基因序列,设计合成了内源性免疫刺激序列(ISS),利用上述结构基因与表位基因分别构建了在大肠杆菌(12种质粒)、毕赤酵母(6种质粒)、哺乳动物细胞(6种质粒)中表达的系列重组质粒,并在大肠杆菌中成功表达了重组N蛋白,在毕赤酵母中表达了病毒的N蛋白和突刺S2蛋白。将DNA疫苗系列质粒免疫小鼠,结果显示除含E蛋白基因的rDNA外,含S、S1、S2、M、N蛋白基因的rDNA均能诱导抗SARS-CoV相应抗体,且使鼠脾T淋巴细胞数量均有增加,表明表达S蛋白、S1蛋白、N蛋白的rDNA作为候选疫苗。将大量制备表达S蛋白和N蛋白的rDNA疫苗注射马体,并应用大肠杆菌中表达的SARS-CoV N蛋白加强免疫,获得了高效价相应抗SARS-CoV血清;获得的抗SARS-CoV马血清经硫酸胺沉淀后粗制成抗体IgG,酶切消化,层析,获得抗SARS-CoV抗体IgG Fab_2片段,其纯度达90%以上。
[Abstract]:In this study, virus collection and virus isolation were carried out in serum and mouthwash of some SARS patients and close contacts with SARS patients in Jilin Province. A large number of clinical samples were detected by fluorescence quantitative RT-PCR and ELISA. SARS virus was isolated from gargle of 2 suspected SARS cases. The gene sequence encoding the structural protein of SARS coronavirus (SARS-CoV) was synthesized and the endogenous immunostimulatory sequence (ISS) was designed and synthesized. A series of recombinant plasmids expressed in 12 plasmids of Escherichia coli, 6 plasmids of Pichia pastoris and 6 plasmids of mammalian cells were constructed using these structural genes and epitope genes. The recombinant N protein was successfully expressed in Escherichia coli and the virus N protein and S 2 protein were expressed in Pichia pastoris. The mice were immunized with DNA vaccine series plasmids. The results showed that, in addition to rDNA containing E protein gene, SnS1 + S2 + M was found in mice. RDNA of N protein gene could induce anti SARS-CoV antibody and increase the number of T lymphocytes in spleen of mice, which indicated that S protein S 1 protein was expressed. RDNA of N protein was used as candidate vaccine. A large number of rDNA vaccines expressing S and N proteins were injected into the horse body, and the SARS-CoV N protein expressed in E. coli was used to enhance the immunization. High titer antiserum against SARS-CoV was obtained. The anti SARS-CoV horse serum was precipitated by sulfate-amine. The antibody was digested and digested by enzyme. The IgG Fab_2 fragment of anti SARS-CoV antibody was obtained by enzyme digestion and chromatography. Its purity is over 90%.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R373;R392

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