SARS-CoV主要结构蛋白基因共表达质粒的构建及其免疫原性研究

发布时间：2019-06-20 09:23

【摘要】： 目的选择编码SARS冠状病毒(SARS coronavirus, SARS-CoV)刺突蛋白(Spike protein, S)和核衣壳蛋白(Nucleocapsid protein, N)的基因,并以鼠粒细胞-巨噬细胞集落刺激因子(mGM-CSF)为基因佐剂,构建S1蛋白和N蛋白、S1蛋白和mGM-CSF、N蛋白和mGM-CSF共表达的三种质粒,并观测它们接种小鼠后的免疫效果,为SARS DNA疫苗的研制奠定基础。方法从灭活的SARS-CoV中提取总RNA,RT-PCR扩增编码S1蛋白和N蛋白的基因片段,PCR扩增编码mGM-CSF的基因片段,经测序正确后分别克隆或亚克隆到真核表达载体pVAX1上,构建质粒并命名为pVAX1/S1-N, pVAX1/S1-mGM-CSF和pVAX1/N-mGM-CSF。三种共表达质粒分别转染COS-7细胞,用免疫细胞化学和Western Blot方法鉴定表达的融合蛋白。采用pVAX1/S1-N与mGM-CSF质粒共免疫,另两种共表达质粒单独免疫小鼠的接种策略, ELISA法检测SARS特异性IgG抗体及IgG亚类的含量,流式细胞仪检测T细胞亚群,ELISPOT检测脾细胞中特异性IFN-γ和IL-2分泌细胞的频数。结果构建出pVAX1/S1-N, pVAX1/S1-mGM-CSF和pVAX1/N-mGM-CSF三种共表达质粒,经DNA序列分析和双酶切鉴定证实质粒构建正确,并均能在COS-7细胞内表达。三种共表达质粒均能诱导小鼠机体产生SARS-CoV特异性IgG,且其滴度随免疫次数的加强而逐步增高,IgG亚类测定显示IgG2a/IgG1的比值均1;三组免疫小鼠的CD4+和CD8+T细胞数量均显著增加(P0.01),而CD4+/CD8+T细胞的比值均明显降低(P0.01),脾细胞中特异性IFN-γ和IL-2分泌细胞的频数均明显增高(P0.01)。三种共表达质粒中,pVAX1/S1-N与mGM-CSF质粒共免疫组的免疫效果又明显优于另两种共
[Abstract]:Objective to select the genes encoding SARS coronavirus (SARS coronavirus, SARS-CoV prickle protein (Spike protein, S) and nucleocapsid protein (Nucleocapsid protein, N), and to construct three kinds of plasmids co-expressed in S1 protein and N protein, S1 protein, mGM-CSF,N protein and mGM-CSF by using mouse granulocyte macrophage colony stimulating factor (mGM-CSF) as gene adjuvant, and to observe their immune effect after inoculating mice. It lays a foundation for the development of SARS DNA vaccine. Methods Total RNA,RT-PCR gene fragments encoding S1 protein and N protein were extracted from inactivated SARS-CoV. The gene fragments encoding mGM-CSF were amplified by PCR and cloned into eukaryotic expression vector pVAX1 or subcloned into eukaryotic expression vector pVAX1. The plasmid was constructed and named pVAX1/S1-N, pVAX1/S1-mGM-CSF and pVAX1/N-mGM-CSF.. The three co-expression plasmids were transfected into COS-7 cells, and the expressed fusion proteins were identified by immunocytochemistry and Western Blot. The co-immunization strategy of pVAX1/S1-N with mGM-CSF plasmid and the other two co-expression plasmids were used to immunize mice. The contents of SARS specific IgG antibody and IgG subclass were detected by ELISA method, T cell subsets were detected by flow cytometry, and the frequency of specific IFN- 纬 and IL-2 secretory cells in spleen cells were detected by ELISPOT. Results three kinds of co-expression plasmids pVAX1/S1-N, pVAX1/S1-mGM-CSF and pVAX1/N-mGM-CSF were constructed. DNA sequence analysis and double enzyme digestion confirmed that the parenchyma was constructed correctly and all of them could be expressed in COS-7 cells. All three co-expression plasmids could induce the production of SARS-CoV specific IgG, in mice, and their titers increased gradually with the increase of immunization times. IgG subclass assay showed that the ratio of IgG2a/IgG1 was 1. The number of CD4 and CD8 T cells in the three groups was significantly increased (P01), while the ratio of CD4 / CD8 T cells was significantly decreased (P01), and the frequency of specific IFN- 纬 and IL-2 secretory cells in spleen cells was significantly increased (P01). Among the three co-expression plasmids, the immune effect of the co-immunized group of pVAX1/S1-N and mGM-CSF plasmid was significantly better than that of the other two kinds of co-expression plasmid.
【学位授予单位】：宁夏医学院
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R373

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