【摘要】： 目的選擇編碼SARS冠狀病毒(SARS coronavirus, SARS-CoV)刺突蛋白(Spike protein, S)和核衣殼蛋白(Nucleocapsid protein, N)的基因,并以鼠粒細胞-巨噬細胞集落刺激因子(mGM-CSF)為基因佐劑,構(gòu)建S1蛋白和N蛋白、S1蛋白和mGM-CSF、N蛋白和mGM-CSF共表達的三種質(zhì)粒,并觀測它們接種小鼠后的免疫效果,為SARS DNA疫苗的研制奠定基礎(chǔ)。 方法從滅活的SARS-CoV中提取總RNA,RT-PCR擴增編碼S1蛋白和N蛋白的基因片段,PCR擴增編碼mGM-CSF的基因片段,經(jīng)測序正確后分別克隆或亞克隆到真核表達載體pVAX1上,構(gòu)建質(zhì)粒并命名為pVAX1/S1-N, pVAX1/S1-mGM-CSF和pVAX1/N-mGM-CSF。三種共表達質(zhì)粒分別轉(zhuǎn)染COS-7細胞,用免疫細胞化學(xué)和Western Blot方法鑒定表達的融合蛋白。采用pVAX1/S1-N與mGM-CSF質(zhì)粒共免疫,另兩種共表達質(zhì)粒單獨免疫小鼠的接種策略, ELISA法檢測SARS特異性IgG抗體及IgG亞類的含量,流式細胞儀檢測T細胞亞群,ELISPOT檢測脾細胞中特異性IFN-γ和IL-2分泌細胞的頻數(shù)。 結(jié)果構(gòu)建出pVAX1/S1-N, pVAX1/S1-mGM-CSF和pVAX1/N-mGM-CSF三種共表達質(zhì)粒,經(jīng)DNA序列分析和雙酶切鑒定證實質(zhì)粒構(gòu)建正確,并均能在COS-7細胞內(nèi)表達。三種共表達質(zhì)粒均能誘導(dǎo)小鼠機體產(chǎn)生SARS-CoV特異性IgG,且其滴度隨免疫次數(shù)的加強而逐步增高,IgG亞類測定顯示IgG2a/IgG1的比值均1;三組免疫小鼠的CD4+和CD8+T細胞數(shù)量均顯著增加(P0.01),而CD4+/CD8+T細胞的比值均明顯降低(P0.01),脾細胞中特異性IFN-γ和IL-2分泌細胞的頻數(shù)均明顯增高(P0.01)。三種共表達質(zhì)粒中,pVAX1/S1-N與mGM-CSF質(zhì)粒共免疫組的免疫效果又明顯優(yōu)于另兩種共
[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- 緯 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- 緯 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.
【學(xué)位授予單位】：寧夏醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R373

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