本文關(guān)鍵詞：SARS冠狀病毒分離鑒定、疫苗構(gòu)建及免疫血清制備研究　出處：《吉林大學(xué)》2006年博士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： SARS冠狀病毒 檢測(cè) 結(jié)構(gòu)蛋白 原核表達(dá) 酵母表達(dá) DNA疫苗 抗血清 IgG

【摘要】：本研究進(jìn)行了吉林省部分SARS病人及與SARS病人密切接觸人員血清和漱口液的病毒收集、病毒分離;采用熒光定量RT-PCR和ELISA方法檢測(cè)了大量臨床樣品;從2例SARS疑似病例漱口液中分離得到了SARS病毒。 人工合成了編碼SARS冠狀病毒(SARS-CoV)結(jié)構(gòu)蛋白的基因序列,設(shè)計(jì)合成了內(nèi)源性免疫刺激序列(ISS),利用上述結(jié)構(gòu)基因與表位基因分別構(gòu)建了在大腸桿菌(12種質(zhì)粒)、畢赤酵母(6種質(zhì)粒)、哺乳動(dòng)物細(xì)胞(6種質(zhì)粒)中表達(dá)的系列重組質(zhì)粒,并在大腸桿菌中成功表達(dá)了重組N蛋白,在畢赤酵母中表達(dá)了病毒的N蛋白和突刺S2蛋白。 將DNA疫苗系列質(zhì)粒免疫小鼠,結(jié)果顯示除含E蛋白基因的rDNA外,含S、S1、S2、M、N蛋白基因的rDNA均能誘導(dǎo)抗SARS-CoV相應(yīng)抗體,且使鼠脾T淋巴細(xì)胞數(shù)量均有增加,表明表達(dá)S蛋白、S1蛋白、N蛋白的rDNA作為候選疫苗。 將大量制備表達(dá)S蛋白和N蛋白的rDNA疫苗注射馬體,并應(yīng)用大腸桿菌中表達(dá)的SARS-CoV N蛋白加強(qiáng)免疫,獲得了高效價(jià)相應(yīng)抗SARS-CoV血清;獲得的抗SARS-CoV馬血清經(jīng)硫酸胺沉淀后粗制成抗體IgG,酶切消化,層析,獲得抗SARS-CoV抗體IgG Fab_2片段,其純度達(dá)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%.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2006
【分類(lèi)號(hào)】：R373;R392

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