【摘要】：SARS-CoV 是一種新型冠狀病毒,核衣殼蛋白(N 蛋白)是重要的結(jié)構(gòu)蛋白,在病毒侵染宿主過程中起重要作用。 本研究中目的基因SARS-N 基因由大連寶生物公司按照SARS 冠狀病毒CUHK-W1 株編碼N 蛋白的基因全序列合成。 選用pET28a 原核表達(dá)載體進(jìn)行表達(dá)。為提高表達(dá)量,在pET28a表達(dá)載體基礎(chǔ)上克隆了分子伴侶chap10 基因,構(gòu)建重組表達(dá)載體。將N 基因克隆于重組表達(dá)載體pET28a-chap10 上,轉(zhuǎn)化大腸桿菌BL21(DE3),經(jīng)IPTG 誘導(dǎo)獲得了高效表達(dá)的分子量約為60KD 的可溶性融合蛋白。Western blot 證實(shí)表達(dá)的目的蛋白是SARS-N 蛋白。 本研究大規(guī)模發(fā)酵培養(yǎng)了重組SARS-N 蛋白,探討了合適的發(fā)酵條件,并嘗試用乳糖代替IPTG 誘導(dǎo)表達(dá)。通過DEAE Sepharose FF、SPSepharose FF 和BLUE Sepharose CL 6B 三步純化,純度達(dá)到95%以上。ELISA 結(jié)果顯示,純化SARS-N 蛋白能與SARS 病人恢復(fù)期血清發(fā)生特異性反應(yīng),進(jìn)而又制備了鼠源性抗SARS-N 蛋白單克隆抗體。
[Abstract]:SARS-CoV is a new type of coronavirus. Nucleocapsid protein (N protein) is an important structural protein and plays an important role in the process of virus infecting host. In this study, the SARS-N gene was synthesized by Dalian Baobio Company according to the complete sequence of N protein encoded by SARS coronavirus CUHK-W1 strain. The prokaryotic expression vector of pET28a was selected for expression. In order to improve the expression level, the molecular chaperone chap10 gene was cloned on the basis of pET28a expression vector, and the recombinant expression vector was constructed. The N gene was cloned into the recombinant expression vector pET28a-chap10 and transformed into E. coli BL21 (DE3). The highly expressed soluble fusion protein with a molecular weight of about 60KD was obtained by IPTG induction. Western blot confirmed that the target protein was SARS-N protein. In this study, recombinant SARS-N protein was cultured on a large scale, and the suitable fermentation conditions were discussed, and the expression was induced by lactose instead of IPTG. The purity of DEAE Sepharose FF,SPSepharose FF and BLUE Sepharose CL 6B was over 95%. The results of Elisa showed that the purified SARS-N protein could react specifically with the convalescent serum of SARS patients. Furthermore, mouse-derived monoclonal antibodies against SARS-N protein were prepared.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R392

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