SARS核衣壳蛋白表达、纯化及单克隆抗体的制备
发布时间：2019-04-08 20:39
【摘要】：SARS-CoV 是一种新型冠状病毒,核衣壳蛋白(N 蛋白)是重要的结构蛋白,在病毒侵染宿主过程中起重要作用。 本研究中目的基因SARS-N 基因由大连宝生物公司按照SARS 冠状病毒CUHK-W1 株编码N 蛋白的基因全序列合成。 选用pET28a 原核表达载体进行表达。为提高表达量,在pET28a表达载体基础上克隆了分子伴侣chap10 基因,构建重组表达载体。将N 基因克隆于重组表达载体pET28a-chap10 上,转化大肠杆菌BL21(DE3),经IPTG 诱导获得了高效表达的分子量约为60KD 的可溶性融合蛋白。Western blot 证实表达的目的蛋白是SARS-N 蛋白。 本研究大规模发酵培养了重组SARS-N 蛋白,探讨了合适的发酵条件,并尝试用乳糖代替IPTG 诱导表达。通过DEAE Sepharose FF、SPSepharose FF 和BLUE Sepharose CL 6B 三步纯化,纯度达到95%以上。ELISA 结果显示,纯化SARS-N 蛋白能与SARS 病人恢复期血清发生特异性反应,进而又制备了鼠源性抗SARS-N 蛋白单克隆抗体。
[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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392
本文编号：2454909
[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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392
【参考文献】
相关期刊论文 前5条
1 石磊,张其鹏,芮伟,卢铭,景霞,尚彤,国强华;SARS冠状病毒推定结构蛋白序列分析[J];北京大学学报(医学版);2003年S1期
2 祝庆余,秦鄂德,王翠娥,于曼,司炳银,范宝昌,常国辉,彭文明,杨保安,姜涛,李豫川,邓永强,刘洪,甘永华;非典型肺炎病例标本中新型冠状病毒的分离与鉴定[J];中国生物工程杂志;2003年04期
3 刘树春,赵雨杰,张学,罗阳;SARS-CoV推测N蛋白功能结构的生物信息学研究[J];中国生物工程杂志;2003年12期
4 张士猛,陈苏红,张敏丽,鲁丹丹,张嵬,杜清友,王升启;重组SARS病毒N蛋白可与SARS患者血清发生特异反应[J];中国生物化学与分子生物学报;2004年04期
5 陈蕴佳,高歌,鲍一明,Rodrigo LOPEZ,吴健民,蔡涛,叶志强,顾孝诚,罗静初;SARS冠状病毒全基因组序列初步分析[J];遗传学报;2003年06期
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