SARS小包膜蛋白表达、纯化及单克隆抗体的制备
发布时间：2018-10-19 15:17
【摘要】：SARS-CoV是一种新型冠状病毒,小包膜蛋白(E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-E基因由大连宝生物公司按照SARS冠状病毒CUHK-W1株编码E蛋白的基因全序列合成。 选用pET28a原核表达载体进行表达。将E基因克隆于重组表达载体pET28a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27KD的可溶性融合蛋白。Western blot证实表达的目的蛋白是SARS-E蛋白。 本研究大规模发酵培养了重组SARS-E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-E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-E蛋白单克隆抗体。
[Abstract]:SARS-CoV is a new type of coronavirus. Small envelope protein (E protein) is an important structural protein and plays an important role in the infection of the virus. In this study, the target gene SARS-E gene was synthesized by Dalian Bao Biological Company according to the gene sequence encoding E protein of SARS coronavirus CUHK-W1 strain. PET28a prokaryotic expression vector was selected for expression. The E gene was cloned into recombinant expression vector pET28a and transformed into Escherichia coli BL21 (DE3). The highly expressed soluble fusion protein. Western blot with a molecular weight of about 27KD was confirmed to be SARS-E protein by IPTG induction. In this study, recombinant SARS-E protein was cultured on a large scale, and suitable fermentation conditions were discussed, and lactose instead of IPTG was used to induce expression. The purity of purified SARS-E protein was over 95% through three steps purification of DEAE Sepharose FF,SPSepharose FF and BLUE Sepharose CL 6B. The results of ELISA showed that the purified SARS-E protein could react specifically with the recovered serum of SARS patients, and then the monoclonal antibody against SARS-E protein was prepared.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392
本文编号：2281523
[Abstract]:SARS-CoV is a new type of coronavirus. Small envelope protein (E protein) is an important structural protein and plays an important role in the infection of the virus. In this study, the target gene SARS-E gene was synthesized by Dalian Bao Biological Company according to the gene sequence encoding E protein of SARS coronavirus CUHK-W1 strain. PET28a prokaryotic expression vector was selected for expression. The E gene was cloned into recombinant expression vector pET28a and transformed into Escherichia coli BL21 (DE3). The highly expressed soluble fusion protein. Western blot with a molecular weight of about 27KD was confirmed to be SARS-E protein by IPTG induction. In this study, recombinant SARS-E protein was cultured on a large scale, and suitable fermentation conditions were discussed, and lactose instead of IPTG was used to induce expression. The purity of purified SARS-E protein was over 95% through three steps purification of DEAE Sepharose FF,SPSepharose FF and BLUE Sepharose CL 6B. The results of ELISA showed that the purified SARS-E protein could react specifically with the recovered serum of SARS patients, and then the monoclonal antibody against SARS-E protein was prepared.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392
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