摘要
通过RT PCR获得SARS冠状病毒N蛋白基因,分别克隆到原核表达载体pET21a, pET32a和pGEX 4T 1中.将3种重组质粒pET21a N,pET32a N和pGEX 4T 1 N分别转化大 肠杆菌BL21(DE3),经IPTG诱导,细菌中分别表达出约46kD的重组N蛋白、约60kD的6 xHis N融合蛋白和约70kD的GST N融合蛋白,表达量分别达总蛋白的45%、40%和30%.进一 步的分析表明:6xHis N融合蛋白在大肠杆菌中为可溶性表达,该可溶性组分占细菌裂解液的 70%左右,且能被6xHis抗体所识别.用蛋白分析软件对N蛋白进行了序列分析和二级结构预测. SARS冠状病毒N蛋白在大肠杆菌中的高效可溶性表达,有助于进一步结晶后进行X射线晶体 衍射分析其结构与功能.
Gene encoding N protein of SARS-CoV was amplified by RT-PCR;and was cloned into expression vector pET21a,pET32a and pGEX-4T-1,respectively.Recombinant plasmid pET21a-N,pET32a-N and pGEX-4T-1-N were transformed into competent cell E.coli strain BL21(DE3) and induced with IPTG;the results of SDS-PAGE indicated that a 46kD band,corresponding to the N protein,was detected in E.coli strain BL21 (DE3) with pET21a-N.The 6xHis-N fusion protein,about 60kD,and GST-N fusion protein,about 70kD,also could be detected in E.coli strain BL21 (DE3) with pET32a-N,pGEX-4T-1-N,respectively.The expression output were up to 45%,40% and 30% in E.coli strain BL21 (DE3) with pET21a-N, pET32a-N and pGEX-4T-1-N.,respectively.The 6xHis-N fusion protein was expressed as soluble form,which was up to 70% in the supernatant of bacteria after sonication;and could be recognized by antibody to 6xHis in Western blot assay.The amino acid sequence was analyzed by Vector NTI 9 and ClustalX.The secondary structure was predicted by GOR4 method on line.The soluble N protein obtained by genetic engineering method can be used for study on X-ray structure analysis.
出处
《三峡大学学报（自然科学版）》
CAS
2005年第1期83-87,96,共6页
Journal of China Three Gorges University：Natural Sciences
基金
广东省非典型肺炎科技攻关资助课题(粤科社字2003[80]号)
