SARS冠状病毒N蛋白的表达及二级结构预测分析

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pET2la-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6xHis-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．

Expression and Secondary Structure Prediction of SARS Coronavirus Nucleocaspid Protein

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.