重组SARS病毒N蛋白的制备及其初步应用

Preparation and Preliminary Application of Recombinant Severe Acute Respiratory Syndrome Coronavirus(SARS-CoV) N Protein

目的在大肠杆菌中表达重组SARS-CoVN蛋白,并进行纯化,为进一步研制SARS早期诊断试剂奠定基础。方法通过RT-PCR获得SARS-CoVN蛋白基因片段,并克隆至大肠杆菌表达载体pQE30中,构建重组质粒。转化E·coliM15,IPIG诱导表达。经SDS-PAGE和Western blot鉴定,采用Chelating-Sepharose Fast Flow纯化融合蛋白,并免疫BALB/c小鼠,检测小鼠血清抗体水平。结果所构建的重组表达载体pQE30-N经诱导可表达重组SARS病毒N蛋白。该蛋白纯化后,纯度可达90%左右。Western blot检测表明,重组SARS病毒N蛋白可与SARS患者恢复期血清呈现特异的免疫反应。免疫BALB/c小鼠可获得高效价抗血清。结论重组SARS-CoV N蛋白具有良好的抗原性,可用于SARS-CoV感染的早期诊断。

Objective To express recombinant severe acute respiratory syndrome coronavirus （SARS-CoV） N protein in E. coli, purify the expressed product and lay a foundation of further development of kit for early diagnosis of SARS. Methods Amplify the gene fragment encoding SARS-CoV N protein by RT-PCR and clone into expression vector pQE30. Transform the constructed recombinant plasmid pQE30-N to E. coli M15 for expression under induction of IPTG. Identify the expressed product by SDS-PAGE and Western blot, then purify by Chelating-Sepharose Fast Flow chromatography. Immunize BALB/c mice with the purified fusion protein and determine the serum antibody. Results The expressed SARS-CoV N protein showed specific reaction with the sera of patients with SARS at recovery stage and reached a purity of about 90% after purification. High titer antisera were obtained by immunization of BALB/c mice with the purified N protein. Conclusion Recombinant SARS-CoV N protein showed good antigenicity and may be used for the early diagnosis of SARS-CoV infection.