Expression and Activity Determination of M Protein Epitopes of SARS Coronavirus

Objective To express the fusion protein of SARS coronavirus(SARS-CoV) M protein epitopes and analyze their antigenic activity.Methods Four primers were designed for synthesis of DNA fragment encoding SARS Co-V M protein epitopes suitable for expression in E.coli.The DNA fragment for the epitopes MKY1(14-22aa,QLLEQWNLV),MKY2(109-117aa,WSFNPETNI),MKY3(127-134aa,VTRPLMES),MKY4(176-182aa,SYYKLGA) and MKY5(186-195aa,VGTDSGFAAY) was cloned into pMD18-T vector,and positive clones were confirmed by DNA sequencing.The insert prepared by digestion with Spe I and Hind III was subcloned into pET-CKS,and the recombinant plasmids were transformed into E.coli BL21.Positive clones were induced with IPTG to express the target protein.The fusion protein was purified by metal chelating affinity chromatography,and the antigenic activity of the protein was analyzed by immunoblotting against the sera from SARS patients.Results The DNA fragment encoding SARS Co-V M protein epitopes was correctly synthesized by PCR,the insert of positive clone was subcloned into pET-CKS,and recombinant protein was highly expressed in the subsequent E.coli BL21 clone when induced with IPTG.The expressed fusion protein was soluble,and accounted for more than 30% of the total proteins.The protein was purified by affinity chromatography and exhibited reaction with the sera from SARS patients.Conclusion The fusion protein of recombinant M protein epitopes was highly expressed in E.coli BL21 and has good antigenic activity against the sera from SARS patients.The result makes the base for further study on developing SARS diagnostic reagent products.