SARS冠状病毒M蛋白B细胞抗原表位的原核表达与抗原活性检测

目的从SARS冠状病毒M蛋白的线性重叠肽链文库中筛选出5个B细胞抗原表位,通过构建原核表达载体,表达抗原表位融合蛋白,并检测其抗原活性.方法应用大肠杆菌高频密码子设计引物,通过PCR方法合成编码SARS冠状病毒M蛋白5个抗原表位(MKY1、MKY2、MKY3、MKY4和MKY5)的DNA片段,经克隆和测序分析,亚克隆至表达载体pET-CKS,转化大肠杆菌BL21;阳性菌株经IPTG诱导,SDS-PAGE分析;大量诱导表达抗原表位融合蛋白,亲和层析予以纯化;Western检测SARS病人阳性血清对融合蛋白的识别.结果成功构建SARS冠状病毒M蛋白抗原表位的表达载体,在大肠杆菌BL21中表达,融合蛋白表达量达到细菌总蛋白30%,经亲和层析纯化,融合蛋白可被SARS病人抗血清识别.结论原核表达的抗原表位融合蛋白具有良好的抗原活性,为下一步进行SARS冠状病毒诊断试剂盒的开发研究奠定基础.

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.