SARS冠状病毒S1蛋白的原核表达与鉴定

Expression and identification of SARS coronavirus S1 protein in prokaryotic cell

目的 克隆SARS冠状病毒 (SARS-CoV)S1基因片段 ,构建原核表达载体 ,并在宿主菌中诱导表达带组氨酸标记的融合蛋白。方法 人工合成SARS-CoVS1基因片段 ,克隆至pUCm T载体 ,经DNA序列分析和酶切后 ,将酶切片段亚克隆至原核表达载体 pET - 2 8b(+) ,重组子pET 2 8b/SARS S1转化大肠杆菌ril,IPTG诱导表达 ,Westernblot鉴定表达产物 ;纯化表达产物并用ELISA检测其抗原特异性。结果 获得了主要以包涵体形式存在的 32kD的目的蛋白 ,Westernblot鉴定其为带组氨酸标记的融合蛋白 ,ELISA证明其能与合成肽的抗体及病人血清中的SARS-CoV的抗体特异结合。结论 成功构建了SARS-CoVS1基因的组氨酸标记表达载体 pET-2 8b/SARS-S1,并在ril中得到了高效表达 ;表达的S1蛋白具有较好的抗原特异性 ,便于免疫动物以获得特异抗体 。

To clone S1 gene fragment of SARS coronavirus (SARS-CoV) and construct the prokaryotic expression vector expressing S1 fusion protein with a 6×His-tag; S1 gene fragment is synthesized and cloned to pUCm-T vector. After sequencing , the S1 gene fragment was digested with BamH I and Sal I and inserted into prokaryotic expression vector pET-28b(+).The recombinant pET-28b(+)/ SARS-S1 was transferred into an expression strain E.coli ril and induced by IPTG to express fusion protein.The protein was identified by Western blot and purified with a kit ,and the specificity of the protein was analyzed by ELISA. It was found that the fusion protein of 32kD was expressed mainly as inclusion body. The presence of the 6×His-tag was confirmed by western blot. The fusion protein had a high affinity to bind to antibodies derived from synthesized peptide and the sera from SARS patients. It concludes prokaryotic expression vector pET-28b(+)/ SARS-S1 is successfully constructed and S1 protein was expressed inE.coli. ril. The protein could be purified by the 6 ×His-tag and the protein-specific antibodies could be produced by immunizing animals. Our work provide foundation for rapid laboratory diagnosis of SARS-CoV.