SARS-CoV N蛋白的原核表达及其免疫原性研究

为了研究SARS-CoV病毒N蛋白基因的原核表达及其免疫原性,为进一步的研究奠定基础,我们以PCR方法在全病毒基因组文库中获得全长N基因,分别克隆到pcDNA3和pET32a载体中获得重组质粒pcDNA3-N和pET32a-N。以亲和层析方法分离纯化pET32a-N转化的BL21细菌裂解液中的重组N融合蛋白,并以ELISA方法检测pcDNA3-N质粒基因免疫小鼠诱导后血清中的特异性抗体。结果pET32a-N转化BL21细菌后可检测到重组SARS-CoVN融合蛋白表达并分离纯化,pcDNA3-N基因免疫能够诱导产生N特异的体液免疫应答。研究的结果表明,SARS-CoVN蛋白可以在原核细胞中有效表达并有良好免疫原性,可以为进一步的功能研究和血清学诊断提供条件。

Expression and immunogenicity of the nucleocapsid protein derived from SARS-CoV

To express SARS-associated coronavirus(SARS-CoV) nucleocapsid(N) protein in vitro and to investigate its immunogenicity in vivo. PCR based gene cloning strategy was applied to isolate and amplify the full length of SARS-CoV N gene from the genome of SARS-CoV virus. The N gene fragment was then cloned into pcDNA3 and pET32a vectors. The recombinant N protein was expressed in E.coli BL21(DE3) and purified by GST-affinity column. ELISA was used to evaluate the specific humoral immune response elicited by the pcDNA3-N gene immunization. Results showed that the N protein of SARS-CoV was successfully expressed and the specific anti-N antibody was elicited in the BALB/c mice, indicating that SARS-CoV virus recombinant N protein obtained by genetic engineering approach might be an efficient way for further functional study and serological diagnosis.