重组SARS冠状病毒棘突蛋白S1区基因片段的克隆、表达及免疫原性研究

Cloning, purification, and antigenic characterization of three recombinant fragments derived from SARS-CoV S1 domain

目的构建SARS冠状病毒(SARS-CoV)棘突(spike)蛋白(S蛋白)S1区基因(40～2001bp)分段表达载体,并研究其免疫原性。方法利用PCR技术扩增S1区基因,将其定向插入pQE30质粒,在pQE30质粒上将S1切成3段(40～751、746～1344、746～2001bp),均插入pQE30质粒,构建质粒在大肠埃希菌M15中表达,表达产物经亲和层析纯化及WesternBlot和ELISA鉴定。结果构建的3种重组质粒(pQE30/S1a、pQE30/S1b、pQE30/S1c)均高效表达,重组蛋白相对分子质量分别为26700、22500和46000,表达量分别占菌体总蛋白质的35%、35%和30%。3种重组蛋白经Ni亲和层析树脂得到成功纯化。经Western-Blot及ELISA证实,重组蛋白片段S1c(746～2001bp)可以被SARS患者血清所识别。结论成功构建了SARS-CoVS蛋白S1区基因分段表达载体,重组蛋白S1c具有较强的免疫原性,它的获得为下一步疫苗的研制奠定了基础。

Objective The present study aimed to clone and express three fragments of genomic RNA derived from SARS associated coronavirus （SARS-CoV） S1 domain and to study its immunogenieity, Methods The SI domain gene was amplified by PCR with specific primers and was inserted into the prokaryotic expression vector pQE-30, Three fragments （40-751, 746-1344 and 746-2001 bp） derived from S1 domain produced after the recombinant plasmht （ pQE-30/S1 ） was digested by restriction endonucleases. The three fragments were cloned into pQE-30 and expressed in M15 strains of Escherichia coli, The expression products, designated S1 a, S1 b and S1c respectively, were purified by Ni affinity chromatography. The immunogenicity was analyzed by Western Blot and ELISA using serologically confirmed sera from SARS patients and the sera from healthy donors was used as control at the same assay. Results Three reeombinant plasmids （pQE-30/S1a, pQE-30/S1b, pQE-30/S1c） were constructed. Fusion proteins with relative molecular mass of 26 700, 22 500 and 46 000 dahon were successfully expressed with amounts of 3.5%, 3.5% and 30% of total cell protein and purified by Ni affinity chromatography, respectively. Western Blot and ELISA analysis showed that the S1c protein could be specifically recognized by the sera from SARS patients. Conclusion The recombinant S1c protein was a good immunogen and has the potential to be used as a vaccine against SARS-CoV infection.