SARS病毒S2蛋白的原核表达与DNA疫苗的构建

SARS-CoV-S2 Protein Expression and DNA Vaccine Development

目的构建SARS冠状病毒(SARS-CoV)S2基因的原核和真核表达质粒,研究该真核表达重组质粒作为DNA疫苗的可行性。方法采用RT-PCR技术从灭活的SARS冠状病毒RNA扩增得到SARS-CoV-S2基因片段,定向克隆入原核表达载体pET-28a和真核表达载体pCDNA3.1+中,构建pET-28a-SARS-CoV-S2和pCDNA3.1-SARS-CoV-S2质粒。用异丙基-β-D-硫代半乳糖苷(Isopropylβ-D-1-thiogalactopyranoside,IPTG)诱导重组质粒pET-28a-SARS-CoV-S2在大肠埃希菌BL21(DE3)plysS中表达融合蛋白;并将重组pCDNA3.1-SARS-CoV-S2质粒免疫BALB/c小鼠,采用ELISA、Western blot的方法检测被免疫小鼠的抗体应答情况。结果应用RT-PCR技术,从灭活的SARS冠状病毒扩增得到大小约为998 bp SARS-CoV-S2基因片段后,构建了含SARS-CoV-S2基因片段的原核表达质粒pET-28a-SARS-CoV-S2和真核表达质粒pCDNA3.1-SARS-CoV-S2;通过IPTG诱导,重组pET-28a-SARS-CoV-S2质粒在表达菌BL21(DE3)plysS内表达S2融合蛋白;用纯化的重组真核表达质粒经基因枪免疫BALB/c小鼠后,获得了高效价的特异性抗体。结论成功构建了可表达SARS-CoV-S2融合蛋白的原核表达质粒pET-28a-SARS-CoV-S2。重组pCDNA3.1-SARS-CoV-S2质粒可作为DNA疫苗使被免疫小鼠产生明显的免疫应答。为建立SARS特异性血清学诊断方法和研制SARS DNA疫苗奠定了一定的基础。

Objective To construct recombinant prokaryotic and eukaryotic expression plasmids of SARS-CoV S2 gene. Methods The fragment of SARS-CoV-S2 was amplified by RT-PCR from inactivated SARS-CoV and cloned into the prokaryotic expression vector pET-28a and eukaryotic expression vector pCDNA-3.1, then the prokaryotic expression plasmid expressing recombinant SARS-CoV-S2 protein in E. coli BL21 （DE3） plysS which was induced by IPTG. BALB/c mice were immunized with the purified eukaryotie expression plasmid as DNA vaccine by using gene gun. The titers and specificity of antibodies were detected by enzyme-linked immuneosorbent assay and Western blot. Results The fragment of SARS-CoV-S2 was amplified by RT-PCR from inactivated SARS-CoV. The prokaryotic expression plasmid pET-28a-SARS-CoV-S2 and eukaryotic expression plasmid pCDNA3.1-SARS-CoV-S2 were successfully constructed. The recombinant protein SARS-CoV-S2 could be expressed in abundance in E. coli BL21（DE3） plysS which was induced by IPTG. After the BALB/c mice were immunized with the purified eukaryotic expression plasmid, high titer polyclonal antibody with a high specificity was obtained. Conclusion The prokaryotic expression plasmid pET-28a-SARS-CoV-S2 which can express recombinant protein SARS-CoV-S2 was successfully constructed. The BALB/c mice, immunized with the purified eukaryotic expression plasmid as DNA vaccine, obtain obviously immune responses.