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目的 构建SARS病毒spike基因片段真核表达质粒DNA疫苗;检测spike基因片段的免疫原性;筛选SARS病毒疫苗构建的理想靶抗原。方法 采用RT PCR从SARS冠状病毒北京0 1(BJ0 1)株cDNA获取了spike基因cDNA的全长D12、编码N末端氨基酸的cDNA片段D14和编码C末端氨基酸的cDNA片段D2 3;构建重组真核表达质粒pcDNA3 /D12、pcDNA3 D14、pcDNA3 D2 /3;用3种重组质粒和pcDNA3空质粒载体(对照)DNA皮下注射免疫Wistar大鼠;ELISA检测免疫后大鼠血清S蛋白特异性抗体IgG的产生;同位素51 Cr实验检测特异性CTL应答;ELISPOT检测单细胞水平IFN -γ分泌。结果 pcDNA3 /D2 3疫苗免疫组大鼠血清有S蛋白阳性抗体IgG产生、抗体滴度>10 0 0 ;脾淋巴细胞可诱导特异的CTL应答和IFN γ分泌,pcDNA3 /D2 3疫苗组与其它组之间统计分析P <0 .0 5。结论SARS冠状病毒S蛋白的C末端具有较强的免疫原性,是疫苗构建的理想候选靶抗原,本研究结果为SARS病毒的疫苗研究提供了资料。
Objective To construct the eukaryotic expression plasmid DNA vaccine of spike gene of SARS virus, detect the immunogenicity of spike gene fragment and screen the ideal target antigen constructed by SARS virus vaccine. Methods The full length cDNA of spike gene D12, cDNA fragment encoding N terminal amino acid D14 and cDNA fragment encoding C terminal amino acid D2 3 were obtained from cDNA of SARS coronavirus Beijing 0 1 (BJ0 1) by RT PCR. The recombinant eukaryotic Western blotting was used to immunize Wistar rats with three kinds of recombinant plasmids and pcDNA3 empty plasmid vector (control) DNA, and the production of S protein specific IgG was detected by ELISA ; Isotope 51 Cr test specific CTL response detected; ELISPOT single-cell level of IFN-γ secretion. Results The sera of mice immunized with pcDNA3 / D2 3 vaccine were positive for IgG with IgG antibody titer> 100%. Specific CTL responses and IFN γ secretion were induced by splenic lymphocytes. The pcDNA3 / D2 3 vaccine group was significantly different from other groups Statistical analysis between P <0. 05. Conclusion The C terminus of SARS coronavirus S protein has strong immunogenicity and is an ideal target antigen for vaccine construction. The results of this study provide information for the vaccine research of SARS virus.