SARS冠状病毒S基因重组DNA诱导的体液免疫反应

Construction and induction of antibody response by recombinant DNA containing SARS coronavirus full-length or truncated S gene

目的以严重急性呼吸综合征冠状病毒(SARS-CoV)密码子优化的S、S1和S2基因分别构建其真核表达质粒,免疫BALBPc小鼠,以初步评价其诱导特异性体液免疫的效果。方法将人工合成密码子优化的S、S1和S2基因克隆入pcDNA4/HisMax-TOPO表达载体,重组质粒转染293T细胞,Westernblot和免疫组化检测其真核表达,重组质粒免疫BALBPc小鼠,酶联免疫(ELISA)检测抗S蛋白抗体,伪病毒中和试验、细胞融合抑制试验检测中和抗体。结果3种重组质粒均可在真核细胞中获得表达,免疫小鼠后可诱导针对S蛋白的特异性抗体,抗体在12周观察期内呈持续上升趋势;其中,仅S和S1蛋白重组质粒能够诱导中和抗体的产生,以S蛋白的效价为高。结论密码子优化S和S1蛋白重组质粒可以有效诱导BALBPc小鼠产生中和抗体,其抗体可能具有阻断SARS-CoV侵袭易感细胞的能力。该结果为进一步研究SARS0Co V DNA疫苗提供了参考依据。

Objective To develop a DNA vaccine inducing specific humoral immunity. Methods The codon-optimized SARS-CoV S, S1 and S2 genes were respectively cloned into pcDNA4/HisMax-TOPO eukaryotic expression vector to get the recombinant plasmids, which were then transfected into 293T cell and their gene eukaryotic expressions were detected by Western blot and immunohistochemistry method. Three recombinant plasmids were then used to immunize BALB/c mice and the specific antibody to S protein was tested by ELISA and that of neutralization was tested by syncytia inhibition assay and SARS pseudovirus neutralization assay. Results Three recombinant plasmids all expressed S protein or its truncated forms of S1 and S2 in eukaryotic cells and induced specific antibody to S protein, the titer of these antibody increased continually in 12 weeks after immunization. Of three forms of S recombinant proteins, only S or S1 protein induced neutralizing antibody responses, antibody titer was increased by S protein immunization. Conclusion The specific humoral immune response is induced by S or S1 proteins in BALB/c mice and the antibody may block SARS-CoV binding to sensitive cells. The results may facilitate the development of SARS-CoV DNA vaccine.