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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 construct eukaryotic expression plasmids of SARS - CoV codon optimized S, S1 and S2 genes and immunize BALB ac c mice to evaluate their specific humoral immunity. Methods Synthesized codon optimized S, S1 and S2 genes were cloned into pcDNA4 / HisMax-TOPO expression vector and transfected into 293T cells. The eukaryotic expression was detected by Western blot and immunohistochemistry. The BALB / c mice were immunized with the recombinant plasmids. Anti-S protein antibodies, pseudovirion neutralization assay, and cell fusion inhibition assay were used to detect neutralizing antibodies. Results All three recombinant plasmids could be expressed in eukaryotic cells. The specific antibodies against S protein were induced after immunization in mice. The antibodies showed an increasing trend during the 12 weeks observation period. Only S and S1 recombinant plasmids Can induce the production of neutralizing antibodies with high titer of S protein. Conclusion The codon optimized S and S1 recombinant plasmids can effectively induce BALBPc mice to produce neutralizing antibodies, and their antibodies may have the ability of blocking the invasion of susceptible cells of SARS-CoV. The results provide a reference for further research on SARS0Co V DNA vaccine.