中东呼吸综合征冠状病毒S1蛋白在昆虫细胞中的重组表达及免疫效果评价

Preparation and Immunization Assessment of Recombinant MERS-CoV S1 Protein Expressed by Insect Cells

目的:利用昆虫杆状病毒表达系统重组表达中东呼吸综合征冠状病毒(MERS-Co V)S1蛋白,并对其免疫效果进行评价。方法:构建含有MERS-Co V S1基因的重组杆状病毒质粒,转染Sf9细胞包装杆状病毒;重组病毒传代3次获得种子病毒,感染Sf9细胞,收获感染上清,通过镍离子亲和层析纯化获得S1重组蛋白;用纯化的S1蛋白免疫BALB/c小鼠,采用ELISA检测免疫小鼠血清抗原特异性的抗体水平;采用假病毒中和试验检测血清中抗体的中和活性。结果:获得了表达MERS-Co V S1蛋白的重组病毒株,在昆虫细胞中表达并纯化了S1重组蛋白;利用重组表达的S1蛋白免疫小鼠3次,血清S1特异性Ig G抗体滴度可达1∶102 400,免疫小鼠血清稀释至1/5120后中和百分比仍达50%以上。结论:利用昆虫细胞重组表达的MERS-Co V S1蛋白具有良好的免疫原性,并能有效诱导产生高滴度中和抗体,为发展MERS-Co V重组蛋白疫苗奠定了基础。

Objective： To prepare the recombinant S1 protein of Middle East respiratory syndrome coronavirus （MERS-CoV） in the baculovirus expression system and evaluate the immunogenicity of purified recombinant S1 protein. Methods： Recombinant bacmid inserted S1 gene of MERS-CoV was constructed, which was followed by transfection into Sf9 cells to package recombinant baculovirus. The Sf9 cells were infected by recombinant baculovirus seed derived from the third passage. Cell cultures were collected and the recombinant S1 protein was purified by nickel column affinity chromatography. Following vaccination with purified recombinant S1 protein, the S1-specific IgG antibody responses in BALB/c mouse sera were measured by ELISA, and the neutralizing antibodies were detected by pseudo-MERS-CoV neutralizing assay. Results： The recombinant baculovirus isolate expressing recombinant S1 protein was obtained and recombinant S1 protein was successfully expressed and purified in insect cells. Following three vaccinations with recombinant S1 protein, titer of S1-specific IgG antibody in mouse sera reaches 1∶102 400, and inhibition percentage against pseudo-MERS-CoV can be still over 50% at 1∶5120 dilutions. Conclusion：Recombinant MERS-CoV S1 protein expressed in insect cells has ability to induce good immunogenicity with high titers of neutralizing activity, which lays a foundation of development of recombinant vaccine against MERS-CoV.