非复制型痘苗病毒NTV修饰株NTV-ΔF1L-C7L的构建及免疫效果评价

Construction of NTV-ΔF1L-C7L modified strain of non-replication vaccinia virus NTV and evaluation of its immunological effects

目的对我国非复制型痘苗病毒NTV进行基因改造,以提高其免疫原性。方法通过基于CRISPR-Cas9技术的痘苗病毒同源重组方法,在痘苗病毒F1L基因缺失的同时回插C7L基因,构建NTV修饰株NTV-ΔF1L-C7L。然后将该重组病毒以10^(7) PFU免疫BALB/c小鼠,采用ELISA和ELISpot方法分别检测修饰株NTV-ΔF1L-C7L诱导产生的体液免疫和细胞免疫水平,并采用噬斑减少中和实验测定中和抗体水平。结果经PCR和western blot鉴定证明构建的NTV修饰株NTV-ΔF1L-C7L的F1L基因缺失,同时C7L基因回插在该区域,且C7L基因能正常表达,说明重组病毒构建正确。NTV-ΔF1L-C7L免疫小鼠后,ELISA结果表明重组病毒NTV-ΔF1L-C7L诱导产生的IgG抗体水平高于NTV;ELISpot结果亦能够表明该重组病毒能够诱导产生水平更高的IFN-γ;噬斑减少中和实验结果表明,该重组病毒比NTV能够诱导产生水平更高的中和抗体。结论正确构建了NTV基因修饰株NTV-ΔF1L-C7L,相比于NTV,其能诱导更强的体液免疫和细胞免疫,为我国天花或猴痘疫苗的换代产品研发提供参考数据。

Objective We genetically modified our non-replicating vaccinia virus NTV to improve its immunogenicity.Methods We constructed NTV-modified strain NTV-ΔF1L-C7L by homologous recombination of vaccinia virus based on CRISPR-Cas9 technology by inserting the C7L gene while deleting the F1L gene.The recombinant virus NTV-ΔF1L-C7L was then immunized with 10^(7) PFU in BALB/c mice,and the levels of humoral and cellular immunity induced by NTV-ΔF1L-C7L were detected by ELISA and ELISpot method,respectively,and the levels of neutralizing antibodies were determined by the phage-reduced neutralization assay.Results The PCR and western-blot identification proved that the F1L gene of the constructed NTV-modified strain NTV-ΔF1L-C7L was missing,while the C7L gene was inserted back in the region,and the C7L gene could be expressed normally,indicating that the recombinant virus was constructed correctly.After immunization of mice with NTV-ΔF1L-C7L,ELISA result showed that the recombinant virus NTV-ΔF1L-C7L induced a higher level of IgG antibody than NTV;ELISpot result also showed that the recombinant virus was able to induce a higher level of IFN-γ;and the result of plaque reduction neutralization test showed that the recombinant virus was able to induce a higher level of IFN-γantibody than that of NTV.Conclusions We correctly constructed the NTV gene-modified strain NTV-ΔF1L-C7L,which induced stronger humoral and cellular immunity compared with NTV,and provided reference data for the research and development of replacement products for smallpox or monkeypox vaccines.