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目的 提高重组痘苗病毒狂犬疫苗的有效性和安全性。方法 利用TK区表达狂犬病毒糖蛋白 (RG)的重组痘苗病毒作为亲本株 ,通过两步同源重组 ,删除痘苗病毒基因组CK片段间与毒力和宿主范围相关的核酸片段 ,同时将狂犬病毒核蛋白 (RN)基因插入C K片段之间 ,获得了含有RG基因和RN基因的非复制型重组痘苗病毒VTKRGΔCKLacZRN。结果 经PCR鉴定 ,RN基因已插入CK区 ;Westernblot结果显示 ,该株病毒能同时表达RG和RN ,相对分子质量 (Mr)分别为 6 5× 10 3 和 5 0 5× 10 3。VTKRGΔCKLacZRN在人源细胞如TK 143细胞中不能正常繁殖 ,在鸡胚成纤维细胞 (CEF)中能正常复制。与非复制型重组病毒VTKRGΔCK相比 ,VTKRGΔCKLacZRN免疫小鼠后能更快地诱生较高滴度的中和抗体 ,效果相当于复制型重组病毒VTKRG免疫组 ;它们均能保护小鼠针对致死剂量狂犬病毒国际标准攻击毒株 (CVS)的攻击。结论 VTKRGΔCKLacZRN具有良好的免疫效果和安全性
Objective To improve the efficacy and safety of recombinant vaccinia virus rabies vaccine. Methods Recombinant vaccinia virus expressing rabies virus glycoprotein (RG) in TK region was used as the parent strain. The two fragments of homologous recombination virus were amplified by two-step homologous recombination. The fragments of virulence and host range of the vaccinia virus genome were deleted, Protein (RN) gene was inserted between the CK segments to obtain a non-replicating recombinant vaccinia virus VTKRGΔCKLacZRN containing RG gene and RN gene. Results The RN gene was inserted into the CK region by PCR. The results of Western blot showed that RG and RN could express RG and RN simultaneously. The relative molecular mass (Mr) was 65 × 103 and 105 × 103, respectively. VTKRG [Delta] CKLacZRN does not propagate normally in human cells such as TK143 cells and replicates normally in chicken embryo fibroblasts (CEFs). Compared with non-replicating recombinant virus VTKRGΔCK, mice immunized with VTKRGΔCKLacZRN induced higher titers of neutralizing antibodies more rapidly, which was equivalent to that of the replication-competent recombinant virus VTKRG; both of them could protect mice against lethal dose Rabies virus international standard challenge strain (CVS) attack. Conclusion VTKRGΔCKLacZRN has good immune effect and safety