【摘要】 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更多还原

【Abstract】 Objective To improve the efficacy and safety of recombinant vaccinia rabies vaccine, the non replicating recombinants co expressing rabies virus glycoprotein and nucleoprotein were constructed. Methods According to the strategy of transient dominant selection, the rabies virus nucleoprotein (RN) gene was introduced into recombinant vaccinia virus(VTKRG) which expresses rabies virus glycoprotein (RG) gene in the TK locus by homologous recombinant. Results A fragment between fragment C and fragment K associated with the virulence and host range of vaccinia virus was substituted by RN gene, resulting in the VTKRGΔCKLacZRN. Western blot showed that VTKRGΔCKLacZRN expressed G protein and N protein with molecular weights of 65×10 3 and 50.5×10 3 respectively. The genetically modified VTKRGΔCKLacZRN possessed a highly attenuated phenotype, attributed to a decreased replicative capacity when grown in human cell lines. Compared with the non replicating recombinant vaccinia virus VTKRGΔCK expressing G protein only, VTKRGΔCKLacZRN, similar to VTKRG, were able to induce higher virus neutralizing antibody titers faster in mice. Mice inoculated with all of the recombinants were protected from a lethal intramuscularly (i.m.) challenge of rabies virus CVS strain which killed all control mice. Conclusion VTKRGΔCKLacZRN possesses excellent immunogenicity and improved safety in view of clinical application.更多还原