Huang Wei, Liu Ying, Duan Dan-li, Li Hai-shan, Liu Yong, Hong Kun-Xue, Zhu Jia-hong, Shao Yi-ming
National Center for STD/AIDS Prevevtion and Control China CDC, Beijing 100050, China.
Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi. 2004 Mar;18(1):43-6.
B8R gene encodes a secreted protein with homology to IFN-gamma receptor, which neutralizes the antiviral and immunological regulation activities of IFN-gamma. To improve the safety of vaccinia virus vector, an attenuated recombinant vaccinia virus with the B8R gene deletion from Tiantan vaccine strain (VTT) was constructed.
The transfer vectors were generated by joining B8R left flank, B8R right flank, vv promoter, LacZ, multicloning site and pBRSK fragments. The recombinant viruses VTTdeltaB8RLacZ (VTT with B8R deletion and LacZ insertion) were constructed by homologous recombination.
The B8R deletion mutants were confirmed by dot blot with B8R gene probe and PCR amplification. The replication ability of VTTdeltaB8RLacZ strain in vitro was similar to that of the VTT. The skin lesions formed by VTTdeltaB8RLacZ (10(6) pfu) were significantly smaller and healed faster than those formed by VTT when injected intradermally to the rabbits,and no visible ulceration occurred. Meanwhile LacZ in VTKgpedeltaB8RLacZ was expressed stably.
The attenuated vector with B8R gene deletion improves the safety of recombinant vaccinia virus vaccine B8R locus may be used as a new site for insertion of foreign genes in vaccinia virus vector.
B8R基因编码一种与干扰素γ受体具有同源性的分泌蛋白，该蛋白可中和干扰素γ的抗病毒和免疫调节活性。为提高痘苗病毒载体的安全性，构建了天坛疫苗株（VTT）缺失B8R基因的减毒重组痘苗病毒。
通过连接B8R基因左侧侧翼、B8R基因右侧侧翼、痘苗病毒启动子、LacZ、多克隆位点和pBRSK片段构建转移载体。通过同源重组构建重组病毒VTTdeltaB8RLacZ（缺失B8R基因并插入LacZ的VTT）。
通过用B8R基因探针进行斑点杂交和PCR扩增证实了B8R缺失突变体。VTTdeltaB8RLacZ株在体外的复制能力与VTT相似。将VTTdeltaB8RLacZ（10⁶ 蚀斑形成单位）皮内注射到兔体内时，其形成的皮肤损伤明显小于VTT形成的损伤，且愈合更快，未出现明显溃疡。同时，VTKgpedeltaB8RLacZ中的LacZ稳定表达。
缺失B8R基因的减毒载体提高了重组痘苗病毒疫苗的安全性，B8R基因座可作为痘苗病毒载体中外源基因插入的新位点。
