摘要
目的 构建缺失IFN γ受体基因的天坛株重组痘苗病毒载体 ,初步研究其毒力的改变及B8R缺失区插入外源基因表达的稳定性。为研制表达多价抗原重组痘苗病毒载体、提高痘苗病毒载体安全性进行探索。方法 采用PCR技术、多步克隆构建带有天坛株痘苗病毒B8R基因外左右侧同源臂、痘苗病毒启动子序列pE L、p7 5及下游LacZ序列的转移质粒pSKB8R、pSKB8RLacZ。将痘苗病毒VTT与转移质粒pSKB8RLacZ共转染CEF细胞进行同源重组 ,经蓝白斑筛选、连续单斑纯化、鉴定获得B8R基因 (IFN γ受体基因同源序列 )缺失为LacZ所取代的重组病毒VTT△B8RLacZ。结果 经酶切、测序鉴定转移质粒构建正确 ,核酸水平、外源基因生物学活性检测表明VTT△B8RLacZ在CEF细胞连续培养传代中B8R基因的缺失和插入外源基因LacZ表达均很稳定。VTT△B8RLacZ在细胞中的繁殖复制水平与VTT相当。兔皮内毒力实验表明B8R基因缺失显著降低VTT的毒力。结论 本研究表明B8R基因缺失可显著降低痘苗病毒天坛株的毒力并可作为外源基因的插入区 ,缺失B8R基因的重组痘苗病毒可能作为新一代的表达多价抗原痘苗病毒载体。
Objective B8R gene encodes a secreted protein with homology to IFN-γ receptor, which neutralizes the antiviral and immunological regulation activities of IFN-γ. 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. Methods The transfer vectors were generated by joining B8R left flank,B8R right flank,vv promoter,LacZ,multicloning site and pBRSK fragments. The recombinant viruses VTTΔB8RLacZ(VTT with B8R deletion and LacZ insertion) were constructed by homologous recombination. Results The B8R deletion mutants were confirmed by dot blot with B8R gene probe and PCR amplification. The replication ability of VTTΔB8RLacZ strain in vitro was similar to that of the VTT. The skin lesions formed by VTTΔB8RLacZ (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 VTKgpeΔB8RLacZ was expressed stably. Conclusions 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.
出处
《中华实验和临床病毒学杂志》
CAS
CSCD
北大核心
2004年第1期43-46,共4页
Chinese Journal of Experimental and Clinical Virology
基金
国家 8 63计划课题资助 (2 0 0 3AA2 1910 0 )
