适用于疫苗株筛选的痘苗病毒载体的构建

为构建适用于疫苗株筛选的痘苗病毒载体,利用标记瞬时稳定的原理,在痘苗病毒单选择标记载体psc65的基础上,构建成带有neo和LacZ双选择标记的痘苗病毒载体pVI75.为检验载体pVI75的有效性,将HIV-1合成基因syngpnef插入到载体pVI75上,构建成转移质粒pVI75-syngpnef,并与天坛株752-1痘苗病毒共转染CEF细胞.筛选得到的重组病毒经PCR和Dot blot检验表明,标记基因已被删除,而目的基因被整合到痘苗病毒基因组上.Westem blot检测结果表明,目的基因的表达正确.痘苗病毒载体pVI75的构建使得疫苗株筛选的工作量大为降低,时间大大缩短,为利用痘苗病毒载体构建重组病毒疫苗株的研究提供了参考.

Construction of Vaccinia Virus Vector for Vaccine Screening

In order to construct an ideal vaccinia virus vector that can facilitate isolation of recombinant viruses and delete the marker genes more effectively at the process of screening recombinant virus,vaccinia virus vector pSC65 with a single selectable marker was rebuilt to construct vaccinia virus vector pVI75 containing neo and lacZ as selectable markers on the basis of transient marker stabilization.To demonstrate the usefulness of vector pVI75 as an expression vector,HIV-1 synthetic gene syngpnef was inserted into the special restriction enzyme site to generate transfer plasmid pVI75-syngpnef,then the pVI75-syngpnef co-transfected CEFs with vaccinia virus Tiantan strain.PCR and Dot blot confirmed the deletion of selectable markers and the insertion of foreign gene HIV-1 syngpnef into the vaccinia virus genome.Western blot ascertained the expression of target protein in CEFs.All results indicate that vaccinia virus vector pVI75 containing two selectable markers facilitates the screening of vaccine and can be used for construction of recombinant vaccinia virus vaccine.