Coupar B E, Andrew M E, Boyle D B
CSIRO Australian Animal Health Laboratory, Geelong, Victoria, Australia.
Gene. 1988 Aug 15;68(1):1-10. doi: 10.1016/0378-1119(88)90593-8.
Plasmid vectors with multiple cloning sites adjacent to a vaccinia virus (VV) promoter were constructed and used to insert a protein coding sequence and a dominant selectable marker into a non-essential region of the VV genome. Recombinant viruses, selected on the basis of expression of the herpes simplex virus (HSV) thymidine kinase gene (tk), were shown to express in infected cells the model gene product, murine major histocompatibility complex (MHC) antigen H-2Kd, by cell-surface binding of antibody and by MHC-restricted recognition by cytotoxic T lymphocytes. Double recombinant VVs with insertions at two sites (in the VV tk gene and in the VV HindIII-F region) were constructed and shown to express influenza A/PR/8/34 haemagglutinin and H-2Kd antigen in addition to the HSV tk gene. The plasmids described allow the construction of recombinant VV expressing two genes of interest under the control of the same VV promoter. Such recombinant VVs can be used to study the interaction of immunologically important antigens simultaneously expressed.
构建了具有多个与痘苗病毒（VV）启动子相邻的多克隆位点的质粒载体，并用于将蛋白质编码序列和显性选择标记插入痘苗病毒基因组的非必需区域。基于单纯疱疹病毒（HSV）胸苷激酶基因（tk）的表达筛选出的重组病毒，通过抗体的细胞表面结合以及细胞毒性T淋巴细胞的MHC限制性识别，在感染细胞中表达了模型基因产物——小鼠主要组织相容性复合体（MHC）抗原H-2Kd。构建了在两个位点（痘苗病毒tk基因和痘苗病毒HindIII-F区域）插入片段的双重组痘苗病毒，并证明其除了表达HSV tk基因外，还表达甲型流感病毒A/PR/8/34血凝素和H-2Kd抗原。所述质粒能够构建在同一痘苗病毒启动子控制下表达两个感兴趣基因的重组痘苗病毒。此类重组痘苗病毒可用于研究同时表达的免疫重要抗原之间的相互作用。
