中文摘要：

目的为研究重组痘苗病毒通用载体，构建融合Zeocin和GFP双筛选标记质粒pcB-Zeo-GFP。方法质粒LeCo-G／Zeo含有Zeo-GFP融合基因片段，通过PCR反应、BamHI酶切后连接替换空白质粒pCB的筛选基因gpt，通过菌落PCR，酶切图谱分析及测序分析鉴定重组质粒，构建成功后将其与野生型痘苗病毒进行位点特异性同源重组，经Zeocin药物筛选2代后，用流式细胞仪观察重组痘苗病毒GFP表达。结果经菌落PCR，1号、5号和10号菌落中扩增出426bp的目的条带，与预期的大小完全一致，经酶切分析和DNA测序进一步验证了重组质粒pCB-zeo-GFP；该质粒与野生型痘苗病毒同源重组，得到了重组的痘苗病毒；Zeocin筛选2代后，病毒上清感染细胞，流式细胞分析7．18％的细胞中有GFP的表达，而阴性对照仅有1．43％；Zeocin药物筛选5代后，通过激光共聚焦可在80％以上的细胞中观察带GFP；提取的重组痘苗基因组DNA也扩增出预期大小目的条带。结论 含Zeocin和GFP双筛选标记的新型重组痘苗病毒不仅具有可观察性且具有药物抗性，非常容易进行筛选鉴定。

英文摘要：

Objective To construct the plasmid pCB-Zeo-GFP fusioned with Zeecin and GFP double selection marker, and construct recombinant vaccinia virus to express the double selection marker. Methods Plasmid LeGo-G/ Zeo which contained Zeo-GFP fusion gene fragment was amplified by PCR, digested by BamH I enzyme, and then ligated with the blank plasmid pCB treated with the same enzyme to replace the original marker gene gpt. Through colony PCR, the recombinant plasmid pCB-Zeo-GFP was identified by restriction map analysis and sequencing analysis, the recombi- nant vaccinia virus was constructed through homologous recombination at homology-bit point TK area. After Zeocin screening of two generations, GFP expression of the cells infected by the viral supematant was analyzed with flow cytome-try. Results The target 426 bp band was amplified on the 1st, the 5th and the lOth colonies by colony PCR, and was fully consistent with the expected size; the recombinant plasmid pCB-Zeo-GFP was verified by restriction enzyme analysis and DNA sequencing. The recombinant vaccinia vires was constructed through homologous recombination and Zeocin screening of the two generations, GFP expression of the ceils infected with the viral supernatant was up to 7.18% by flow cytometry while the control was up to 1.43%. Through Zeocin screening of the five generations, GFP was observed on more than 80% cells by laser confocal and the target band was amplified from recombinant vaccinia virus genomic DNA. Conclusions The recombinant vaccinia virus is not only observable but also resistant to drug resistance, so it is easy to be screened and identified.