表达红荧光蛋白的重组痘苗病毒的包装与鉴定  

Packing and identifcation of recombinant vaccinia virus expressing the RFP gene

作　　者：沈百庆 程乾[1] 罗砚曦[2] 阎辉[2] 郑骏年[1] 

机构地区：[1]徐州医学院肿瘤研究所,江苏徐州221002 [2]浙江省医学科学院药物所,浙江杭州310013

出　　处：《徐州医学院学报》2015年第5期286-290,共5页Acta Academiae Medicinae Xuzhou

基　　金：国家自然科学基金面上项目（81172137）;江苏省高校自然科学基金面上项目（13KJB320028）;江苏省肿瘤生物治疗协同创新项目

摘　　要：目的包装并鉴定表达红荧光蛋白（RFP）基因的重组痘苗病毒（VV—RFP）。方法以质粒pDsRed-express—C1为模板，PCR扩增出RFP基因，并克隆至痘苗病毒转移载体pCB中，经酶切分析和DNA测序鉴定正确，得到阳性重组质粒pCB—RFP。将该重组质粒用脂质体转染293细胞后再用野生型痘苗病毒感染，通过同源重组获得重组痘苗病毒，利用RFP和gpt抗性标志筛选和噬斑纯化获得重组痘苗病毒VV—RFP。结果成功包装出表达RFP基因的重组痘苗病毒。结论包装的VV—RFP为进一步构建携带特定治疗基因的重组痘苗病毒奠定了基础。Objective To pack and identify the recombinant vaccinia virus （VV -RFP） expressing red fluorescent protein （RFP） gene. Methods The full - length sequence of RFP gene was PCR - amplified using plasmid pDsRed - express - C1 as the template. The RFP fragment was cloned into pCB vaccinia expression vector and the resulting recombinant plasmid pCB - RFP was confirmed correct by restriction analysis and DNA sequencing. After 293 cells were transfected with the recombinant plasmid using liposome then infected with wild - type vaccinia virus, the VV - RFP was packed through homologous recombination and subsequently selected and screened by plaque - purified, taking advantage of RFP marker and gpt resistance. Results The recombinant vaccinia virus VV - RFP expressing RFP gene was constructed successfully. Conclusion The packing of VV - RFP will provides a useful tool for the construction of recombinant vaccinia virus carrying therapeutic genes.

关 键 词：红荧光蛋白 痘苗病毒 同源重组 

分 类 号：R730.54[医药卫生—肿瘤]