Gateway技术构建小鼠血管生成素-1慢病毒表达载体及其病毒包装  

Construction of mice angiopoietin-1 gene lentiviral expression vector by Gateway technology and its virus packa-ging

作　　者：李秋平[1] 马新娜[1] 张小英[1] 许靖[1] 章晟[1] 王春枝[1] 封志纯[1] 

机构地区：[1]北京军区总医院(第二军医大学临床医学院)附属八一儿童医院,北京100700

出　　处：《临床儿科杂志》2013年第9期866-870,共5页Journal of Clinical Pediatrics

基　　金：国家自然科学基金青年基金项目(No.81000265);国家自然科学基金项目(No.81270059)

摘　　要：目的构建携带小鼠血管生成素-1(Ang-1)基因的慢病毒载体并进行慢病毒包装。方法利用Gateway技术构建携带DsRed荧光蛋白的Ang-1慢病毒载体,经PCR及基因测序鉴定后,与辅助质粒pLV/helper-SL3、pLV/helper-SL4及pLV/helper-SL5混合采用脂质体法制备DNA-Lipofectamine 2000复合物,并共同转染293FT细胞进行慢病毒包装,产生相应慢病毒颗粒,通过荧光表达情况来测定病毒滴度和感染效率。结果通过PCR、基因测序证实,Ang-1慢病毒表达载体PLV.Ex3d.P/puro-CMV>Ang-1>IRES/DsRed-Express2构建成功。经转染293FT细胞包装出具高效感染力的慢病毒,经测定病毒滴度为5×108TU/ml。结论成功构建Ang-1慢病毒表达载体,并包装出具高效感染力的慢病毒颗粒,为进一步研究Ang-1基因功能及基因治疗提供实验基础。Objective To construct a lentiviral vector carrying angiopoietin-1 （Ang-1） and DsRed gene, and to package a virus particles. Methods The Ang-1 lentiviral vector with DsRed （PLV.Ex3d.P/puro-CMV〉Ang-l〉IRES/DsRed-Express2） was constructed by Gateway technology, and identified by PCR and gene sequencing. The lentiviral vector was mixed with helper vector pLV/helper-SL3, pLV/helper-SL4 and pLV/helper-SL5 by Lipofectamine 2000 to prepare DNA-LipofeetamineTM 2000 complexes. The complexes were then added to transfect 293FT cells and package virus. The virus titers and infection ef- ficiency were determined by fluorescence expression. Results Ang-1 lentiviral vector PLV.Ex3d.P/puro-CMV〉Ang-1〉IRES/ DsRed-Express2 was constructed successfully as identified by PCR and gene sequencing. Lentivirus with high-efficiency infection was produced by transfection to 293FT cells and the virus titer was 5×10^8 TU/ml. Conclusions The recombinant len- tiviral vector for Ang-1 was successfully constructed by Gateway technology and the lentivirus with high-efficiency infectionpackaging can be used for further experiment orang-1 gene.

关 键 词：血管生成素-1 慢病毒 载体 GATEWAY技术 

分 类 号：R363[医药卫生—病理学]