Rac1及其突变体重组腺病毒载体的构建与鉴定
Construction and identification of recombinant adenovirus vector of rac1 and its mutants
康乃馨 1朱爱思 1曲静 1徐晓静 1张焕相1
作者信息
- 1. 苏州大学基础医学与生物科学学院;江苏省干细胞研究重点实验室,江苏苏州215123
- 折叠
摘要
目的 构建大鼠Rac1及其突变体Rac1Q61L、Rac1G12V、Rac1T17N腺病毒表达载体,并制备出病毒,为进一步研究Rac1在间充质干细胞(MSCs)迁移中的作用奠定基础.方法 以pMD-19-T-Rac1、pMD-19-T-Rac1Q61L、pMD-19-T-Rac1G12V、pMD-19-T-Rac1T17N为模板,扩增Rac1及其突变体Rac1Q61L、Rac1G12V、Rac1T17N,酶切连接到带有GFP标记的pAdTrack-CMV上,PmeI线性化重组质粒pAdTrack-CMV-Rac1及其突变体重组质粒,与腺病毒骨架质粒pAdEasy-1共转化BJ5183细菌,获得重组腺病毒载体,经PacI线性化后转染QBI-293A细胞包装病毒,收获腺病毒重组病毒子,检测病毒滴度并进行间充质干细胞病毒感染复数的鉴定.结果 测序结果显示,Rac1及其突变体Rac1 Q61L、Rac1G12V、Rac1T17N序列正确,成功包装出重组病毒子后经2～3次扩增得到约为107 pfu/ml高效价重组病毒子,感染间充质干细胞后发现150 MOI为最适病毒感染复数.结论 成功构建了Rac1及其突变体重组腺病毒载体pAdEasy-1-pAdTrack-CMV-Rac1、pAdEasy-1-pAdTrack-CMV-Rac1Q61L、pAdEasy-1-pAdTrack-CMV-Rac1G12V、pAdEasy-1-pAdTrack-CMV-Rac1T17N,获得了Rac1重组病毒子Ad-Rac1、Ad-Rac1Q61L、Ad-Rac1G12V、Ad-Rac1T17N.
Abstract
Objective The recombinant adenovirus vector of Racl and its mutants were constructed for study the role racl gene plays in the migration of MSCs. Method The pAdTrack-CMV-Racl was constructed by PCR with pMD-19-T-Racl recombinant plasmid as template, enzyme digestion and ligation. The pAdTrack-CMV-Racl lineared by Pmel was co-transformed into BJ5183 with pAdEasy-1. The pAdEasy-1-pAdTrack-CMV-Racl recombinant adenovirus vector was lineared with Pad and then transfected into the package cell QBI-293A. The Racl and its mutants recombinant adenovirus were obtained and the virus multiplicity of infection ( MOI) of mesenchymal stem cells were identified after the detection of the virus titer. Results The Racl and its mutants were confirmed by sequencing, and about 107 pfu/ml of high titer recombinant virus was successfully packaged after 2-3 amplification. For mesenchymal stem cells infection, the optimal virus of Ad-Racl was 150MOI. Conclusion The pAdEasy-1-pAdTrack-CMV-Racl, pAdEasy-l-pAdTrack-CMV-RaclQ61L, pAdEasy-1- pAdTrack-CMV-RaclG12V, pAdEasy-1-pAdTrack-CMV-RaclT17N vectors were constructed and the Racl and its mutants recombinant adenovirus ( Ad-Racl, Ad-RaclQ61L, Ad-RaclG12V, Ad-RaclT17N) were obtained successfully.
关键词
Rac1/腺病毒载体/构建/鉴定Key words
Rac1/adenovirus vector/construction/identification引用本文复制引用
基金项目
国家自然科学基金(30671041)
国家自然科学基金(30870642)
国家自然科学基金(31071220)
江苏省高校自然科学重大基础研究项目(06KJA18025)
江苏省自然科学基金资助项目(BK2009119)
出版年
2012
NETL
NSTL
维普
万方数据