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

作　　者：康乃馨[1,2] 朱爱思[1,2] 曲静[1,2] 徐晓静[1,2] 张焕相[1,2] 

机构地区：[1]苏州大学基础医学与生物科学学院 [2]江苏省干细胞研究重点实验室,江苏苏州215123

出　　处：《苏州大学学报（医学版）》2012年第4期494-498,509,共6页Suzhou University Journal of Medical Science

基　　金：国家自然科学基金资助项目(30671041;30870642;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)

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Racl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,Racl及其突变体Rac1Q61L、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107pfu/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Racl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Racl重组病毒子Ad-Rac1、Ad-Rac1Q61L、Ad-Rac1G12V、Ad-Rac1T17N。Objective The recombinant adenovirus vector of Rac1 and its mutants were constructed for study the role rac1 gene plays in the migration of MSCs.Method The pAdTrack-CMV-Rac1 was constructed by PCR with pMD-19-T-Rac1 recombinant plasmid as template,enzyme digestion and ligation.The pAdTrack-CMV-Rac1 lineared by PmeI was co-transformed into BJ5183 with pAdEasy-1.The pAdEasy-1-pAdTrack-CMV-Rac1 recombinant adenovirus vector was lineared with PacI and then transfected into the package cell QBI-293A.The Rac1 and its mutants recombinant adenovirus were obtained and the virus multiplicity of infection（MOI） of mesenchymal stem cells were identifited after the detection of the virus titer.Results The Rac1 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-Rac1 was 150MOI.Conclusion The pAdEasy-1-pAdTrack-CMV-Rac1,pAdEasy-1-pAdTrack-CMV-Rac1Q61L,pAdEasy-1-pAdTrack-CMV-Rac1G12V,pAdEasy-1-pAdTrack-CMV-Rac1T17N vectors were constructed and the Rac1 and its mutants recombinant adenovirus（Ad-Rac1,Ad-Rac1Q61L,Ad-Rac1G12V,Ad-Rac1T17N） were obtained successfully.

关 键 词：RAC1 腺病毒载体 构建 鉴定 

分 类 号：R394.3[医药卫生—医学遗传学]