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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 To construct rat Rac1 and its mutants Rac1Q61L, Rac1G12V and Rac1T17N adenovirus expression vector and prepare the virus, which lays the foundation for further study on the role of Rac1 in mesenchymal stem cell (MSCs) migration. Methods Rac1 and its mutants Rac1Q61L, Rac1G12V and Rac1T17N were amplified using pMD-19-T-Rac1, pMD-19-T-Rac1Q61L, pMD-19-T-Rac1G12V and pMD-19-T-Rac1T17N as templates The recombinant plasmid pAdTrack-CMV-Rac1 and its mutants were ligated with pAdTrack-CMV with GFP tag to co-transform BJ5183 with adenovirus backbone plasmid pAdEasy-1 to obtain recombinant adenoviral vector. PacI was linearized and then transfected into QBI-293A cell-packaging virus. Recombinant adenoviruses were harvested and virus titers were detected and multiplexed for infection with mesenchymal stem cells. Results The sequencing results showed that the sequences of Rac1 and its mutants Rac1Q61L, Rac1G12V and Rac1T17N were correct. After the recombinant virus was successfully packaged, the recombinant virus with high titer of 107pfu / ml was obtained after 2 or 3 times of amplification. After infection with mesenchymal stem cells The MOI of 150 was found to be the most suitable for infecting the virus. Conclusion The recombinant adenoviral vector pAdEasy-1-pAdTrack-CMV-Rac1, pAdEasy-1-pAdTrack-CMV-Rac1Q61L, pAdEasy-1-pAdTrack-CMV-Rac1G12V and pAdEasy-1-pAdTrack- -Rac1T17N, Rac1 recombinant virus Ad-Rac1, Ad-Rac1Q61L, Ad-Rac1G12V and Ad-Rac1T17N were obtained.