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目的构建小鼠Sema4D慢病毒过表达载体。方法采用Gateway技术将体外合成小鼠全长Sema4D基因插入并重组到预先已经插入荧光标记基因片段IRES的质粒中,阳性菌落由Ampicillin筛选后,大量繁殖,获得大量转染后的质粒,经过测序鉴定后,经慢病毒包装,转染293T细胞,经由Neomycin筛选,获得高滴度的病毒液。结果测序结果显示Sema4D基因共有2 586 bp,重组慢病毒载体共有11 966bp,其中Sema4D插在CMV启动子和IRES标记序列之间,位置为2569-5144。测序结果表明小鼠Sema4D基因已经成功构建到过表达慢病毒载体中。结论小鼠Sema4D基因慢病毒过表达载体Lenti-CMV-sema4D-IRES-PGK-neo能够正确构建,该载体为研究小鼠Sema4D基因在特定细胞内过表达株的筛选奠定基础,为其作用机制的研究提供用力工具。
Objective To construct mouse Sema4D lentivirus overexpression vector. Methods The full length Sema4D gene of mouse in vitro synthesis was inserted and recombined into the plasmids which had been inserted into the IRES of fluorescent marker gene in advance by Gateway technology. The positive colonies were screened by Ampicillin and then multiplied to obtain a large number of plasmids after transfection. After sequencing, After the lentiviral packaging, 293T cells were transfected and screened by Neomycin to obtain a high titer virus solution. Results The sequence of Sema4D gene was found to be 2 586 bp in length. A total of 11 966 bp of recombinant lentiviral vector was obtained. Among them, Sema4D was inserted between the CMV promoter and IRES marker and located at 2569-5144. Sequencing results show that mouse Sema4D gene has been successfully constructed into lentiviral vector. Conclusion Lenti-CMV-sema4D-IRES-PGK-neo, a lentiviral vector expressing mouse Sema4D gene, can be constructed correctly. This vector lays the foundation for screening the mouse Sema4D gene for screening of overexpressing cells in specific cells, Research provides hard tools.