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SynGAP (1-700aa)中670-685aa缺失突变质粒的构建及表达
作者:张清秀  魏秀娥  高红  荣良群 
单位:徐州医科大学第二附属医院 神经内科, 江苏 徐州 221006
关键词:SynGAP 重叠延伸聚合酶链式反应 缺失突变 
分类号:Q782
出版年·卷·期(页码):2016·35·第六期(832-835)
摘要:

目的:缺失突变SynGAP(1-700aa)-Flag中的670-685aa片段,为后续实验中新的药物靶点的鉴定提供可靠理论依据。方法:以前期构建好的pCMV-SynGAP(1-700aa)-Flag质粒为模板,重叠延伸PCR技术缺失突变670-685aa片段,扩增出目的片段;限制性内切酶将载体线性化,然后分别纯化目的片段与线性化载体;利用同源重组技术连接目的片段与线性化载体,获取重组质粒;PCR技术鉴定重组质粒构建成功,测序进一步验证质粒碱基正确;将质粒转入293T细胞,免疫印迹鉴定能否成功表达。结果:重叠PCR成功缺失突变670-685aa片段,成功构建重组质粒,且测序正确。免疫印迹结果表明,SynGAP(1-700aa)缺失突变670-685aa片段后仍可成功表达。结论:成功缺失突变SynGAP(1-700aa)中的670-685aa片段,并且在细胞株中稳定表达。该质粒的成功构建,为我们的后续研究奠定实验基础。

Objective: To mutant deletion 670-685aa fragments of SynGAP(1-700aa)-Flag plasmid,to provide a reliable theoretical basis for subsequent experiments on identification of new drug targets. Methods: The constructed pCMV-SynGAP(1-700aa)-Flag plasmid as template,mutanted deletion 670-685aa and amplified fragment by overlap extension PCR(SOE PCR); then made vector linearized with restriction endonuclease,and purified amplified fragment and the linearized vector; connected the linearized vector and amplified fragment by homologous recombination technology to obtain recombinant plasmid; PCR technology was used to identify if recombinant plasmid was successfully constructed,sequence technology was used to further verify bases in constructed plasmid; and finally transfected the mutanted plasmid into 293T cells,collected cells after 48 hours,Western blot was used to identify whether it could express sucessfully. Results: 670-685aa was mutanted deletion successfully by SOE PCR,and recombinant plasmid was successfully constructed,and the sequence results was correct.Western blot analysis showed that SynGAP(1-700aa) plasmid mutanted deletion 670-685aa could express successfully. Conclusion: SynGAP(1-700aa) plasmid mutantes deletion 670-685aa successfully,and it can be expressed stably in cell lines.The successfully constructed plasmid will be an experimental basis for our follow-up study.

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