SynGAP(1-700aa)中670-685aa缺失突变质粒的构建及表达

Construction and expression of SynGAP(1-700aa) plasmid mutanted deletion at 670-685aa residues

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p CMV-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 670-685 aa 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 p CMV-SynGAP（ 1-700aa）-Flag plasmid as template,mutanted deletion 670-685 aa 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 293 T cells,collected cells after 48 hours,Western blot was used to identify whether it could express sucessfully. Results： 670-685 aa 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 670-685 aa could express successfully. Conclusion： SynG AP（ 1-700aa） plasmid mutantes deletion 670-685 aa successfully,and it can be expressed stably in cell lines. The successfully constructed plasmid will be an experimental basis for our follow-up study.