PSD-95酪氨酸突变体真核表达载体的构建及其表达

目的构建突变型PSD-95的重组表达质粒(Y432F、Y439F、Y523F和Y533F,酪氨酸突变为苯丙氨酸),并在COS-7细胞中表达。方法以野生型PSD-95-pcDNA3.1为模板,采用重叠延伸PCR法获得突变型PSD-95的局部或全长cDNA片段,并克隆至PSD-95-pcDNA3.1或pcDNA3.1载体;以脂质体法将构建好的重组质粒转染COS-7细胞;通过免疫印迹法鉴定PSD-95及其突变体的表达。结果限制性内切酶酶切和测序结果表明,扩增出的突变型PSD-95基因完全正确。免疫印迹显示,转染的突变型重组质粒在相对分子质量9.5×104处均有相应条带出现。结论成功构建了4个PSD-95酪氨酸残基突变体的真核表达质粒,重组质粒在COS-7细胞中高效表达。

Construction and expression of the eukaryotic expression plasmids of PSD-95 tyrosine mutants

Objective To construct the recombinants of PSD-95 tyrosine residue mutants(Y432F,Y439F,Y523F and Y533F)and to express the recombinants in COS-7 cells.Methods With PSD-95-pcDNA3.1 as the template,the PSD-95 mutant genes were amplified by overlap extension PCR,and then were cloned into the plasmids of PSD-95-pcDNA3.1 or pcDNA3.1.These recombinants were transfected into COS-7 cells through the mediation by lipofectamine reagents.Immunoblot method was used to determine the expression of wild and mutant PSD-95 proteins.Results The target genes were confirmed by restriction enzyme digestion and sequencing.The bands corresponding to PSD-95 were detected in the wild-and mutant-type of recombinant-transfected COS-7 cells.Conclusion The PSD-95 tyrosine mutants were constructed successfully and the recombinants were highly expressed in COS-7 cells,which provides a base for identifying the function of PSD-95 tyrosine residues.