中文摘要：

目的构建野生型和突变型PSD-95（postsynaptic density protein95）的真核表达质粒载体，并在COS-7细胞系中表达。方法采用RT-PCR扩增大鼠PSD-95的cDNA，以PSD-95-GW1为模板，以PCR扩增PSD-95序列的突变体Myc-△SG（-SH3-GK）和Myc-△G（-GK），分别克隆到真核表达载体peDNA3．1（＋）。以脂质体法将重组质粒转入COS-7细胞，免疫印迹法鉴定PSD-95及其突变体的蛋白表达。结果限制性内切酶酶切和测序结果表明，扩增出的野生型，突变型PSD-95基因完全正确；免疫印迹法检测表明，转染的重组质粒能在COS-7细胞中高效表达。结论构建的PSD-95-peDNA3．1（＋）、Myc-ASG-peDNA3．1（＋）和Mye．AG-peDNA3．1（＋）重组载体在COS-7细胞系中高效地表达，为深入研究PSD-95在缺血性脑损伤中的作用及机制打下基础。

英文摘要：

Objective To construct a expression system for wild/mutant postsynaptie density protein 95 （PSD-95） and express the vector in COS-7 cells. Methods The full-length gene was amplified by RT-PCR. With PSD-95-GW1 （gift from Sheng M） as a template, Myc- △SG and Myc-AG PSD-95 mutant genes were amplified by PCR. The wild/mutant PSD-95 genes were cloned into the expression vector pcDNA3.1 （ ＋ ）, and then the right recombinants were transformed into COS-7 cells by lipofectamine reagent. The immunoblotting method was used to determine the expression of wild/mutant PSD-95 proteins. Results The correct clonings of PSD-95-pcDNA3.1 （ ＋ ）, Myc-ASG-pc DNA3.1（ ＋ ）, and Myc-AG-pcDNA3.1（ ＋ ） were confirmed by restriction enzyme digestion and sequencing. PSD-95, Myc-ASG, and MycAG proteins were highly expressed in COS-7 cells. Conclusion The expression of PSD-95 proteins and mutants will provide a base for investigating the role and the mechanism of PSD-95 in ischemic brain damage.