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-ΔSG(-SH3-GK)和Myc-ΔG(-GK),分别克隆到真核表达载体pcDNA3.1(+)。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-pcDNA3.1(+)、Myc-ΔSG-pcDNA3.1(+)和Myc-ΔG-pcDNA3.1(+)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。
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.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2007年第1期20-22,共3页
Immunological Journal
基金
国家自然科学基金(30300069)
教育部新世纪优秀人才支持计划(NCET-04-0520)
江苏省高校自然科学研究计划(02KJB310011)资助
