摘要
目的:为制备重组小鼠Pem(以下简称mPem)-谷胱甘肽巯基转移酶(GST)融合蛋白,作为研究mPem蛋白功能的材料.方法:根据携带小鼠Pem基因编码序列的模板质粒pEGFP/mPem设计合成特异性引物,PCR扩增小鼠Pem基因编码序列,并插入融合蛋白原核表达载体pGEX-4T-3中,得到重组表达质粒pGEX-4T-3/mPem,用此重组质粒转化大肠杆菌BL21细胞,IPTG诱导重组菌表达mPem蛋白,SDS-PAGE及WesternBlot鉴定表达产物.结果:重组菌株明显诱导表达出预期相对分子质量49000的融合蛋白.结论:成功构建了mPem-GST原核表达质粒,并在大肠杆菌中表达出mPem-GST融合蛋白,为mPem蛋白功能的研究打下了基础.
Aim:To obtain GST-mouse Pem(mPem) fusion protein as material for the study of function of mPem. Methods: According to the template plasmid pEGFP/mPem, a pair of specific primers were designed and synthesized, then the entire mouse Pem gene (mPem) cDNA coding sequence was amplified by PCR. The amplified product was cloned into prokaryotic GST fusion protein expression plasmid pGEX-4T-3 and a recombinant plasmid marked pGEX-4T-3/mPem was obtained. The recombinant plasmid was transformed to E. coli BL21 and the GST-mouse Pem fusion protein was induced to express with IPTG. GST-mPem fusion protein was detected by SDS-PAGE and Western blot assays. Results: A 49 000 Dalton fusion protein, as expected, was obtained evidently. Conclusion: A prokaryotic system capable of expressing GST-mPem fusion protein was successfully constructed. This will facilitate our study of the function of mPem.
出处
《暨南大学学报（自然科学与医学版）》
CAS
CSCD
2003年第1期68-72,共5页
Journal of Jinan University(Natural Science & Medicine Edition)
基金
国家自然科学基金(30070422)
