摘要
目的构建抗癫痫肽(anti-epilepsy peptide,AEP)/GST融合表达载体,原核表达GST-AEP融合蛋白。方法从克隆载体质粒pGEM-T/AEP中双酶切,得到AEP片段;通过中间载体puc18,转换酶位点;最后将AEP基因克隆入表达载体pGEX-4T-1质粒中,构建融合表达载体pGEX-4T-1/AEP;转化感受态大肠杆菌DH5α,经异丙基-β-D硫代半乳糖苷(IPTG)诱导表达,获得GST-AEP融合蛋白;SDS-PAGE分析表达产物,确定蛋白质高表达条件;超声裂解法鉴定蛋白质可容性;凝胶薄层扫描确定蛋白质相对表达量。结果经酶切鉴定、测序证明,AEP基因已正确插入到pGEX-4T-1中,经IPTG诱导后,表达出相对分子质量约为36 000的融合蛋白,该融合蛋白以包涵体形式存在,约占总蛋白质的70%左右。结论pGEX-4T-1/AEP载体构建成功,融合蛋白在大肠杆菌中获得高效表达。
Purpose To clone the anti-epilepsy peptide（AEP）gene into pGEX-4T-1 plasmid and to express the corresponding fusion protein. Methods The vector pGEM-T/AEP was digested with BamH I/EcoR I and the product recovered was cloned into expression vector P GEX-4T-1 after the restriction sites was changed finally. The protein GST-AEP was expressed in E. coli-DH5α as a fusion protein induced by IFTG. The fusion protein was identified as inclusion body by 12% SDS-PAGE and thin-layer scanning analysis. Results AEP cOding region was cloned into pGEX-4T-1 vector and the sequence was confirmed to be right. The fusion protein GST-AEP was correctly expressed and identified as inclusion body. The purity of GST-AEP was about 70 %. Conclusion Fusion expressing vector of pGEX-4T-1/AEP was successfully constructed and expressed.
出处
《中国生化药物杂志》
CAS
CSCD
2007年第3期148-151,共4页
Chinese Journal of Biochemical Pharmaceutics
基金
国家自然科学基金资助项目(No.30671764)
关键词
抗癫痫肽
GST
载体
融合蛋白
原核表达
anti-epilepsy peptide
GST
vector
fusion protein
prokaryotic expression
作者简介
张德安（1975．），男，辽宁辽阳人，主治医师，硕士研究生
王宗仁，通信作者，Tel：029-82376992，E-mail：zongren@fmmu．edu．cn。
