摘要
目的构建并表达出PTK重组蛋白,以鉴定酪氨酸蛋白激酶(PTK)的活性,筛选酪氨酸激酶的抑制剂。方法从PTK重组克隆载体上切下目的基因片段Abl-PTK使其连接到表达载体pGEX4T-2上,转化大肠杆菌DH5a,筛选鉴定出正确的转化子。转化菌株经IPTG诱导后进行表达并进行SDS-PAGE分析。结果经酶切和诱导表达鉴定,重组质粒pGEX4T-2-PTK构建成功,并高效表达了58KD的GST-PTK融合蛋白。结论PTK基因被成功地重组至融合蛋白表达载体中,并在大肠杆菌中获得高效表达。该研究为进一步纯化、鉴定PTK的活性,筛选PTK的抑制剂奠定了基础。
Objective To obtain the recombinant protein tyrosine kinase （PTK） in order to identify the activity of PTK and screen the inhibitors of PTK . Method to digest the target gene PTK from cloning plasmid containing PTK and cloned into expression vector pGEX4T - 2. The recombinant plasmid pGEX4T - 2 - PTK was identified with restriction analysis to choose the right recombinant transformant E. coli DH5a containing pGEX4T - 2 - PTK. The engineering E. coliDH5a/pGEX4T - 2 - PTK were induced to express by IPTG and the expressed products were analysis with SDS - PAGE. Result Restriction analysis showed that the plasmid pGEX4T - 2 - PTK was constructed successfully. SDS - PAGE analysis result showed special band of the induced product was observed at the position of 58KD. Conclusion The fusion protein GST - PTK was successfully expressed in E. coli. This study established a good foundation for the purification of PTK and identification of PTK inhibitors.
出处
《上海生物医学工程》
CAS
2006年第1期31-34,共4页
Shanghai Journal of Biomedical Engineering
关键词
PTK
表达
PTK活性
Abl - PTK cloning expression PTK activity
