摘要
通过质粒提取、PCR扩增构建F3-PE40表达载体转化大肠杆菌,然后进行表达纯化。以pEKH-F3质粒为模板,PCR扩增F3片段,将F3插入克隆质粒PQE80L,转化大肠杆菌,筛选获得含有F3的PQE80L重组载体。提取绿脓杆菌菌株染色体DNA为模板,PCR扩增绿脓杆菌外毒素A催化区(PE40)。然后将PQE80-F3和PE40重组,得到表达PQE80L-F3-PE40载体。转化至大肠杆菌DH5a,BL21,表达融合蛋白F3-PE40。大肠杆菌表达了融合蛋白F3-PE40,表达的融合蛋白量占菌体总体蛋白量的20%。成功地表达了融合蛋白F3-PE40,为进一步大规模表达、纯化F3-PE40功能奠定了基础。
Through plasmid extraction,PCR amplification and construction,the expression vector of F3-PE40 was tranformed to E.coli,then expressed and purified.Taken the plasmid pEKH-F3 as the template,PCR was amplificated F3,then inserted into PQE80Lcarrier.PQE80L-F3 recombinated plasmid was obtained.Taken extraction pseudomonas aeruginosa strain chromosome DNA as template,PCR amplificated PE40 which is the pseudomonas aeruginosa exotoxin A catalysis area.Then recombination of PQE80-F3 with PE40,PQE80L-F3-PE40 carrier was obtained.The DH5a and BL21 were transformated and the F3-PE40 was induced.F3-PE40 fusion protein was expressed and purified.The quantity of expressed fusion protein accounted for probably 20% of total bacterial proteins.The F3-PE40 was successly expressed for further research.
出处
《生物学杂志》
CAS
CSCD
2010年第4期49-52,共4页
Journal of Biology
