基因工程构建表达融合蛋白GST-FCoAT的益生大肠杆菌EcN-Frc

Construction of probiotic Escherichia coli Nissle-Frc expressed GST-FCoAT fusion protein by genetic engineering

目的:将产甲酸草酸杆菌代谢草酸基因Frc克隆至表达载体PGEX-4T-2,转化大肠杆菌EcN,稳定表达甲酰辅酶A转移酶。方法:采用PCR方法从产甲酸草酸杆菌基因组中获得Frc基因,插入到载体PGEX-4T-2,转化EcN,IPTG诱导表达GST-FCoAT融合蛋白,产物行western-blot鉴定分析。结果:EcN-Frc可稳定表达可溶性融合蛋白GST-FCoAT。结论:改造后的EcN能诱导表达甲酰辅酶A转移酶,将为下一步体内外代谢草酸实验和临床治疗高草酸尿和防治草酸结石的益生菌制剂的研究奠定基础。

Objective To clone oxalate-degredation gene Frc of oxalobacter formigenes （OF） into vector PGEX-4T-2, and stably express Formyl-CoA transferase （FcoAT） in probiotic E. coli Nissle （EcN）. Methods Gene Frc was amplified by PCR and verified by sequencing, then it was extracted and inserted into the prokaryotic expression vector PGEX-4T-2; finally the recombinant expression plasmid PGEX-4T-2-Frc was transformed into EcN and induced to express the GST-FCoAT fusion protein with IPTG. The product was identified and analyzed by Western blot. Results GST-FCoAT fusion protein was stably expressed in EcN-Frc. Conclusions The altered EcN can be induced to express FCoAT, which will lay the foundation for the further study of in vitro-in vivo oxalate metabolism and clinical treatment of hyperoxaluria and prevention of oxalate stones.