基于大肠杆菌受体菌DH5α △asd缺失株的构建及作为基因转化、表达操作系统的评估

The Construction of E.coli DH5α △asd Deletion Mutant and the Evaluation on Its Potential on Gene Transformation-expression System

基于大肠杆菌(E.coli)染色体上asd基因的已知序列,利用λ噬菌体的Red同源重组系统一步法构建E.coliDH5α的asd基因缺失突变株DH5α△asd::cat,在二次重组中利用携带能够表达FLP位点特异性重组酶的质粒pCP20介导二次同源重组,以去除上述缺失突变株中氯霉素抗性筛选基因。结合PCR扩增和测序结果,证明DH5α△asd缺失突变株的正确构建。该缺失突变株失去了在普通LB培养基上生长的能力,只有添加DAP或导入表达asd基因的质粒(asd基因互补试验)才能在LB培养基上生长,与原型DH5α比较,其生长速度和生长对数期、接受不同拷贝数质粒的转化效率几乎相一致。基于该缺失突变株构建出以asd营养基因为标志的大肠杆菌染色体-质粒平衡致死系统。体外培养连续传代50代次,pnirBMisL-fedF-asd质粒不丢失,并功能性表达F18大肠杆菌黏附素FedF。

E. coli DH5α △asd deletion mutant was constructed by using Red recombination system. First, the chloramphenicol resistance（cat）gene flanked by homology extensions of asd gene was ampli- fied by PCR. The PCR products were electro-transformed into E. coli DHSα strain, with the help of Red recombinant system, the most part of asd gene was in vivo replaced by homology extensions connected with cat gene. E. coli DH5α （Aasd：：cat）deletion mutant with cat gene was selected by LB plate with DAP and chloramphenicol. The cat gene was then eliminated by using a helper plasmid, pCP20, encoding the FLP recombinase; the mutant for the recombinant E. coli was named E. coli DH5α △asd which lost the capability of growth on LB plate. The deletion mutant recovered the capability of growth on LB plate when added with DAP component. The function of the asd deletion mutant also could be compensated by the plasmid expressing asd gene. There were no significant difference in the key characters of growth speed, growth log phase, accepting different origin plasmids with high efficiency between E. coli DH5α and E. coli DH5α △asd mutant. Based on the DH5α △asd mutant, the chromosome-plasmid balanced-lethal system was set up successfully, which was stable for 50 generations of passage culture in vitro and expressed the FedF adhesin without antibiotic resistance gene.