Construction Of QseC Gene Deletion Mutant Of Chicken Pathogenic Escherichia Coli And Its Primary Biological Characteristics

Colibacillosis refers to local or systemic infectious diseases caused entirely or partly by avian pathogenic Escherichia coli (APEC), including septicaemia, granuloma (Hjarre’S disease), air sac disease, chronic respiratory disease(CRD), avian cellulitis, swollen head syndro, peritonitis, salpingitis, osteomyelitis/synovitis, panophthalmitis, and omphalitis/yolk sac infection. Colibacillosis is a very common infectious disease in poultry industry and can cause the significant economic loss over the world.391samples were collected among2005-2010in four large-scale chicken farms in Henan province.235Escherichia coli strains were isolated and then identificated by API bacterial identification system. Firstly, qseC genes were detected by PCR. Then, the challenge tests in mice were performed to determine the pathogenicity of these E. coli strains.Consequently, the qseC gene deletion mutant was constructed by double-crossover homologous recombination. Firstly, qseC gene was amplified by PCR and ligated it into the T-Easy vector. Then, zeocin resistance gene was inserted into the target gene qseC using the molecular cloning method. After this, the vector T-qseC-zeo with zeocin resistance gene marker was constructed. After PCR and digest confirmation, the positive plasmid was subjected to sequencing. The fragment qseC-zeo was amplified by PCR, and then transformed into avian pathogenic Escherichia coli isolate E401. The mutant E401ΔqseC::zeo was screened according to the homologous recombination. After PCR confirmation, the positive plasmid was subjected to sequencing to confirm whether the mutant is successful. The genomic DNA of wild-type strain and the mutant was extracted, and digested by EcoRl. With the zeocin resistance marker gene-labeled probe, whether the qseC gene was knock out was confirmed by Southern blot.Then, the primary biological characteristics of the wild-type strain and the mutant, such as the growth curve and the vitro competition ability, were analyzedPCR results showed65qseC-positive strains were screened. All of them were resistance against the most antibiotics, but susceptible to zeocin by the drug. Through the changellege tests in mice,12qseC-positive strains were screened.After the multiple molecular cloning, transformation, PCR amplification and plasmid restriction map analysis, a qseC gene mutant was successfully screened, designated E401ΔqseC::zeo. This mutant was further confirmed by Southern blot, qseC was successfully replaced by the zeo gene.The growth curve of the wild-type strain and the mutant revealed that the growth capacity of the mutant was significantly reduced after the deletion of qseC gene. In vitro competition tests showed that the competition ability of mutant was mildly impaired. These data inferred that the pathogenicity of the mutant was probably slightly attenuated compared to that in the wild-type strain.In summary, the qseC gene was prevalent among these E. coli strains isolated from four large-scale chicken farms in Henan Province in May2005-November2010. Parts of these strains were pathogenic, inferring that the qseC gene may play an important role in the pathogenicity of these strains. By the double-cross homologous recombination method, the qseC gene deletion mutant E401ΔqseC::zeo was successfully constructed and further confirmed by Southern blot. The growth curve and in vitro competition tests of the wild-type strain and the mutant revealed that the growth capacity of the mutant was significantly reduced and the competition ability of mutant was mildly impaired after the deletion of qseC gene, infering that the pathogenicity of the mutant was probably slightly attenuated compared to that in the wild-type strain. This study provided a new strategy and means to search for new antibacterial drug targets for prevention and control of animal pathogens.