| Goose yolk peritonitis,caused by avian pathogenic Escherichia coli (APEC), is one of the commonest diseases for avian infectious diseases , which usually onsets and breaks out a in breeding geese at egg- laying peak periods, it always leads to high mortality in the infected ones with low fertility rate and natality in the developed eggs. APEC is often complicated with combination of other pathogen infection such as bacteria or viruses, and it has also caused great economic losses to the poultry industry worldwide. So far, there is no commercialized vaccine for the prevetion of the disease because of the numerous serotypes of APEC as we know, and the optimal antibiotics control is aslo more stricted due to the multi-drug resistance bacteria. So it is a hot topic and urgent project in which how to obtain a kind of live attenuated vaccine candidate with good immunogenicity, low or no virulence against APEC infection.It has been demonstrated that the loss of some host-keeping genes would effectively attenuate the virulence of bacteria, such as genes cya and crp, which are responsible for synthesis of cAMP and its receptor protein CRP respectively. Both of the two genes are essential for the regulatory genes playing important role in the transportation and degradation of nutrient substance. cAMP receptor protein(CRP) is the one of the seven general regulatory proteins which are identified in E.coli so far, the most important one. The initiation of about 50% of transcription of genes is regulated by CRP. With the further study on the function mechanism of cya and crp, the association between the two genes and virulence factors will also become a hot topic in the future research. It has been demonstrated that successful constructs of△cya△crp mutants originating from Salmonella typhimurium and APEC(serotype O2 and O78) were effectively atteuated in the animal model tests.Based on the original sequences of cya and crp gene clusters in the GenBank.The wild type APEC isolates G8107(Serotype O2:K89),G803(Serotype O39:K89,G8102(Serotype unidentified)and J5(E.coli lipopolysaccharide mutant)were selected as the prototype of cya and crp, and the two genes mutant were constructed by using Red recombination system. First, the chloramphenicol resistance (cat) gene flanked by homology extensions of cya and crp gene was amplified by PCR respectively. The PCR products were introduced into wild type strainG8107,G803,G8102 and J5 by electroporation, with the help ofλRed-mediated recombination system, the target gene was replace by homology extensions connected with cat gene, the strains expressing chloramphenicol resistance gene were selected by chloramphenicol agar, the chloramphenicol resistance gene was then eliminated by using a helper plasmid, pCP20, encoding the Flp recombinase. Using this system, cya gene in chromosome of Escherichia coli was deleted. G8107△cya,G803△cya,G8102△cya and J5△cya mutants obtained and further confirmed by PCR verification and sequencing. In the same way ,we can continue to construct the△cya△crp mutants.Based on the△cya,△cya△crp mutants, some assays related to the biochemical characteristics and function , such as glucolysis , growth , animal challenge and so on ,were carried out. We found that the mutants could not get an effective use of lactose, maltose and sucrose sugar, and have smaller growth velocity compared with wild type. Through 1 day-old chicken intratrachea inoculation, we found that the inoculated mortalityfor wild isolate , mutant△cya and△cya△crp double mutants reached 60%,20%,0% respectively.All the results described above implies the virulence of mutants was great attenuated. In this study, the APEC mutants△cya,△cya△crp were successfully constructed and showed that the mutants lead to atteuated virulence to some great extent. All these data provided a new idea to make futher study on the relationship among genes cya, crp and virulence,and also established basis for exploit high-performance live attenuated vaccine to APEC.
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