Identification Of Specific Proteins Of Escherichia Coli Associated With Salpingitis-peritonitis In Geese And Preparation Of Monoclonal Antibody

Goose salpingitis and peritonitis, also known as "goose egg-plague" in China, mainly occurs in egg-laying geese. The pathogen of the disease is E.coli which could infect the genital of geese. When the reproductive system of goose is infected by the E.coli, especially infected tubal, the mature ovum can’t enter the fallopian tube, which fall into the abdominal cavity, eventually leading to the yolk peritonitis. If the disease can not be diagnosed and treated in time, it would cause goose egg production drop or not produce eggs, even die from the disease. Goose salpingitis and peritonitis is one of important diseases in goose industrySince the disease was first reported in the sixty’s of last century, its clinics and preventions have been discribed in Jiangsu, Hubei, Guangdong, Henan, Anhui, Guangxi, Sichuan and other provinces. However there are few reports abroad about the disease. Only a few scholars have reported salpingitis and peritonitis in egg-laying hens and its coinfection pathogens. In this study, we applied proteomics to investigate the pathogenesis of E.coli causing salpingitis and peritonitis and explore the mechanism of yolk peritonitis, its clinical prevention and control.1. Idendification of specific proteins of E.coli caused salpingitis and peritonitisIn order to identify the specific proteins of E.coli caused salpingitis and peritonitis, the whole proteins were extracted from the experimental bacteria and other control strains, then the proteins were separated by two-dimensional electrophoresis. The2-DE maps of total proteins were analyzed by PDQuest8.0.1. The results showed that42spots expressed only in E.coli caused salpingitis and peritonitisd.25of these spots were further analyzed by MALDI-TOF-MS.21spots were successfully identified.According to the results of MALDI-TOF-MS and bioinformatics analysis,11identified specific proteins were detected with PCR and RT-PCR. The nucleotide sequences of the proteins were searched and the primers for the11proteins were synthesized. PCR and RT-PCR analyzed the genes encoding of these proteins and transcripton. The results showed that the encoding genes of most proteins were found in normal E.coli, but30S ribosomal protein S6was found expressed only in goose pathogenic E.coli.2. Prokaryotic expression of the RPS6geneAccording to the RPS6gene sequence （gi|218707811|ref|YP₀02415330.1）, a pair of specific primers was designed and synthesized.The genomic DNA was extracted from pathogenic E. coli strains from geese and used as template to amplify the RPS6gene by PCR. The RPS6fragment was cloned into the T vector and sequenced. The result showed that RPS6fragment was396bp. Comparing with the sequences of the RPS6gene published in GenBank, the homology was99.7%to other E.coli. RPS6fragment was digested from the recombinant plasmid pGEM-T-RPS6by BamHⅠ and HindⅢ enzymes and subcloned into the expression vector pET-32a（+）, and then transformed into competent E.coli DE3/BL21cells. The positive recombinant pET-32a （+）-RPS6/DE3clones were identified by double enzyme digestion and then expressed by IPTG inducer. SDS-PAGE analysis result indicated that the expressed fusion protein was soluble, and the molecular weight of the fusion protein was about34kDa. Using the HisTrap FF Ni2+column, the protein His-RPS6was purified.3. Development of monoclonal antibodies to RPS6Three monoclonal antibodies against RPS6, named RPS6-2C2, RPS6-3B1and RPS6-5H7were developed by fusions between SP2/0cells and spleen cells from BALB/C mice immuned with His-RPS6fusion protein expressed in E.coli, Western-blot results showed that the monoclonal antibodies could react with the recombinant His-RPS6fusion protein expressed by E. coli but didn’t react with the His protein. The monoclonal antibodies could react only with the pathogenic E.coli from goose but didn’t react with other phthogenic E.coli. The immunoglobulin subtypes of monoclonal antibodies were identified with acommercial capture-ELISA kit. The heavy chain types of the three monoclonal antibodies were IgG1and the light chains all were Kappa. The ascite titer of2C2monoclonal antibody was1:409600and its cell supernatant titer was1:2560. The ascite and supernatant titers for3B1and5H7monoclonal antibodies were1:51200,1:640and1:204800,1:12800respectively. These monoclonal antibodies would be potential tools for the functional research of RPS6.