Isolation And Identification Of Actinobacillus Pleuropneumoniae And Screening Of In Vivo Induced Antigen Of AAP Serotype7

The Porcine Contagious Pleuropneumonia （PCP） is composed of Actinobacillus pleuropneumoniae （APP）,caused a highly contagious disease, the development of the disease threatening the world pig industry, bring huge economic losses to the pig industry.In this study we Isolation and Identification of Actinobacillus pleuropneumoniae and Screening of In vivo induced antigen of AAP serotype7.1. Isolation and Identification of Actinobacillus pleuropneumoniae1 strain Actinobacillus pleuropneumoniae（APP） serotype 7 G- coccobacillus was isolated from samplesof sick pig suspected Porcine Contagious Pleuropneumonia infection collected from a pig farm in Sichuan province, and was indentified through the bacterical colony,bacteria morphological observation, biochemistry reaction,PCR identification,serological test and drug test. Like the mosts Biochemistry reaction results, the strain can ferment with Mannose, Glucose、Urease et al.PCR results display as expected there have stripe in 450bp. Double diffusion in two dimmention assay （DDTA） with positive serum that isolates this strain belonged to serotyope7.The drug susceptibility test results showed that the APP isolates were sensitive mosts antibiotic, just resistance lincomycin.2. Establishment of APP7 genome librarypET-28a/b/c vehicle expression system were adopted in this study. Genome DNA of AAP serotype7 stain was extracted, with 0.5-2 kb DNA segments recycled after incomplete digestion with Sau3A I. Segments were inserted into pET-28 a/b/c expression vehicle system digested by its isocaudarner BamH I and CIAP enzyme dephosphorylated; the product were electro transformed into DH5acompetent cells, and positive recombinant were screened by using kanamycin LB plates. Positive clones were randomly selected for double endonuclease identification and PCR identify after plasmid extraction, the results demonstrate that most insertions were 0.5～2 kb, and total collection of positive recombinant clones were 26000 approximately, higher than the requirement of a library, indicating the success of library establishment.3. Screen of genome library by immunoblottingGenome library transformed organism fluid were evenly distributed on LB K+ plates, each plate have about 300～500 colonies. Positive colonies were 10h 37℃ cultured and printed on NC film.The NC films were transferred on LB plates with Kana and IPTG for induced culture 4 h, and 15 min over chloroform for protein from organism lysates. NC film was blocked with 10% nonfat skim milk overnight at 4℃. Each NC membrane was reacted with adsorbed convalescent sera and horseradish peroxidase labeled sheep anti pig IgG for 1～2 h in RT. Those with apparent denser color than background were determined as positive clones in primary screen,a second screen was performed with empty pET-28 a/b/c vehicles as negative control for eliminating false positives. A total of 6 positive clones were determined, with three of the Fts A, Fts K and APP7₁224 possible ivi gene.