Cloning And Expression Of OMPA Gene Of Pasteurella In Rabbit And Establishment Of Rapid Diagnostic Method

Rabbit pasteurellosis is respiratory infectious disease of rabbits caused by Pasteurella multocida. The disease could happen in different age of rabbits with high morbidity and mortality. Serious economic losses in large production units could induce by the disease. A specific PCR assay was developed for diagnosis of Pasteurella multocida as well as an ELISA procedure was established for the antibody detection.A pair of primers was designed according to 16S rRNA gene of Pasteurella multocida published in Ganbank. The conditions of the PCR reaction were optimized. The minimum detection amount of PCR was 60 cfu/mL. A specific fragment of 643bp could be amplified in Standard and Isolated strains of Pasteurella multocida, Escherichia. coli and Bordetella bronchiseptica couldn't be amplified. PCR method is sensitive, specific and reliable and deserves for further detection of rabbit pasteurellosis.A pair of specific primers to amplify Pasteurella multocida outer membrane protein A (OmpA) gene based on reported gene sequences were designed.The OmpA gene was amplified by PCR and cloned into the prokaryotic expression vector pET28a(+) which named pET-OmpA. The pET-OmpA was then transformed into host strain BL21(DE3), the expression conditions were optimized and the recombinant protein was purified. Western blot analysis showed that the expression protein could be recognized by positive sera of rabbit Pasteurella multocida. The successful expression of OmpA supplies a good background for researching the function of OmpA gene and serological detection of the Rabbit pasteurellosis.An indirect ELISA was established and optimized to detect Pasteurella multocida antibody of rabbits using recombinant multi-Pasteurella multocida OmpA protein as coating antigen. The optimal procedure was shown as follows, concentration of antigen was 8μg/mL, blocked overnight at 4℃, sample serum dilution is 1:200 and conjugated antibody working dilution is 1:10000, incubated at 37℃for 60min and 90min respectively, the substrate solution developed for 15 minutes at 37℃. Specificity and reproducibility results of experimental demonstrated that the established ELISA was specific and reproducible.