Cloning And Expression Of Fimn Gene Of Bordetella Bronchiseptica In Rabbitry And Establishment Of Rapid Detection Method

Bordetella bronchiseptica infection in rabbits is a respiratory infectious disease caused by Bordetella bronchiseptica. It is described mostly as an endemic, chronic, acute and septic disease. It is difficult to be cured for the disease lasts long and occurs repeatedly. Therefore, serious economic losses are produced in rabbitry by the disease. The immunological and virulent character of Bb's pilin has been proved.The study contains four parts:the first part is the amplification of fimN gene of Bb by PCR; the second part is the expression of fimN gene in prokaryotic vector and the detection by western blot; the third part is the establishment of PCR method; the last part is the development of ELISA method.A pair of primers was designed to amplify a fragment of 648bp in length to the target gene according to the sequence of the fimN gene. The PCR products were linked with pMD-19T vector, and the positive plasmid was digested with EcoRl and HindⅢ. meanwhile it was sequenced. The result indicated that comparison of the finzN gene sequence of the amplified fragment with the published sequence of gene fimN show that they were 99%homologous, finally.the products were cloned into the vector successfully.The positive plasmid pETBb-fimN was produced by digesting with EcoRl and HindⅢand linking together with pMD 19T-fimN and pET-28a(+). The insert position and the orientation were right by digestion and sequence detection. The 27 KD target protein was produced by inducing IPTG, Western-blot which showed that the expressed protein could be recognized by the positive serum of Bb. The research established the basis for establishing ELISA method using the purified recombination protein as coating antigen.A pair of primers was designed to amplify the 387bp fragment according to the sequence of the fimN gene of Bordetella bronchiseptica, the condition of PCR reaction was optimized. A PCR assay was developed successfully for rapid detection of Bordetella bronchiseptica infection. The specificity and sensitivity assays revealed that the assay didn't react crossly with Escherichia coli, Staphylococcus, Salmonella, Clostridieum Welchii and Pasteurella.moreover, the minimum detection concentration was 10pg, consequently, the method was with high sensitivity and strong specification and deserved for further clinical detection of Bordetella bronchiseptica infection.Purified protein was used as coating antigen, rabbits were immunized with purified recombination protein and serum samples were collected for positive control.Reaction conditions were optimized includ choosing the optimal concentration of antigen and serums, selecting the best reacting time for sealing fluid as well as second antibody incubation, meanwhile, the negative critical value 0.267 was calculated. The optimized ELISA method was highly sensitive and specific and could be applied for rapid detection of Bordetella bronchiseptica infection. Finally, the method was used to analyze 50 serum samples those were collected randomly from rabbit farm. Twenty-nine of them were positive. The establishment of ELISA provided another new way for serological detection of Bb.