Study On Diagnostic Method Of Bordetella Caniculum In Rabbit And Cloning And Prokaryotica Expression Of FimN Gene

The paper has four parts. The first is the establishment of ELISA method, the second is the establishment of PCR method,the third is nucleotide sequencing of fimN gene of the Bb, the last one is the expression of the fimN gene of the Bb in prokaryotic vector. The results are:1. 50 rabbits seras were collected randomly from Yangling as samples.Sterilized vaccine made by standard Bordetella bronchiseptica was immunited rabbits, then we collected seras as positive seras. We use clinical health rabbits seras or somatic inhibited agglutination test negative rabbits cesarean section fetus sera as negative sera.Bordetella bronchiseptica CUCC718 was made into outer membrane protein. Selecting appropriate consistency of antigen, antibody and enzyme-labelled,we did ELISA for examine sera and obtained positive determining valure, setting blank, positive and negative well.If OD valure of samples were more than positive determining valure,it was positive, other than it was negative. We use ELISA and inhibited agglutination examine 50 seras, results in 21 positive seras, positive percent is 42%.2.With a pair of primers designed to amplify fimbrial gene of Bordetella bronchiseptica according to the sequence of the gene, a fragment of 648bp in length on the target gene was amplified by PCR and a standard PCR assay was developed for quick detection of Bordetella bronchiseptica infection. Specificity and sensitivity assays revealed that the assay did not cross react with Escherichia coli, Staphylococcus and Pasteurella. 50 samples of nasal mucus from rabbits from Yangling was examined by the developed PCR assay and 70% were positive.Comparison of the assay with the conventional bacterioscopy and the micro-agglutination test revealed that the sensitivity of the PCR assay was 3.89 times higher than that of the bacterioscopy and 1.94 times higher than that of the micro-agglutination test.3. The fimN gene was amplified from Bordetella bronchiseptica chromosomal DNA by PCR. The PCR products about 648bp in length was cloned into a pMD-19T vector and then was digested with EcoRâ… a nd Hindâ…¢. The same fragment 648bp was got. The positive plasmid was sequenced. Comparing the DNA sequence of the amplified fragment with the published sequence of gene fimN showed that they were 99% homologous.4.The recombinant plasmids pETBb-fimN were obtained after being cloned pMD19T-fimN into expression vector pET-32a(+). The insert position and the orientation were right by digestion and sequence analysis. The 41.71 ku target proteins were produced by inducing with IPTG. Western-blot showed that the expressed proteins could be recognized by positive serum of Bb. Then, the recombinant fimN protein makes the basis for establishing ELISA method.