Screening And Identification Of The Differential Genes Of Genomic DNAs Between Bovine Brucella Virulent And Vaccine Strain

Brucellosis is an important zoonosis caused by bacterial of genus Brucella. Since 1887, Brucellosis as an infectious disease has been proposed for the first time so far over a hundred years. Every year Brucellosis caused economic losses amount to billions, and posed a serious hazard on human health and livestock production. So the prevention and diagnosis of Brucellosis are vital. At present the most effective measure of prevention and control of Brucellosis is vaccine inoculation. However,we can not distinguished vaccination from the infected animals,which has seriously affected the diagnosis,quarantine and the prevention of Brucellosis. Deeply understanding of Brucella virulent and vaccine strain genome differential gene is an essential to establish differential diagnosis. This study carried out screening and identification of the differential genes of genomic DNAs between Bovine Brucella virulent and vaccine strain, using representational difference analysis of the serious hazards of bovine Brucella virulent strain with the current widespread application of attenuated vaccine strains for differences in levels of genomic DNA analysis and identification.1. Screening and identification of the differential genes of genomic DNAs between Bovine Brucella virulent 544 and vaccine strain 104MBy extracting bovine Brucella virulent 544 genomic DNA and vaccine strain 104M genomic DNA, got a clear genomic DNA bands. The genome digested with the enzyme Dpnâ…¡, we obtained 250~500bp small DNA fragments. Purified product of enzyme digestion and connected specific R12/24 connector. Taq DNA polymerase filled level and used the appropriate connectors R24 as primers for PCR amplification to be a representative amplicon, the tester amplicon and driver amplicons.Digested representative amplicon with Dpnâ…¡to out both ends of the same sticky ends. Tester DNA connected with a new connector, and added excessive amount of driver DNA, and then carried out denaturation and renaturation. The tester DNA's 5 'end added oligonucleotide primers for selective PCR. Amplification digested with Mung bean nuclease to remove single-stranded DNA for PCR amplification,that is the product of the first round of subtractive hybridization.After three rounds of subtractive hybridization we obtained hybrid product of 544 and 104M.Connected the purification of hybrid products with the cloning vector. After transformed, picked the single colonies identified by PCR and sequenced. Through Southern blot verification, five fragments are specific to virulent strain 544 and ten fragments are unique to vaccine strains 104M.2. Screening and identification of the differential genes of genomic DNAs between Bovine Brucella virulent 544 and vaccine strain S2By extracting bovine Brucella virulent 544 genomic DNA and vaccine strain S2 genomic DNA, we have got a clear genomic DNA bands. Digested the genome with the enzyme Dpnâ…¡, we obtained 250~400bp small DNA fragments. Purified product of enzyme digestion in turn, prepared 544 and S2 amplicons,544 and S2 subtractive hybridization and differences in genetic screening. After three rounds of subtractive hybridization we obtained hybrid products of 544 and S2.The purification of hybrid product connected cloning vector. Connected the purification of hybrid products with cloning vector. After transforming, identified the single colonies by PCR and sequenced. Through Southern blot verification, six fragments were unique to virulent strain 544 and twelve fragments is strain-specific of vaccine S2.3. Bioinformatics analysis and PCR identification of differences fragments between Brucella virulent 544 and vaccine strains 104M and S2In order to obtain more extensive bovine brucella virulent with the current widespread application of attenuated vaccine strains for differences in genomic DNA, we obtained the virulent strain 544 and 104M subtractive hybridization differential gene fragments, and the virulent strain 544 and S2 subtractive hybridization differential gene fragments bioinformatics analysis respectively showed that 544 and 104M variance analysis of gene fragments obtained, virulent strain 544-specific differences 1-16,1-67,1-70 in gene fragments in three of all cattle have been sequenced virulent strains of Brucella have 99%~100% homology, but has no homology with the Brucella vaccine strain S19 which has been only sequenced vaccine strain. In 544 and S2 gene fragments obtained by variance analysis, S2-specific differences 2-21#,2-34#,2-76# in gene fragments in three of all Bovine Brucella virulent strain which have been sequenced has no homology. Sequenced and compared this six differential gene fragments, confirmed the virulent strain 544-specific differences in gene fragments for the 544 and 104M vaccine strain differences exist in the genomic DNA,S2 vaccine strain-specific differences in gene fragments for the 544 and S2 differences exist in the genomic DNA. Eventually gene fragments identified by PCR with virulent strains of 544 and vaccine strains of 104M, and identified gene fragments with virulent strains of 544 and vaccine strains of S2. All of these laid the foundations for establising differential diagnosis to distinguish animals of Brucella infection and vaccination 104M and S2.