The Cloning Of Bp26 And OMP10 Gene And Study Of PCR Detection Brucella In Hohhot

Primers were designed and synthesized according to the nucleotide sequence of bp26 gene and OMP10 genes of Brucella reported in GenBank. With the specific primers, target fragments were amplified by Polymerase Chain Reaction depending on the template of total DNA isolated from Hohhot region.The target fragments were inserted into the plasmid vector PMD18-T respectively. By PCR analysis, the recombinant plasmids were proved to be true. The recombinant plasmids were extracted and were sequenced. The result showed that the length of the bp26 gene was 995bp, containing a complete Open Reading Frame of 753bp which encoding a protein of 250 amino acids. Sequence homology analysis showed that the gene sequence homology of 11 isolates was 100% each other respectively , and was 100%, 99.9%, 99.9% and 100% with that of M5, S2, S19 and 870 respectively.The length of the OMP10 gene was 531bp, containing a complete Open Reading Frame of 381bp which encoding a protein of 126 amino acids. Sequence homology analysis showed that the gene sequence homology of 11 isolates was 100% each other respectively, and was 100%, 100% and 99.7% with that of M5, S2, 544 respectively.For making up some deficiencies of serologic diagnosis and microbiological examination, the author also set up a method to detect Brucellosis by Polymerase Chain Reaction (PCR). The expected gene with 350bp was amplified based on the primer which designed according to the sequence of gene encoding the outer membrane protein (31KDa). The result showed that this method has a high specificity and sensitivity, and the minimum detection limit for DNA was 0.42pg. The Polymerase Chain Reaction has a great significance for detect and diagnose of Brucellosis.