Isolation And Identification Of Brucella Melitensis And Analysis Of Bp26Gene

Brucellosis caused by Brucella is the commonest zoonotic disease worldwide. Brucella invadesbody to cause infect and disease through the skin mucous membrane and respiratory. The prevalence ofbrucellosis displays the increased trend in Inner Mongolia in recent years. It caused great economiclosses in productivity and husbandry industry. So, it is key to control the occurrence and development ofBrucellosis by developing effective early diagnosis technology. BP26, soluble peripheral proteinreleased by intra-cellular, has good antigenicity compared with the fixed membrane protein, which iseasy to be tested. Therefore, BP26is suitable diagnostic antigens of brucellosis with higher similarity inall brucellosis strains. The5683samples of serum from the Siziwang county of Ulanqab and Damaocounty of Baotou were detected with RBT and iELISA for preliminary screening, and SAT and cELISAfor final check.373samples were positive in the5683samples（6.56%）by RBT,395samples werepositive（6.95%）by iELISA,355samples were positive（6.25%）by SAT;337samples were positive（5.93%）by cELISA. The results showed that different degree of sheep brucellosis had happened.Samples of2sheep aborted fetuse and26whole blood were detected by SAT. The result was that4strains of brucella were isolated. Its were identified as Br melitensis by traditional methods and namedas B1、B2、B3and B4.Strain B1、B2、B3and B4(isolated by this study), strain M5, strain A19and strain S2wereidentified by PCR method. One fragment of1390bp of isolated strains was amplified by16SrDNA-PCR,and it was identical with the reference strains of M5、A19and S2. The result of DNA sequence analysisshowed that the evolutionary relationships of the four isolates was nearest with NR074149; The880nucleotide mutated into threonine (T) by sequence contrast. To confirm further isolated strains as Brmelitensis, omp10gene was amplificated in this study. The result showed that fragment of513pb sizedof isolated strains was amplified, and it was identical with the three reference strains.100%ofhomology was showed for4isolated strains each other. Open reading frame of the amplified gene was396bp, encoding131amino acids correspondingly. The protein molecular weight was13.9KD, and itsisoelectric point was7.87. The strain of KF780864had a variants at144C-T, but didn’t alter amino acid.The results showed that the gene is very conservative and vital for the growth of brucella.The bp26gene of the isolated strains and the3reference strains was cloned successfully, thefragment sized is900bp. The sequence analysis revealed that open reading frame of the amplified bp26gene was753bp, which encoded250amino acids. The protein molecular weight was26.6KDa, and itsisoelectric point was6.781. The bp26gene of isolated strain was nearest in genetic evolutionaryrelationship with M5.Sequence alignment showed that there is4mutation points, The304th A-G and405th C-Tmutations points was located in CDS region of bp26gene at A19; The498th C-T and727th G-Amutation points was located in bp26at S2.304A-G mutation point caused the change of the encoded amino acid occurred from asparagines (N) into aspartic acid (D),727G-A mutation point caused thechange of encoded amino acid occurred from valine (V) into isoleucine (I). These changes of aminoacids is likely to cause changes in the protein structure, which affects the bp26protein’s function.compared with A19and S2, the isolated strains and M5was very stable, has the great potential todevelop a new diagnosis method.