Abstract

Brucella melitensis is a Gram negative coccobacillus bacterium from the Brucellaceae family. Brucellosis is an infectious zoonotic disease affecting most species of domestic animals, but sheep and goats, particularly milking breeds, are the most susceptible. Cattle may occasionally be affected and the disease may appear in pigs. vacB gene is one important gene of B. melitensis that encoded a protein (RNase R) and in B. melitensis andBrucella abortus has no impact on bacterial virulence. The aim of the present study wasto construct a novel recombinant vector as B. melitensis vacB gene knockouts candidate.B. melitensis were collected from Microbiology laboratory of Islamic Azad University of Shahrekord Branch and cultured into Brucella agar. Genomic deoxyribonucleic acid (DNA) was extracted and the polymerase chain reaction (PCR) were performed using designed primers for amplification of upstream and downstream regions of vacB gene of B. melitensis and kan gene of pET-28 vector. Then, amplified fragments were cloned using T/A cloning technique and the construction was transformed into competent Escherichiacoli Top10F' strain in LB media. The final construction was confirmed by digestion withXhoI, KpnI, XbaI, and BamHI restriction enzymes. PCR amplified products for flanking regions of vacB gene and kan gene on 1% agarose gel revealed 609, 595, and 870 bp, fragments, respectively. The results showed that vacB gene and kan gene were cloned inE. coli successfully. Sub-cloning of all fragment into pET-32 vector were done successfully and pET-32-Up-Kan-Down recombinant vector was generated. The findings of this study showed that the designed novel recombinant construct (pET-32-Up-Kan-Down recombinant plasmid) is useful for genetic engineering and for manipulate of vacBgene from B. melitensis.

Key words: Virulence-associated gene (vacB), kan gene, Brucella melitensis, the polymerase chain reaction (PCR) and cloning.