RESUMO
OBJECTIVE@#To clone the virB12 gene in pET28a expression vector for production of recombinant protein to be used as antigenic component for future serological test development.@*METHODS@#Brucella melitensis (B. melitensis) 16M strain was cultured and bacterial DNA was extracted by Bioneer AccuPrep® Genomic DNA Extraction Kit. Oligonucleotide primer pair was designed based on Brucella virB12 gene sequence with BamHI and HindIII restriction site at 5' end of the forward and reverse primers, respectively. DNA amplification was performed using PrimSTAR® HS DNA polymerase and the PCR product was purified by DNA AccuPrep®Gel Purification Kit. Purified DNA was cloned into pJET1.2 cloning vector. VirB12 gene fragment was excised from pJET1.2 using BamHI/HindIII and subsequently subcloned into pET28a (+).@*RESULTS@#Brucella virB12 gene was successfully cloned in pJET1.2 and then in pET28a (+) plasmids. PCR and restriction enzyme digestion confirms the procedure.@*CONCLUSION@#We cloned and expressed the Brucella virB12 gene which could be used as antigenic component for specific serological assay development.
Assuntos
Antígenos de Bactérias , Genética , Proteínas de Bactérias , Genética , Brucella melitensis , Genética , Clonagem Molecular , Métodos , DNA Bacteriano , Genética , Vetores Genéticos , Genética , Técnicas de Amplificação de Ácido Nucleico , Reação em Cadeia da Polimerase , Métodos , Proteínas Recombinantes , Genética , Testes Sorológicos , MétodosRESUMO
Among the members of legionellaceae, Legionella Pneumophila is involved in 95% of cases of severe pneumonia. Isolation of the causative agent from bronchoalveolar lavage [BAL] fluid specimen is a delicate process and also time-consuming. Moreover, it has been shown that some Legionella strains may be viable but cannot be cultured. The aim of this study was comparison of culture and PCR for detection of Legionella Pneumophila from bronchoalveolar lavage [BAL] fluid specimens. In this study, 70 BAL fluid specimens were collected from patients suspected to Legionnaires' disease. These samples were cultured on selective buffered charcoal-yeast extract agar [BCYE] and then tested with specific L. Pneumophila primers for mip gene. Among 70 BAL samples, three [4.2%] were positive with culture and six [8.4%] of specimens were positive by PCR. The three culture positive samples were all positive after specific DNA amplification. Among 63 culture-negative samples, 3 were positive after amplification. The clinical features of the patients were in accordance with legionellosis. The accurate diagnosis of Legionella Pneumophila has an important implication for the treatment of infection. Analysis of the results showed that PCR is faster and more sensitive for isolation and identification of L. pneumophila to apply on BAL fluid specimens than culture. Therefore, specific Legionella PCR can be a good option for isolation and identification of Legionella Pneumophila from bronchoalveolar lavage [BAL] fluid specimens in patient of severe pneumonia