[Optimization of gene expression and purification of Legionella pneumophila peptidoglycan associated lipoprotein recombinant protein]

There are many problems with most of the available diagnostic tests used to diagnose Legionella pneumonia, including inadequate sensitivity and specificity, and inability to provide a result in a clinically useful time period. Legionella pneumophila PAL protein has been considred as a target for detecting of Legionella infection from urine specimen, because it is conserved sequence and is secreted into the urine. The aim of this study was to optimize expression and purification of L. pneumophila PAL protein. In this experimental study, optimizing of 5 parameters [cell density, induction time, growth temperature, IPTG concentration and type of medium] was performed. After expression, periplasmic extract was prepared and recombinant PAL protein purified using Ni2+-charged resin column. Finally, recombinant PAL protein was verified by Western blotting. In terrific broth medium, the optimum condition of r-PAL protein induction was occurred at an OD600 of 0.6, 1mM IPTG concentration and 15 hours incubation at 25°C Recombinant periplasmic PAL protein was highly purified [>80%] using Ni-NTA column. Western blotting analysis showed that recombinant PAL protein was also specifically recognized by anti-His6-peroxidase antibody. By purification of recombinant PAL protein in purity greater than 80% it can be used to evaluate its capacity in diagnosis of Legionella infection and preparation of diagnostic kit

[Determination of Brucellamelitensis biotypes in human and sheep isolates]

Brucellosis is one of the most important zoonsis. Because of the importance of this disease and the effect of strain variation on strategic planning for the control of this disease, sheep and human Brucella isolates were evaluated for strain variation. Five human blood Brucella isolates and 25 sheep embryo Brucella isolates were collected from hospitals and veterinary centers in Isfahan. Total genomic DNA was extracted from the collected samples using method of SDS and proteinase K treatment, phenol/chloroform extraction and ethanol precipitation. omp2a and omp2b fragments of all isolates were amplified using 2 pairs of specific primers [omp2a R, F and omp2b R, F] and the PCR products were electrophoresed and stained. These PCR products were then restricted using Alu I, Hinf I and TaqI restriction endonuclease. The PCR products of all human and sheep isolates had the same size 1100bp for omp2a and 1200bp for omp2b. The banding pattern of PCR-RFLP for all of the isolates was similar to banding pattern of the Brucellamelitensis biotype 1. Based on the banding pattern of PCR-RFLP of the omp2a and omp2b fragments, it can be concluded that all the studied samples in Isfahan are Brucellamelitensis biotype 1. This study should be repeated regularly. This will inform us if new species or biotype of Brucella have entered to the region. This is important in planning for the control of the disease