[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

serological study of influenza subtype A/H9N2 by ELISA and hemagglutination inhibition among the poultry workers in Ahwaz, Iran [2003-4]

Background and Purpose: Type a influenza virus causes infection in man, avian and primates. It has recently been reported that avian H5N1 and H9N2 subtypes were transferred to man. The purpose of the study is, therefore, to investigate the probability of H9N2 transmission from poultry to people engaged in poultry farming industries

Methods and material: In this study, serological tests were carried out on 100 blood samples of contacted cases and special antibody of H9N2 subtype was measured by hemagglutination inhibition. Also, the sensitivity and specificity of the prepared ELISA test kit were measured in comparison with the standard method of hemagglutination inhibition. Out of 100 HI tests, 45 cases were normally selected and measured by ELISA

Results: 66 percent of contacted cases had the specific H9N2 antibody. ELISA specificity and sensitivity, in comparison with HI, were 20% and 87.5% respectively

Conclusion: The Presence of H9N2 antibody in the subject's serum suggest the prevalence of H9N2 virus among poultry, its transfer to people exposed and the possibility of their infection. Also, diagnosis influenza H9N2 antibody

[Isolation and purification of Ag 85 complex from mycobacterium bovis [BCG] and assessment of its cell proliferation response in whole blood]

Introduction: Tuberculosis [TB] caused by Mycobacterium tuberculosis remains a major worldwide health problem. The only TB vaccine currently available is an attenuated strain of mycobacterium bovis termed Bacillus Calmette -Guerin [BCG]. The efficacy of BCG remains controversial. Mycobacterium secretory proteins are generally considered important antigens for protection against TB. A major protein component of mycobacterial culture filtrate is Antigen 85 complex which is a promising potential vaccine candidate

Material and Methods: Antigen 85 complex was purified from Mycobacterium bovis [BCG] culture filtrate by sequential chromatography on phenyl Sepharose 4B, DEAE-Sephacel and Superdex G75. Purification was confirmed by SDS-PAGE and immunoblotting with a specific monoclonal antibody. The in vitro ability of Ag 85 complex to stimulate cell proliferation was compared with that of Purified Protein Derivative [PPD] and the polyclonal T cell mitogen PHA in a whole blood assay in which the target cells were derived from 25 healthy PPD-positive and 25 healthy PPD-negative subjects

Results: Antigen 85 complex was found to have a molecular weight of 30-32 KDa by SDS-polyacrylamide gel electrophoresis and reacted strongly by immunoblotting with the monoclonal antibody specified against Ag 85. The responses to Ag 85 and PPD were significantly higher in cells of PPD- positive than in cells of PPD-negative donors. Eighty eight percent [22/25] of the PPD- positive cells responded to Ag 85 whereas only 16% [4/25] of the PPD-negative cells responded

Conclusion: The cell proliferation response to Ag 85 complex is significantly different between cells of skin-test positive and skin- test negative subjects and may suggest an immuno-protective role for Ag 85 complex against M. tuberculosis infection

Studies on hepatitis B vaccines

HBs vaccines were prepared by three different methods: 1] Heat inactivation [Krugman, et. al; 1971]; 2] Ammonium sulphate precipitation followed by column chromatography [Sephadex G-200]; 3] PEG-6000 [polyethylene glycol] precipitation followed by column chromatography using Sephadex G-200. Their efficacy was studied in guinea pigs, and the results compared with commercial Japanese vaccine [Green Cross Corporation, Osaka, Japan]. We conclude that vaccine prepared by PEG-6000 precipitation gives better results