[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

[Study of exon deletion in the dystrophin gene in individuals being diagnosed with duchenne muscular dystrophy in Ahvaz]

Duchenne muscular dystrophy is an X-linked genetic disorder resulting from mutation or deletion in the Dystrophin gene. The aim of this study was to evaluate the primary diagnosis of affected individuals that have been referred to the genetic lab of the Shafa hospital in Ahvaz. Progressive muscle weakness was present in all the patients. DNA from peripheral blood was extracted from affected patients and subsequent multiplex-PCR was performed to determine putative deletions in the Dystrophin gene. in 53% of cases were deletions identified in exons 44-51 in the Dystrophin gene and therefore the clinical diagnosis could be confirmed. On the other hand, we found no deletion in 47% of cases. it seems that the patients suffering Duchenne muscular Dystrophy in Ahvaz show, independent to their ethnicity, the gene inactivating deletion in the end part of the Dystrophin gene. These results would be used for the differential and for the prenatal diagnosis in the Khuzestan province

[ study on mutations in hotspot sites of exon 15 of apc gene in familial adenomatous polyposis patients and their relatives in Iran Khuzestan province]

The aim of this study was to estimate mutation frequency in exon 15 of APC gene in Khuzestan FAP patients and their relatives. We have analyzed 12 patients and 11 individual among their relatives. DNA extraction from EDTA treated whole blood was performed by routine salting out method. Three hotspot regions of axon 15 from APC gene were amplified separately with three primer pairs by PCR and the fragments have been analyzed for putative mutations by single stranded conformation polymorphism [SSCP] according to standard protocols and subsequent sequencing. We found aberrant bands by SSCP method in seven patients and two related members, that compared with data from sequence analysis yield following five patients [24 to 35 years old] carried novel frame shift mutation [3195-3196insT] leading to premature protein. In two patients whose SSCP were positive, no mutations in their two relatives were identified. In this report we have used SSCP as pre-screening method for mutation analysis of our samples and found putative mutations in nine of them. After sequencing, only in five samples "true mutation" could be confirmed It is intersting that approximately 50% of Khuzestan FAP patients carry mutation in axon 15 of APC gene. Noteworthly, in this work only a small region of the APC gene was subject of the investigation and interestingly, all patients showed the same new mutation. Studies with large number of samples can help us to know, whether this new mutation is predominant or even exclusive within Khuzestan FAP patients.

Evaluation of RT-PCR to detect translocations in children diagnosed with acute lymphoblastic leukemia

Acute lymphoblastic leukemia [ALL] is the most common subtype of childhood cancer. Chromosomal abnormality, specially the replacement of chromosomal material is one of the main reasons in generating leukemia, wherein the kind of translocation play a key role in managing the remedy. The goal of the present study was to develop a reliable, rapid, and cost effective method to detect translocations, which are the main sources of leukemia. Twenty seven samples were collected from leukemia affected individuals that were referred to the Shafa Hospital in Ahwaz from summer 2007 to spring2008. Total RNA was extracted from one milliliter whole blood, and then reversely transcribed using reverse transcriptase. Finally, multiplex RT-PCR was performed for each sample. Cell lines [K562, Jurkat E 6.1] that are harboring known translocations were used as positive control, with additional internal control to prove false negative results. Translocations t [9; 22], t [12; 21], t [1; 19] and t [4; 11] were observed in patients that have been diagnosed with the ALL, respectively. No Translocation has been seen in individuals suffering lymphoma. Multiplex RT-PCR assay is an effective, sensitive, accurate, and cost-effective diagnostic tool, which can improve the ability to accurately and rapidly risk-stratify patients that were diagnosed with acute lymphoblastic leukemia

TTV prevalence in Ahwaz blood donors by using semi-nested PCR

Occurrence of new infectious agents threatens access to zero risk in blood transfusion and enhancement of blood safety. Although sensitive methods are available for diagnosis of hepatitis, yet some hepatitis cases do not have a known etiology. In 1997, the novel DNA virus was isolated from post-transfusion serum samples of patients affected by non-A-G hepatitis. Nowadays this novel virus is known as transfusion-transmitted virus. This circular single stranded unenveloped and virucidally resistant virus is the first human circovirus and has universal distribution. It is believed that TTV may cause hepatitis and aplastic anemia. This study was conducted to determine the prevalence of TTV in healthy blood donors in Ahwaz and set up N22 PCR for subsequent first-time viral studies in south region in Iran. In 2003, We studied the presence of TTV DNA by using Okamoto primers with PCR in plasma of blood donors in whom serologic tests for hepatitis A-C and HIV-Ab were negative. Our study showed that the virus prevalence in blood donors was 23.7% [60/253] and there was not any significant differences between prevalence of TTV and background variables. Our findings showed the same prevalence rate as in neighboring countries; however, in comparison with thalassemic patients that were studied in parallel with the present research, the difference was significant [143/250; 57.3%]. It shows the importance of blood transfuison in transmission of the virus