Manoochehr Rasouli; Ahmad Zavaran Hoseini; Bahram Karimi; Abdolvahab Alborzi; Simin Kiany
Volume 6, Issue 2 , June 2009, , Pages 75-86
Abstract
Background: Heat shock protein 70 (HSP70) is present in all organisms studied so far, and is a major immunogen in infections caused by pathogens including Leishmania spp. Objective: The aim of this study was to clone and express HSP70 from L. infantum strain MCAN/IR/96/LON-49 and evaluate antibody response ...
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Background: Heat shock protein 70 (HSP70) is present in all organisms studied so far, and is a major immunogen in infections caused by pathogens including Leishmania spp. Objective: The aim of this study was to clone and express HSP70 from L. infantum strain MCAN/IR/96/LON-49 and evaluate antibody response against HSP70 in visceral leishmaniasis (VL). Methods: The L. infantum HSP70 gene segment was amplified by specific primers. It was cloned into pTZ57R vector and subcloned into pET32a (+) expression vector. The new construct was transformed in the E.coli Rosetta strain, and HSP70 protein was expressed in the presence of 1 mM IPTG and purified using a HiTrap chelating column. Antibody responses against HSP70 were determined by ELISA in 37 patients with visceral leishmaniasis and 63 healthy controls. Results: Expression of HSP70 protein was confirmed using SDS-PAGE electrophoresis and dot blot with an anti-His tag antibody. There was no difference between the sequence of nucleotides of the HSP70 gene in the present study and other reported sequences. The ELISA results indicated that the sera of 81.1% (30/37) of the patients and 6.3% (5/63) of controls reacted to L. infantum HSP70. Conclusion: The conservative nature of the HSP70 molecule is an advantage in vaccine studies, because of minor differences (6%) between the nucleotide sequences and consequently the similarity in amino acid sequences in various strains of L. infantum. It could therefore be used in vaccine research against leishmaniasis and also as a tool for serodiagnosis.
Roghayeh Rahimi; Ahmad Zavaran Hosseini; Fatemeh Yari
Volume 5, Issue 4 , December 2008, , Pages 207-211
Abstract
Background: HLA-G gene contains 15 alleles including a null allele, HLA-G*0105N. Previous studies have shown that HLA-G*0105N does not encode the complete HLA-G1 or HLA-G5 isoforms but encodes a functional HLA-G protein with the ability to in-hibit NK cell cytolysis. Thus, although the biological functions ...
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Background: HLA-G gene contains 15 alleles including a null allele, HLA-G*0105N. Previous studies have shown that HLA-G*0105N does not encode the complete HLA-G1 or HLA-G5 isoforms but encodes a functional HLA-G protein with the ability to in-hibit NK cell cytolysis. Thus, although the biological functions of HLA-G1 and HLA-G5 proteins are abrogated, other isoforms such as HLA-G2 can replace their roles. Stud-ies on the null allele of HLA-G gene could be useful in understanding the genetic vari-ants of HLA-G alleles in ethnic groups. Objective: The goal of this research was to de-termine the frequency of HLA-G*0105N null allele in Iranian healthy subjects. Meth-ods: The frequency of HLA-G*0105N null allele was evaluated in Iranian healthy sub-jects by PCR-RFLP method. Genomic DNA was isolated from the whole blood of 100 randomly selected, healthy, unrelated Iranian individuals using salting-out technique followed by PCR amplification of the exon 3 of HLA-G gene. PCR products were di-gested with PpUM-1 and the resulted fragments were analyzed using gel electrophore-sis. Results: In this study the restriction enzyme digestion confirmed homozygous HLA-G*0105N null allele for 9 % of the population. Furthermore obtained results indi-cated that the total frequency of HLA-G*0105N null allele was 20 % in the studied population of Iran. Conclusion: The final data analysis showed that the total frequency of this allele in Iranian people was higher than other ethnic groups that have been stud-ied so far.