Cloning, expression and characterization of zebrafish ferroportin in Hek293T cell line

Ferroportin [Fpn], a regulator of iron homeostasis is a conserved membrane protein that exports iron across the enterocytes, macrophages and hepatocytes into the blood circulation. Fpn has also critical influence on survival of microorganisms whose growth is dependent upon iron, thus preparation of Fpn is needed to study the role of iron in immunity and pathogenesis of micoorganisms. To prepare and characterize a recombinant ferroportin, total RNA was extracted from Indian zebrafish duodenum, and used to synthesize cDNA by RT-PCR. PCR product was first cloned in Topo TA vector and then subcloned into the GFP expression vector pEGFP-Nl. The final resulted plasmid [pEGFP-ZFpn] was used for expression of Fpn-EGFP protein in Hek 293T cells. The expression was confirmed by appearance of fluorescence in Hek 293 T cells. Recombinant Fpn was further characterized by submission of its predicted amino acid sequences to the TMHMM V2.0 prediction server [hidden Markov model], NetOGlyc 3.1 and NetNGlyc 3.1 servers. The obtained Fpn from indian zebrafish also contained eight transmembrane domains with N- and C-termini inside the cytoplasm and harboured 78 O-glycosylated amino acids. The recombinant Fpn from Indian zebra fish was successfully expressed in Hek 293 cell line. Although the discrepancy in two amino acids was observed in our produced Fpn and resulted in an additional O-glycosylation site, but had no effect on the topology of the protein compared to other Fpn described by other researchers. Therefore this construct can be used in future iron studies

problem of mixing up of leishmania isolates in the laboratory: suggestion of ITS1 gene sequencing for verification of species

Leishmaniasis is endemic in Iran. Different species of Leishmania [L.] parasites are causative agents of this disease. Correct identification of Leishmania species is important for clinical studies, prevention, and control of the diseases. Mix up of Leishmania isolates is possible in the laboratory, so there is need for verification of species for isolates of uncertain identity. Different methods may be used for this purpose including isoenzyme electrophoresis and molecular methods. The isoenzyme electrophoresis, due to its drawbacks, is feasible only in specialized laboratories while molecular methods may be more feasible. The aim of this research was to study the application of the internal transcribed spacer 1 [ITS1] sequencing method, in comparison to isoenzyme electrophoresis method, for verification of Leishmania species. Six Leishmania isolates were received from different research institutions in Iran. The species of these isolates were known by donating institution according to their isoenzyme profile. The species of these isolates were re-identified in Pasteur Institute of Iran by PCR amplification of ITS1 followed by sequencing and comparison of these sequences with Leishmania sequences in GenBank. Isoenzyme electrophoresis confirmed the confirmation of the results of ITS1. ITS1 sequence showed that some isolates were mixed up or contaminated with Crithidia. Isoenzyme electrophoresis confirmed the results of ITS1 sequences. ITS1 Sequencing is relatively more feasible than the traditional isoenzyme electrophoresis method and is suggested for verification of Leishmania species

Molecular epizootiology of rodent leishmaniasis in a hyperendemic area of Iran

Zoonotic cutaneous leishmaniasis [ZCL] is an expanding disease and public health problem in Iran. In the current study, natural Leishmania infection rate and seasonal fluctuation of the infection in Rhombomys opimus population of a hyperendemic focus of ZCL in Iran was investigated. The study was conducted from October 2006 to October 2008 in Esfahan Province, central part of Iran. An extensive sampling of rodents using Sherman traps was done in different seasons. Nested PCR assay was used for detection and identification of Leishmania species and the results were confirmed using PCR-RFLP. Leishmania infection rate was 58.6% [34 of 58] using nested PCR. 44.8% of the gerbils were infected only with L. turanica and 1.7% with L. gerbilli alone. A mixed natural infection with L. major and L. turanica was seen in 12.1% of the rodents. L. major infection alone was not seen in R. opimus population in the study area. The highest and lowest Leishmania infection rates were observed in fall and spring respectively. L. turanica infection was observed throughout the year whereas mixed infections with L. major and L. turanica was not seen in spring. It is concluded that in the study area, L. major, L. gerbilli and L. turanica circulate in the population of R. opimus. Leishmania major infection usually accompanied by L. turanica in naturally infected gerbils with the highest rate in fall. It is recommended that the role of L. turanica in the epidemiology and transmission of ZCL be revisited

Preparation of monoclonal antibody against the common determinants of HBsAg

Subtype-specific monoclonal antibodies are valuable tools for structural analysis of hepatitis B virus surface antigens and epidemiological investigations. Nineteen hybridoma clones producing monoclonal antibodies [MAb] specific for different epitopes of hepatitis B surface antigens [HBsAg] were established from mice immunized with HBsAg of either ayw or adw subtype. The immunizing antigens were purified from serum of carrier individuals or extracts of transgenic plants by affinity chromatography using polyclonal anti-HBs antibody. The specificity of MAbs was determined by enzyme-linked immunosorbent assay and immunoblotting using a panel of purified HBsAg of given subtypes. Out of the 19 MAbs, 9 were found to be specific for the common "a" determinant, and the remainders were specific for either "d" [n=2], "y" [n=5] or "w" [n=3] subtypic determinants. None of the subtype-specific MAbs displayed cross-reactivity with the other major subtypic epitopes. These MAbs have potential as monospecific reagents for subtyping HBsAg in carrier individuals for epidemiological and experimental investigations

Skin testing with stsndard leishmanin of Iran

Delayed-type hypersensitivity [DTH] reaction measured by skin testing is an important indicator for evaluation of cellular immune response in leishmaniasis. Skin testing in leishmaniasis, known as leishmanin or Montenegro test, is used normally for immunoepidemiological studies and for diagnosis. In cutaneous leishmaniasis [CL], the skin test is positive during the active phase of disease and remains reactive in almost 100% of recovered cases. The present paper describes the preparation and evaluation of the standard leishmanins produced from a well characterized strain of Leishmania major, and their applications in human CL, Different batches of leishmanin preparations were prepared in standard conditions and their reactivities were tested in different areas. Specificity of reagents was evaluated in healthy individuals in non endemic area of Tehran. Sensitivity and potency of antigens were evaluated in recovered cases from cutaneous leishmaniasis [CL] in endemic areas for both urban CL [north - west of Tehran] and rural CL [Isfahan area].The skin testing was carried out by intradermally injection of 0.1 ml of antigen or control regent into forearm of the individuals. Data obtained showed that specificity is 100% for all preparations. In recovered cases, different batches of leishmanin showed high sensitivity in different foci of leishmaniasis [> 93% in urban CL foci, and > 95.83% in rural CL foci]. Also different batches of leishmanin demonstrated a reliable potency in cured cases from urban CL, with mean diameter of indurations between 11.85 +/- 3.78mmand 14.21 + 5.0 mm. Moreover, strong potency was found in healed cases of rural CL with mean indurations between 15.18+ 3.93 mm and 18.04 + 5.22 mm. Finally, the reactivity of leishmanin was tested and proved stable after eight years study in recovered cases, This standard leishmanin, has also been tested in research centers of the country and abroad, and compared with other reagents, and selected by FDR as a reference reagent for vaccine trials