In Vitro Infectivity Assessment by Drug Susceptibility Comparison of Recombinant Leishmania major Expressing Enhanced Green Fluorescent Protein or EGFP-Luciferase Fused Genes with Wild-Type Parasite

Leishmaniasis is a worldwide uncontrolled parasitic disease due to the lack of effective drug and vaccine. To speed up effective drug development, we need powerful methods to rapidly assess drug effectiveness against the intracellular form of Leishmania in high throughput assays. Reporter gene technology has proven to be an excellent tool for drug screening in vitro. The effects of reporter proteins on parasite infectivity should be identified both in vitro and in vivo. In this research, we initially compared the infectivity rate of recombinant Leishmania major expressing stably enhanced green fluorescent protein (EGFP) alone or EGFP-luciferase (EGFP-LUC) with the wild-type strain. Next, we evaluated the sensitivity of these parasites to amphotericin B (AmB) as a standard drug in 2 parasitic phases, promastigote and amastigote. This comparison was made by MTT and nitric oxide (NO) assay and by quantifying the specific signals derived from reporter genes like EGFP intensity and luciferase activity. To study the amastigote form, both B10R and THP-1 macrophage cell lines were infected in the stationary phase and were exposed to AmB at different time points. Our results clearly revealed that the 3 parasite lines had similar in vitro infectivity rates with comparable parasite-induced levels of NO following interferon-gamma/lipopolysaccharide induction. Based on our results we proposed the more reporter gene, the faster and more sensitive evaluation of the drug efficiency.

Cloning of coagulant VII factor from hepatoma cell line

Factor VII, is a coagulant protease; it begins the proteolytic cascade reactions and produces thrombin. The use of recombinant human factor VII, [rhFVII] is effective for the treatment of patients with hemophilia A or B. It is a target for gene therapy. This study was done to clone factor VII from HepG2 cell line. RNA was extracted from the hepatoma, [HepG2], cell line. On reverse transcription FVII cDNA was amplified by RT-PCR. PCR product was cloned into the pTZ57R/T vector and transported into the E-coli cells. By amplification of the FVII gene, the PCR band was observed and cloning into the vector was confirmed by restriction analysis. In this paper we report the cloning of factor VII from HepG2 cell line

Insulin receptor gene mutations in Iranian patients with type II diabetes mellitus

Patients with diabetes mellitus type II suffer from hyperglycemia because they are not able to use the insulin that they produce, often due to inadequate function of insulin receptors. There are some evidences that this deficiency is inherited in a dominant autosomal manner and leads to the malfunction of the pancreatic beta cells resulting in insulin excretion disorders. In this study, we sought to identify mutations in the insulin receptor [INSR] gene, which can cause insulin resistance in type II diabetic patients. DNA was extracted from peripheral blood cells of the patients [n = 128] diagnosed with type II diabetes. All 22 exons of the INSR gene of the patients were analyzed for mutations running PCR, conformation-sensitive gel electrophoresis and DNA sequencing, consecutively. Approximately 26% of the patients had genetic mutations; however, most of them were not reported. These mutations include exon 2 [His171Asn, Ile172Ser, Cys196Ser and Ser210Arg], exon 3 [Gly227Asp and Gly232Ser], exon 8 [Thr543Ser], exon 9 [a heterozygote was observed with no change in phenylalanine at position 669], exon 13 [two heterozygotes: Arg890Pro with Asn865 remaining unchanged], exon 14 [Ala906Gly and Pro918Trp with Arg902 unchanged], exon 17 [Val1086Glu] and exon 19 [His1157Gln with Thr1172 unchanged]. The lack of similar mutation records in literature and genetic data banks may suggest a geographic pattern for these INSR gene variants in our population

Cloning, expression and purification of truncated Chlamydia trachomatis outer membrane protein 2 [Omp2] and its application in an ELISA assay

Although a simple and direct method does not exist for the detection of chlamydial infections, there are situations in which reliable serological tests, with sensitivity related to a specific antigen, could be helpful. The aim of this study was to clone the first 1100 bp of the C. trachomatis outer membrane protein 2 [omp2] gene in order to prepare a recombinant protein for use in an ELISA system designed to recognize the anti- C. trachomatis antibody in patient sera. The PCR product of the chlamydial omp2 gene was cloned in pBluescript and its first 1100 bp was subcloned in the pQE-30 expression vector and induced by IPTG. The recombinant protein was purified by affinity chromatography and its purity was confirmed by SDS-PAGE, gel diffusion and western blot analyses. The purified protein was coated onto a polystyrene microplate and tested by ELISA using patient serum. We have cloned, over-expressed and purified biologically functional recombinant truncated Omp2 from C. trachomatis for use, as a species-specific recognition antigen, in an ELISA system. In this study we determined a cut-off value of 0.345 for this ELISA system using 55 negative sera and measured six positive sera at dilutions of 1:20-1:2560. As a species-specific recognition antigen, the over-expressed and purified recombinant truncated Omp2 from C. trachomatis performed well in an ELISA system

Production of mutant streptokinase recombinant protein

Streptokinase [SK] is most widely used for treatment of myocardial infarction, however, it is the most expensive thrombolytic agent. A major drawback to SK use is the widespread presence of anti-streptokinase antibodies [Abs]. These Abs cause allergic reactions and neutralize streptokinase therapeutic effects. To produce an engineered variant of streptokinase being functional and less antigenic than the native molecule, we cloned and expressed streptokinase mutant gene lacking the C - terminal 42 amino acids. Recombinant protein was confirmed by western blot analysis with anti T7 monoclonal antibodies. pGEMEX-1 expression vector contains T7 gene 10 protein as fusion protein immediately down stream of T7 promoter and before multiple cloning site, streptokinase mutant gene was cloned after fusion protein. We cloned and expressed mutant streptokinase gene, lacking the C-terminal 42 amino acids. If mut-C42 activity was less affected by neutralizing antibodies compared with native streptokinase, this engineered variant could be a preferred alternative to native streptokinase for thrombolytic therapy

Using recombinant chlamydia major outer membrane protein [MOMP] in ELISA diagnostic Kit

Chlamydia trachomatis is one of the main causes of Sexually Transmitted Diseases [STDs] such as prostatitis and epididymitis in men and cervicitis, endometriosis, vaginitis and ureogenital tract infections in women. Serological tests with sensitivities related to specific antigens are commonly used as routine laboratory tests for diagnosis of Chlamydia. In this research the Chlamydia Major Outer Membrane Protein gene was coloned in order to prepare a specific recombinant protein for use in the ELISA diagnostic kit. DNA was extracted from cultured C. tachomatis. PCR reaction was carried out and the resulting PCR product was cloned into the pGemex-1 expression vector and induced by IPTG [Isopropyl beta-DThiogalactopyrano side]. Recombinant protein was confirmed by gel diffusion, dot blot and western blot, using patient's serum. The use of recombinant protein for diagnosis of Chlamydia by ELISA is therefore recommended

Assessment of anti-streptokinase antibody in patients with heart diseases and normal subjects

Background: Streptokinase, which is injected intravenously with a standard dose of 1.5 MIU, is the most widely used thrombolytic agent around the world. What is so important about this bioproduct is the level of anti-streptokinase [anti-sk] antibody in the population, which is directly correlated to the incidence of streptococcal infections in that population

Objectives: Since Iran is an endemic area for streptococcal infections, this study was conducted to assess the anti-sk level in an Iranian population

Materials and Methods: 97 males and 47 females referred to Modarress Hospital of Tehran for coronary angiography and cardiac catheterization were included. 10 ml of venous blood was taken before angiographies from each patient. According to the angiography reports, the patients were divided into three groups: Coronary Artery Diseases [CAD, n=95], Rheumatic Heart Disease [RHD, n=19] and normal coronaries [n=30]. The anti-sk antibody level was assessed in the serum samples of all patients using Enzyme Linked Immunosorbant Assay

Results: In 23.2% of patients with CAD, 40% of normal coronaries and 73.7% of patients with RHD, the serum samples contained more than 2 arbitrary units [AU] of anti-SK antibody which regarded as high levels. There was no significant difference between the anti-sk level of patients with CAD and normal coronaries [2.03 +/- 3.02 AUs vs. 2.52 +/- 2.23 AU], but the level of antibody in RHD group [8.16 +/- 10.1 AU] was significantly higher than other groups [p<0.05]. No significant correlation was observed between antibody levels and the age or gender of patients

Conclusion: We concluded that the level of anti-sk antibody is high in Iranian population as compared to other endemic areas for streptococcal infections. Also we found no relation between the level of antibody and sex and age of patients. This study accentuated the necessity of assessment of drug efficacy in endemic areas for streptococcal infections especially in those patients with valvular heart disease