Immunoreactivity analysis of Toxoplasma gondii recombinant antigen rSAG3 in sera from immunized BALB/c mice and tox-oplasmosis patients

Background: The coccidian protozoa Toxoplasma gondii is an obligate intracellular parasite of humans and other warm-blooded animals. Diagnosis of toxoplasmosis is of considerable medical importance for human, especially pregnant women and immunocompromised individuals. The apply of an Escherichia coli recombinant antigen(s) would be signifi-cantly useful in developing standardization of the diagnostic tests and reducing their costs. In this study, immunoreac-tivity of recombinant SAG3 against sera from immunized mice and human anti-T. gondii IgG positive patients was evaluated by western-blotting and enzyme immunoassay (EIA) in Department of Parasitology and Mycology, School of Medicine, Shiraz University of Medical Sciences in 2013. Methods: Three inbreed BALB/c female mice were obtained. Two mice were injected with rSAG3 and one was re-mained untreated, as control. Sera from immunized mice and also pooled sera from IgG positive toxoplasmosis cases were evaluated with western-blotting. IgG antibody responses to recombinant SAG3 was measured by indirect ELISA against the negative control group. Results: The rSAG3 protein reacted with sera of immunized mice and sera from patients with anti-Toxoplasma IgG antibodies in western-blot analysis. The result of ELISA showed that, there was marked differences in the absorbance values between the recombinant SAG3 immunized mice and control group. Conclusion: The rSAG3 showed IgG reactivity with sera from immunized mice and anti-Toxoplasma IgG patients. © 2016, Iranian Journal of Public Health. All rights reserved

Study on ITS1 Gene of Iranian Trichomonas vaginalis by Molecular Methods

Background: Trichomoniasis is a worldwide protozoan parasitic disease and metronidazole is a choice drug for its treatment. Because of disease importance in public health and its controversial ideas about the prevalence of drug resistance, this study was carried out.Methods: Fifty-two suspected vaginal samples were collected from 2006 to 2007 in Gynecology Maryam Hospital, Tehran, Iran. All isolates were examined by microscopic, culture and PCR tech­niques. The PCR products were analyzed by RFLP and CSGE methods and two suspected samples were sequenced.Results: Trichomonas vaginalis was identified from all 52 samples. Of 52 isolates, 45 samples were successfully cultured and amplified by PCR except one. Seven were positive only by PCR. Finally, ITS1 fragment was successfully amplified in 51 of 52. CSGE analysis and PCR products di­gestion by MspI followed by sequencing showed nucleotide mutation at position 209 (C209T) of the ITS1 fragment in two (3.9%) of them.Conclusion: The results showed mutation in ITS1 fragment of T. vaginalis in two (3.9%) of Ira­nian isolates which may be related to metronidazole resistance

Serological Evaluation of EgAgB16 kDa, a Recombinant Antigen from Echinococcus granulosus for Diagnosis of Human Hydatidosis

Background: Regarding that accurate diagnosis of human hydatidosis still needs more investigations, the present study was conducted to clone, express, and evaluate the gene encoding AgB subunits (EgAgB16 kDa) from Echinococcus granulosus (Iranian G1 strain) and its evaluation by ELISA test.Methods: DNA was extracted from protoscoleces and was utilized by PCR for strain identification. Total RNA was prepared with RNeasy protect mini kit from E. granulosus (Iranian G1 strain) protoscoleces collected from naturally infected sheep with hydatid cyst. Recombinant AgB16 kDa was produced using pETDuet as vector and evaluated by ELISA method. A panel of sera including hydatid cyst-infected individu­als (n=72), healthy individual (n=48), toxoplasmosis (n=4), strongyloidosis (n=4), kala-azar (n=5) and tuberculosis (n=5) were examined using this recombinant antigen.Results: Recombinant protein was purified by affinity chromatography using His-Tag column. After purifica­tion, recombinant protein was confirmed by western blot analysis using His Tag monoclonal anti­body or hydatid positive human serum. The sensitivity, specificity; positive and negative predictive values were calculated as 93.5%, 95.6%, 96% and 92.9%, in that order. The cut-off point was detected 0.3 for rAgB16. Conclusion: While the produced recombinant AgB16 kDa showed promising results in diagnosing human hy­datidosis, but more investigations should be implemented to reach an accurate gold standard

Development of Sensitive Detection of Cryptosporidium and Giardia from Surface Water in Iran

Background: The protozoan parasites Cryptosporidium spp. and Giardia are known to occur widely in both raw and drinking waters. They are two of the causative agents of waterborne out-breaks of gastroenteritis throughout the world. In the present study, a PCR assay and FA were developed for detection of Cryptosporidium oocysts and Giardia cyst in environmental samples. Methods: We have detected Cryptosporidium spp. oocysts and Giardia cysts in seeded and un-seeded environmental water samples by PCR method. Water samples were spiked with oocysts (50, 100,300,500) and filtrated with a 1.2-µm pore size cellulose nitrate and follow by DNA extrac¬tion and purification by QIAamp DNA mini kit. Nested-PCR assay amplified an 850 bp fragment of 18s rRNA gene specific for Cryptosporidium and 435 bp fragment of glutamate dehydrogenase (GDH) target gene for Giardia. Also many river water from north of Iran, be checked by these methods. Results: Cryptosporidium and Giardia DNAs were detected in seeded water sample and Giardia was detected in all 5 water samples from river in north of Iran by nested- PCR and FA. Also in one river water sample, Cryptosporidium was detected.Conclusion: This protocol is effective for detection of these waterborne parasites in treated and untreated water samples. This study can also serve as a platform for further investigations and research water source in Iran

Investigation of genetic marker (Luciferase gene) production for detection and stigmatize Cyprinus carpio or Rutilus frisii kutum

In this study lusiferse gene extracted from Vibrio fischeri. V. fischeri strain was kindly provided by Iranian Research Organization for Science and Technology (IROST). Genomic DNA extraction was carried out by Phenol-chloroform. A DNA fragment encoding the luxA and LuxB was amplified by PCR using sense and antisense primers, specific restriction sites for BamH1 and Kpn1 were introduced into 5´ end of forward and reverse primer, respectively. The PCR product was purified from agarose gel and ligated into cleaved PT257R cloning vector. Following the confirmation of the cloned Luciferase transformed into E. coli. Recombinant clones were confirmed by specific PCR and restriction enzyme digestion analysis. The luxA and LuxB fragment was released subcloned in to the PcDNA3.1\hug and PcDNA3.1\neo expression vectors, respectively. recombinant plasmid was confirmed through restriction digestion using BamH1 and Kpn1 enzymes and subsequently, transformation procedure continued into NIH3T3 eukaryote cells by specific kit. Luminescence ability of recombinant clones was tested by NIH3T3 cells and dechanal (substrate) and Neomysin and hygromysin. The results showed that luminescence start after 2 hours and then increase after 6 hours. Inaddition, the protein identity was verified by western blot analysis, the protein bands 76 kD were detected which indicates protein expression of luxB, luxA

Identification of white spot syndrome virus in cultured Penaeus indicus by polymerase chain reaction (PCR) in I.R. IRAN

The WSD is the important viral shrimp disease in past decade. The detection of virus in each country was investigated by polymerase chain reaction for sensitivity and accurate. In this research study we collected 23 samples from shrimp suspected to WSD. The DNA from samples collected and extracted and design two kind of primer from VP24 identified in gene bank by DNAsis software. A primer also designs for Housekeeping gene for positive and negative samples in all examined. The results showed the gene colon for wssv is the similar with others and 97% is consistency. The product of PCR was colon in plasmid and confirmed and this plasmid used for internal control

Molecular cloning, expression and enzymatic assay of pteridine reductase 1 from Iranian lizard Leishmania

Background: Currently, there are no effective vaccines against leishmaniasis, and treatment using pentavalent antimonial drugs is occasionally effective and often toxic for patients. The PTR1 enzyme, which causes antifolate drug resistance in Leishmania parasites encoded by gene pteridine reductase 1 (ptr1). Since Leishmania lacks pteridine and folate metabolism, it cannot synthesize the pteridine moiety from guanine triphosphate. Therefore, it must produce pteridine using PTR1, an essential part of the salvage pathway that reduces oxidized pteridines. Thus, PTR1 is a good drug-target candidate for anti-Leishmania chemotherapy. The aim of this study was the cloning, expression, and enzymatic assay of the ptr1 gene from Iranian lizard Leishmania as a model for further studies on Leishmania. Methods: Promastigote DNA was extracted from the Iranian lizard Leishmania, and the ptr1 gene was amplified using specific primers. The PCR product was cloned, transformed into Escherichia coli strain JM109, and expressed. The recombinant protein (PTR1 enzyme) was then purified and assayed. Results: ptr1 gene was successfully amplified and cloned into expression vector. Recombinant protein (PTR1 enzyme) was purified using affinity chromatography and confirmed by Western-blot and dot blot using anti-Leishmania major PTR1 antibody and anti-T7 tag monoclonal antibody, respectively. The enzymatic assay was confirmed as PTR1 witch performed using 6- biopterin as a substrate and nicotinamide adenine dinucleotide phosphate as a coenzyme. Conclusion: Iranian lizard Leishmania ptr1 was expressed and enzymatic assay was performed successfully

Suicide gene therapy for breast cancer with a suicide-inducing vector carrying the mammaglobin-1 promoter

BACKGROUND: Breast carcinoma is the most frequent cancer among women and finding a therapeutic approach is necessary for the treatment of this carcinoma. Gene therapy is a promising therapeutic approach for cancer. Targeted expression of the desired therapeutic proteins within the tumor cells is the best approach to reduce toxicity and improve survival. The mammaglobin-1 gene encodes a novel, breast cancer-associated glycoprotein. Mammaglobin-1 expression is restricted to the mammary gland and the function of the mammaglobin-1 protein is unknown. No mammaglobin-1 expression has been reported in various types of benign tissue or neoplasia other than the breast carcinomas. Over expression of mammaglobin-1 gene was reported in breast cancer tissues by some scientists. In this study, for the first time, the mammaglobin-1 promoter was introduced as a cancer specific promoter with a high efficacy. METHODS: A suicide-inducing vector that expresses the BAX suicide gene under the transcriptional control of the mammaglobin-1 promoter was constructed and evaluated against breast cancer in vitro. RESULT S: These data show that the mammaglobin-1 promoter is active in the breast cancer cell lines. Under the control of the mammaglobin-1 promoter, BAX expression in MD-MBA 438 cell line was five-fold higher than in human embryonic kidney (HEK) cell line and no expression was found in A549 cell line. With the help of mammaglobin-1 promoter, a wonderful enhancement was observed in the apoptosis in breast cancer cell lines. CONCLUSIONS: Concerning the expression of BAX gene under the control of mammaglobin-1, it can be used to specify suicide gene therapy in the treatment of breast tumor. © 2017 EDIZIONI MINERVA MEDICA