Effects of human chorionic gonadotropin-producing peripheral blood mononuclear cells on the endometrial receptivity and implantation sites of the mouse uterus / 대한생식의학회지

Objective@#This research investigated the effects of human chorionic gonadotropin (HCG)-producing peripheral blood mononuclear cells (PBMCs) on the implantation rate and embryo attachment in mice. @*Methods@#In this experimental study, a DNA fragment of the HCG gene was cloned into an expression vector, which was transfected into PBMCs. The concentration of the produced HCG was measured using enzyme-linked immunosorbent assay. Embryo attachment was investigated on the co-cultured endometrial cells and PBMCs in vitro. As an in vivo experiment, intrauterine administration of PBMCs was done in plaque-positive female mice. Studied mice were distributed into five groups: control, embryo implantation dysfunction (EID), EID with produced HCG, EID with PBMCs, and EID with HCG-producing PBMCs. Uterine horns were excised to characterize the number of implantation sites and pregnancy rate on day 7.5 post-coitum. During an implantation window, the mRNA expression of genes was evaluated using real-time polymerase chain reaction. @*Results@#DNA fragments were cloned between the BamHI and EcoRI sites in the vector. About 465 pg/mL of HCG was produced in the transfected PBMCs. The attachment rate, pregnancy rate, and the number of implantation sites were substantially higher in the HCG-producing PBMCs group than in the other groups. Significantly elevated expression of the target genes was observed in the EID with HCG-producing PBMCs group. @*Conclusion@#Alterations in gene expression following the intrauterine injection of HCG-producing PBMCs, could be considered a possible cause of increased embryo attachment rate, pregnancy rate, and the number of implantation sites.

Anacardic acid reduces acetylation of H4K12 in mouse Oocytes during vitrification

Objective: Over the last years, vitrification has been widely used for oocyte cryopreservation, in animals and humans; however, it frequently causes minor and major epigenetic modifications. The effect of oocyte vitrification on levels of acetylation of histone H4 at lysine 12 [AcH4K12], and histone acetyltransferase [Hat] expression, was previously assessed; however, little is known about the inhibition of Hat expression during oocyte vitrification. This study evaluated the effect of anacardic acid [AA] as a Hat inhibitor on vitrified mouse oocytes

Materials and Methods: In this experimental study, 248 mouse oocytes at metaphase II [MII] stage were divided in three experimental groups namely, fresh control oocytes [which were not affected by vitrification], frozen/thawed oocytes [vitrified] and frozen/thawed oocytes pre-treated with AA [treatment]. Out of 248 oocytes, 173 oocytes were selected and from them, 84 oocytes were vitrified without AA [vitrified group] and 89 oocytes were pretreated with AA, and then vitrified [treatment group]. Fresh MII mouse oocytes were used as control group. Hat expression and AcH4K12 levels were assessed by using real-time quantitative polymerase chain reaction [PCR] and immunofluoresce staining, respectively. In addition, survival rate was determined in vitrified and treatment oocytes

Results: Hat expression and AcH4K12 modification significantly increased [4.17 +/- 1.27 [P.0.001] and 97.57 +/- 6.30 [P<0.001], respectively] in oocytes that were vitrified, compared to the fresh oocytes. After treatment with AA, the Hat mRNA expression and subsequently H4K12 acetylation levels were significantly reduced [0.12 +/- 0.03 [P.0.001] and 89.79 +/- 3.20 [P.0.05], respectively] in comparison to the vitrified group. However, the survival rate was not significantly different between the vitrified [90.47%] and treatment [91.01%] groups [P>0.05]

Conclusion: The present study suggests that AA reduces vitrification risks caused by epigenetic modifications, but does not affect the quality of vitrification. In fact, AA as a Hat inhibitor was effective in reducing the acetylation levels of H4K12

Design and development of modified mRNA encoding core antigen of hepatitis C virus: a possible application in vaccine production

Introduction: Hepatitis C virus [HCV] is a ?blood-borne pathogen, resulting in liver cirrhosis and liver cancer. Despite of many efforts in development of treatments for HCV, no vaccine has been licensed yet. The purpose of this study was ?to design and prepare a specific mRNA, without 5' cap and poly [A] tail transcribed in vitro capable of coding core protein and also to determine its functionality

Methods: Candidate mRNA was prepared by in vitro transcription of the designed construct consisting of ??5' and 3' untranslated regions of heat shock proteins 70 [hsp70] mRNA, T7 promoter, internal ribosome entry site [IRES] sequences of eIF4G related to human dendritic cells [DCs], and the ?Core gene of HCV. To design the modified mRNA, the ??5' cap and poly [A] tail structures were not considered. DCs were transfected by in vitro-transcribed messenger RNA [IVT-mRNA] and the expressions of green fluorescent protein [GFP], and Coregenes were determined by microscopic examination and Western blotting assay

Results: Cell transfection results showed that despite the absence of ??5' cap and poly [A] tail, the structure of the mRNA ?was stable. Moreover, the successful expressions of GFP and Coregenes were achieved

Conclusion: Our findings indicated the effectiveness of a designed IVT-mRNA harboring the Core gene of HCV in transfecting and expressing the antigens in DCs. Considering the simple and efficient protocol for the preparation of this IVT-mRNA and its effectiveness in expressing the gene that it carries, this IVT-mRNA could be suitable for development of an RNA vaccine against HCV

Interleukin-1 and Keratinocyte growth factor/Fibroblast growth factor-7 gene expression in skin experimental irritant contact dermatitis mouse model treated with Aqueous extract of Trachyspermum copticum [L.] link seeds

Background: In our previous study, the extract of Trachyspermum copticum [L.] Link seeds on gene expression of IFN-alpha and TGF-beta1 in mouse model with irritant contact dermatitis [ICD], in comparison with cutaneous corticosteroids were evaluated. In that study, in addition to significantly increase of IFN-alpha and TGF-beta1 genes expression levels in skin samples of [mice with ICD] groups treated with extract in comparison to other groups, histopathologic findings showed substantial improvement of skin color, texture and thickness, and also significant increase in hair follicle number. Therefore, we have decided to study the levels of Interleukin-1 [IL-1] gene expression, which plays a major role in inflammation responses, and Keratinocyte Growth Factor/Fibroblast Growth Factor-7 [KGF/FGF-7], which has growth effect on cells and is an important endogenous mediator of hair follicle growth and development

Materials and Methods: We used autopsy samples of skin lesions obtained from [mice model with irritant contact dermatitis [ICD]] from the previous study. In that study, [mice with ICD] divided in 9 groups and were treated with three concentrations of Trachyspermum Copticum [L.] Link dried seeds, cutaneous hydrocortisone, and fluocinolone acetonide. Then from the first day until the 10th day of treatment, clinical signs and histopathologic investigations were investigated. In the present study, using Real-Time PCR, the levels of IL-1 and KGF/FGF-7 genes expression in skin samples of inflammation site in above mice groups were studied. Statistical analysis, using one -way ANOVA, were performed. Level of significance was set at 0.05

Results: The IL-1 gene expression showed a significant difference between groups: IL-1 gene expression levels in mice with ICD treated with extract and corticosteroids were higher than the other groups [p=0.0001]. While in untreated [mice with ICD], no significant differences were observed. Also, during the treatment, there was a considerable increase in levels of IL-1 gene expression in groups treated with the extract at a rate of at least 2 to 3-fold in comparison with the [healthy untreated mice] group. The levels of KGF/FGF-7 gene expression in [mice with ICD] groups treated with the extract showed significance difference [p=0.014]; also there was a meaningful difference in [mice with ICD] groups treated with cutaneous corticosteroids [p=0.004]. While, in [untreated mice with ICD] group there were a significant decrease in the levels of KGF/FGF-7 gene expression in comparison with [healthy untreated mice] group [p=0.0001]. Also, changes in the levels IL-1 and KGF/FGF-7 gene expressions in each group in different days were seen

Conclusion: In this study, significant changes in the IL-1 and KGF/FGF-7 genes expression levels in the skin samples with inflammation, were associated with an increase in the rate and speed of improvement of contact dermatitis, more favorable conditions of the healed skin [in terms of color, consistency, and thickness], and a remarkable increase in the number of hair grown on the site of dermatitis [compared with control groups, and even groups with corticosteroid therapy]

CRISPR-Cas9 mediated capsule gene silencing in Escherichia coli

Background: Genomic engineering of Escherichia coli is applied to design and produce recombinant proteins as the new drugs. The aim of this study was to CRISPR-Cas9 mediated capsule gene silencing in E. coli

Materials and Methods: We suppressed genes involved in capsule expression of E.coli by CRISPR cas9 process. The constructed E.coli was confirmed by microscopic smear, transmission electron microscopy and T7 phage influence assay

Results: The results were shown that the inhibition of capsule production was carried out successfully and there was not any capsule layer around the bacteria

Conclusion: E. coli without any capsule around may proper for replacement of it with other molecules in future

In silico analysis of immunologic regions of surface antigens [Sags] of Toxoplasma gondii

Background: Surface antigens [SAGs] of Toxoplasma gondii are known candidates for diagnostic tests and vaccines. The present study argues about the main necessary properties for determination and prediction of T-cell agretopes and B-cell epitopes of surface antigens of Toxoplasma gondii

Materials and Methods: Primary, secondary and tertiary structures of the proteins were analyzed by different methods. The three-dimensional structures were determined by use of ab initio method for prediction of discontinues epitopes. The agretopes and epitopes were predicted via several various web servers with different methods employed

Results: The results of in silico analyses showed that the regions 129-GAPAGRNNDGSSAPT-143 for protein p22, 234-SENPWQGNASSD-245 for protein p30 and 348-PGTEGESQAGT-358 for protein p43, have the highest immunogenic potential

Conclusion: We reached to three antigenic epitopes for cloning and protein expression. In following the purified polypeptide will be applied for diagnosis of Toxoplasma gondii

Evaluation of in vivo bioactivity of a mutated streptokinase

Background: Immunogenicity of Streptokinase, as a thrombolytic drug, has limited its clinical use. Elimination of the amino acid residues that are responsible for immunogenicity while don't affect the bioactivity of streptokinase is worthy. Recently, we modified the streptokinase through the elimination of 42 amino acids from its' C-terminal and assessed its bioactivity in vitro. In this study, bioactivity of the mutated-streptokinase determined and compared with those of commercially available streptokinase [Heberkinase] in rabbits with induced blood clot

Materials and Methods: Recombinant mutated streptokinase was purified and its lipopolysaccharide contained remove and evaluated by LAL test. Thrombolytic activity of drug was evaluated by rabbit jugular vein as in vivo thrombosis model. The thrombolytic property of the drug was evaluated with determining of D-dimer in plasma

Results: The results showed in vivo bioactivity of both truncated and commercial streptokinase [p<0.05]. This study showed an important influence of the 42 amino acids of C-terminal in bioactivity of the streptokinase

Conclusion: Clinical use of the r-streptokinase requires more modification to restore its' activity in vivo. This product may be a promising choice for clinical use after confirmation of its stability and non-immunogenicity

Suppressive effect of constructed shrnas against apollon induces apoptosis and growth inhibition in the hela cell line

Background: Cervical cancer is the second most common female cancer worldwide. Inhibitors of apoptosis proteins [IAPs] block apoptosis; therefore, therapeutic strategies targeting IAPs have attracted the interest of researchers in recent years. Apollon, a member of IAPs, inhibits apoptosis and cell death. RNA interference is a pathway in which small interfering RNA [siRNA] or shRNA [short hairpin RNA] inactivates the expression of target genes. The purpose of this study was to determine the effect of constructed shRNAs on apoptosis and growth inhibition through the suppression of apollon mRNA in HeLa cell line

Methods: Three shRNAs with binding ability to three different target sites of the first region of apollon gene were designed and cloned in pRNAin-H1.2/Neo vector. shRNA plasmids were then transfected in HeLa cells using electroporation. Down-regulation effects of apollon and the viability of HeLa cells were analyzed by RT-PCR, lactate dehydrogenase assay, and MTT assay, respectively. Also, the induction and morphological markers of apoptosis were evaluated by caspase assay and immunocytochemistry method

Results: The expression of shRNA in HeLa cells caused a significant decrease in the level of apollon mRNA1. In addition, shRNA1 effectively increased the mRNA level of Smac [as the antagonist of apollon], reduced the viability of HeLa cells and exhibited immunocytochemical apoptotic markers in this cell line

Conclusion: Apollon gene silencing can induce apoptosis and growth impairment in HeLa cells. In this regard, apollon can be considered a candidate therapeutic target in HeLa cells as a positive human papillomavirus cancer cell line

Cloning and expression of luteinizing hormone subunits in Chinese hamster ovary cell line

Background: Luteinizing hormone [LH] was secreted by the stimulating cells of the testes and ovaries in the anterior pituitary gland. The application of this hormone is in the treatment of men and women with infertility and amenorrhea respectively

Materials and Methods: In the present study the alpha and beta subunits of human LH gene were cloned into the pEGFP-N1 expression vector and produced the recombinant LH hormone in Chinese hamster ovary [CHO] eukaryotic system

Results: Alpha and beta subunits of LH hormone were cloned between NheI and BamHI cut sites of pEGFP_N1 expression plasmid and confirmed by PCR. Hormone expression was evaluated in CHO cell line by Western blotting using the specific antibody

Conclusion: Alpha and beta subunits of LH hormone were expressed in CHO cell line perfectly

Cloning and expression of EBI3 and p28 subunits of human interleukin 27 in E. coli

Interleukin [IL]-27 is a heterodimeric cytokine belonging to IL-12 and IL-23 families, secreted by antigen presenting cells [APCs]. The IL-27 is composed of 2 subunits: Epstein-Barr virus induced gene 3 [EBI3] and p28. IL-27 is an anti-inflammatory cytokine which has an inhibitory effect on Th17 population and suppress the IL-17 expression. It is suggested that IL-27 could be a potent drug candidate for treating auto immune diseases. The EBI3 and p28 subunits of human interleukin 27 were constructed into plasmid vectors; they sub-cloned into pETDuet-1 expression vector in restriction sites of BamHI, SacI and NotI. Subsequently the induction was carried out by 1mM IPTG and the recombinant proteins were confirmed by SDS-PAGE and Western Blot analysis using anti His-tag antibody. The EBI3 and p28 subunits of human interleukin 27were cloned into plasmid vectors. The 28 and 25kDa protein bands were observed on SDS-PAGE and finally confirmed by Western blot technique. In this research p28 and EBI3 proteins were produced successfully

Mycoplasma infection in pyospermic infertile and healthy fertile men

Infections are one of the correctable causes of infertility with low cost and cost effective treatment. The 50% of infertile cases is related to men in some way, and 30% of them are absolutely related to them. Mycoplasmas are the smallest microorganisms with capability of DNA replication. Present study is planned to compare the mycoplasma infection in infertile men and men with established fertility. 45 Semen samples were collected from case and control persons who referred to Royan Infertility and Fertility Institute between 2004 and 2005 and stored in -20 degree°C until time of test. DNA was extracted from semen using phenol chloroform. PCR reaction was done by mycoplasma specific primers. Mycoplasma genitalium gene was amplified in 6 [40%] cases from 15 infertile semen samples and 11 [36.6%] from 30 control semen samples. Probability of genital infection, at least, in these studies group, is very lower than other communities' reports

Cloning and expression of TNF related apoptosis inducing ligand in Nicotiana tabacum

Molecular farming has been considered as a secure and economical approach for production of biopharmaceuticals. Human TNF Related Apoptosis Inducing Ligand [TRAIL] as a promising biopharmaceutical candidate has been produced in different expression hosts. However, little attention has been paid to molecular farming of the TRAIL in spite of numerous advantages of plant expression systems. Therefore, in this study the cytoplasmic production of the TRAIL was tackled in Nicotiana tabacum using Agrobacterium tumefaciens LBA 4404. Initially, the desired coding sequence was obtained using PCR technique on the constructed human cDNA library. Afterward, the necessary requirements for expression of the TRAIL in plant cell system were provided through sub-cloning into 35S-CaMV [Cauliflower Mosaic Virus] helper and final 0179-pGreen expression vectors. Then, the final TRAIL-pGreen expression vector was cloned into A. tumefaciens LBA 4404. Subsequently, the N. tabacum cells were transformed through co-culture method and expression of the TRAIL was confirmed by western blot analysis. Finally, the recombinant TRAIL was extracted through chromatographic technique and biological activity was evaluated through MTT assay [Methylthiazol Tetrazolium Assay]. The result of western blot analysis indicated that only monomer and oxidized dimer forms of the TRAIL can be extracted from the N. tabacum cells. Moreover, the lack of trimeric assembly of the extracted TRAIL diminished its biological activity in sensitive A549 cell line. In conclusion, although N. tabacum cells can successfully produce the TRAIL, proper assembly and functionality of the TRAIL were unfavorable

Periplasmic expression of TNF related apoptosis inducing ligand [TRAIL] in E.coli

Tumor necrosis factor-related apoptosis-inducing ligand [TRAIL], a member of TNF family, is an interesting ligand which selectively induces apoptosis in tumor cells and, therefore, it has been developed for cancer therapy. This ligand has been produced by various hosts such as E.coli. However, protein expression in E.coli cytoplasm leads to problems such as incorrect folding, reduction in biological activity, inclusion body formation, and sophisticated downstream. The aim of this study is to develop an expression system for the production of recombinant TRAIL secreted to the E.coli periplasm instead of cytoplasm. By using Overlapping Extension PCR, an OmpA signal sequence was fused to TRAIL cDNA and OmpA-TRAIL fragment was then cloned in pET-22b plasmid. This construct was confirmed by PCR and DNA sequencing. Promoter was induced in E.coli BL21 [DE3] and periplasmic expressed proteins were released using osmotic shock procedure. SDS-PAGE analysis showed that about 37% of recombinant TRAIL was transferred into the periplasm and its identity was confirmed by western blot analysis. Finally, the cytotoxic activity of TRAIL against HeLa cell line was confirmed by using MTT assay. The results demonstrate that our expression system may be useful for the production of TRAIL in the periplasmic space

Cloning and expression of recombinant nucleoprotein of influenza H1N1

Influenza virus is the major cause of lower respiratory tract illnesses on the worldwide. Vaccination can be an effective tool to prevent its outbreak. Highly conserved viral nucleoprotein is an effective vaccine candidate to provide heterosubtypic immunity, offering resistance against various influenza virus strains. In present research NP gene was inserted in pET-22b expression vector. New construct [pET-22b/NP] was transformed into E. coli BL21 [DE3] strain and the expression of nucleoprotein was induced by IPTG. It was analyzed by SDS-PAGE and confirmed by Western blotting. Western blotting confirmed the expression and production of recombinant Influenza nucleoprotein. These results suggest that the codon-optimized influenza A virus NP gene can be efficiently expressed in E. coli

Fibrinolytic activity of recombinant mutant streptokinase

Streptokinase is a bacterial protein produced by different beta hemolytic streptococci and widely used in thrombolytic treatment. The main disadvantage of using Streptokinase is antibody formation which causes allergic reaction to neutralize effects of Streptokinase therapy. Aim of this study was investigate of recombinant mutant Streptokinase fibrinolytic activity. In this study recombinant mutant Streptokinase without 42 amino acids from the C terminal region was purified by affinity S-Tag column chromatography and its fibrinolytic activity was studied. The concentration of expressed and purified protein was 10 mg/ml. Its enzyme activity was assayed using zymography, radial caseinolytic activity and fibrin plate test methods and estimated quantitatively by casein digestion method compared to a commercial form. It was found that this product had the more volume and more enzymatic activity

Histone modification of embryonic stem cells produced by somatic cell nuclear transfer and fertilized blastocysts

Nuclear transfer-embryonic stem cells [NT-ESCs] are genetically identical to the donor's cells; provide a renewable source of tissue for replacement, and therefore, decrease the risk of immune rejection. Trichostatin A [TSA] as a histone deacetylase inhibitor [HDACi] plays an important role in the reorganization of the genome and epigenetic changes. In this study, we examined whether TSA treatment after somatic cell nuclear transfer [SCNT] can improve the developmental rate of embryos and establishment rate of NT-ESCs line, as well as whether TSA treatment can improve histone modification in NT-ESCs lines. In this experimental study, mature oocytes were recovered from BDF1 [C57BL/6xDBA/2] mice and enucleated by micromanipulator. Cumulus cells were injected into enucleated oocytes as donor. Reconstructed embryos were activated in the presence or absence of TSA and cultured for 5 days. Blastocysts were transferred on inactive mouse embryonic fibroblasts [MEF], so ESCs lines were established. ESCs markers were evaluated by reverse transcription-polymerase chain reaction [RT-PCR]. Histone modifications were analyzed by enzyme linked immunosorbent assay [ELISA]. Result of this study showed that TSA treatment after SCNT can improve developmental rate of embryos [21.12 +/- 3.56 vs.8.08 +/- 7.92], as well as establishment rate of NT-ESCs line [25 vs.12.5]. We established 6 NT-ESCs in two experimental groups, and three embryonic stem cells [ESCs] lines as control group. TSA treatment has no effect in H3K4 acetylation and H3K9 tri-methylation in ESCs. TSA plays a key role in the developmental rate of embryos, establishment rate of ESC lines after SCNT, and regulation of histone modification in NT-ESCs, in a manner similar to that of ESCs established from normal blastocysts

design of the constructs of cpsD and simA genes of Streptococcus iniae

Streptococcosis is one of the bacterial infections in fish, especially rainbow trout which infects brain and nervous systems offish and is caused by S. iniae. Estimation of the impact of disease prevalence by S. iniae in fish fanning in some countries is reported about 100 million dollars per year. Some of the most effective proteins in pathogenicity of these bacteria are SimA and CpsD. In order to design new and effective vaccine, in this study cloning of two genes of Streptococcus was performed into pNZ8148 vector and expressed in Escherichia coli. simA and cpsD genes were subcloned into pNZ8148 vector. Obtained constructs were transformed to expressing E. coli BL21 strain. After induction with nisin, SDS PAGE electrophoresis and Western blotting were used to confirm the procedures. Using PCR with specific and universal primers, the accuracy of cloning was confirmed. Final verification of expressed protein was carried out by SDS-PAGE and western blotting. With regard to the obtained results, it seems that the generated gene construct in this study can be used as a vaccine against Streptococcosis in future researches

Genotyping and phylogenetic analysis of fasciola spp. isolated from sheep and cattle using PCR-RFLP in Ardabil province, Northwestern Iran

The aim of this study was to detect the genotype of Fasciola spp. in Meshkin-Shahr, Ardabil Province, northwestern Iran in different hosts using PCR-RFLP. The parasite hosts included cattle, and sheep. Overall, 70 adult flukes from livers of slaughtered animals were collected from the abattoirs of aforementioned area. The included 35 samples from infected sheep and 35 samples from 35 infected cattle. PCR-RFLP and sequence analysis of the first nuclear ribosomal internal transcribed spacer [ITS 1] region from Fasciola species were used to conduct the study. The fragment of approximately 700bp in all of the Fasciola samples was amplified. PCR products of ITS 1 were subjected for digestion by restriction enzyme. RsaI restriction enzyme was selected for RFLP method that caused the separation specifically of Fasciola species. Amplicons with the sequences of F. hepatica had a pattern of about 360, 100, and 60 bp band size, whereas F. gigantica worms had a profile of 360, 170, and 60 bp in size, respectively. Results based on PCR-RFLP analysis were confirmed by sequence analysis of representative ITS 1 amplicons. No hybrid forms were detected in the present study. All sheep were infected with F. hepatica but cattle were infected with both species. Both species of Fasciola are present in Ardabil. The method described here can be valuable for identification of Fasciola species in endemic parts for fasciolosis, regions with intermediate species and in that overlapping distribution area

Molecular detection and identification of theileria species by PCR-RFLP method in sheep from Ahvaz, Southern Iran

The present study was carried out to investigate the accurate status of ovine Theileria infection in sheep from Ahvaz and surrounding region, a tropical area southwest Iran. A PCR-RFLP method based on 18S ribosomal RNA gene was designed which could detect and differentiate Theileria and Babesia spp. and also differentiate main Theileria species in sheep at the same time. 119 sheep blood samples were collected from Ahvaz and surroundings. Microscopic examination of blood smears revealed 69.7% [83/119] infection with Theileria spp. Of the total samples subjected to PCR, 89% [106/119] were found to be positive, all of which were identified as Theileria by RFLP analysis using enzyme Hind II. In enzymatic digestion of PCR products by Vsp I, 91.5% [97/106] of Theileria positive samples were identified as T. ovis while mixed Theileria infections were found in 9 samples. The samples with mixed infections were analyzed with an additional nested PCR-RFLP method, by Hpall enzyme digestion. 3 samples with T, lestoquardi infection, 1 sample with T. ovis and T. annulata., 1 sample with T. lestoquardi and T. annulata, and 4 samples with T. ovis, T. lestoquardi and T. annulata mixed infections were detected. Ovine theileriosis caused by T. ovis is highly prevalent in southwest Iran while T. lestoquardi and T. annulata infection can be detected in a lesser proportion of sheep in this region. The new PCR-RFLP method that was designed in this study, can serve as a beneficial diagnostic tool, especially in T. ovis prevalent regions

Evaluation of Recombinant SAG1, SAG2, and SAG3 Antigens for Serodiagnosis of Toxoplasmosis

Serologic tests are widely accepted for diagnosing Toxoplasma gondii but purification and standardization of antigen needs to be improved. Recently, surface tachyzoite and bradyzoite antigens have become more attractive for this purpose. In this study, diagnostic usefulness of 3 recombinant antigens (SAG1, SAG2, and SAG3) were evaluated, and their efficacy was compared with the available commercial ELISA. The recombinant plasmids were transformed to JM109 strain of Escherichia coli, and the recombinants were expressed and purified. Recombinant SAG1, SAG2, and SAG3 antigens were evaluated using different groups of sera in an ELISA system, and the results were compared to those of a commercial IgG and IgM ELISA kit. The sensitivity and specificity of recombinant surface antigens for detection of anti-Toxoplasma IgG in comparison with commercially available ELISA were as follows: SAG1 (93.6% and 92.9%), SAG2 (100.0% and 89.4%), and SAG3 (95.4% and 91.2%), respectively. A high degree of agreement (96.9%) was observed between recombinant SAG2 and commercial ELISA in terms of detecting IgG anti-Toxoplasma antibodies. P22 had the best performance in detecting anti-Toxoplasma IgM in comparison with the other 2 recombinant antigens. Recombinant SAG1, SAG2, and SAG3 could all be used for diagnosis of IgG-specific antibodies against T. gondii.