[Evaluating the immunogenicity for plasmid encoding GRA5 antigen of Toxoplasma gondii in BALB/c mice]

Severe or lethal damages of toxoplasmosis clearly indicate the need for the development of a more effective vaccine. Immunization with recombinant plasmid encoding protective proteins is a promising vaccination technique. Therefore, this study aimed to evaluate the immunization with plasmid encoding GRA5 antigen of Toxoplasma gondii in BALB/c mice. In this experimental study, three groups of BALB/c mice [n=10 in each group] were selected using simple random sampling. GRA5 gene was cloned into pcDNA3 plasmid and purified by plasmid purification kits and then the product was injected [IM]. To determine the status of cellular and hurnoral immunity, the 11-4, IFN- gamma and IgG; IgG2a, IgG subtypes were evaluated respectively using the ELISA-based assay. The group immunized with pcGRA5 indicated a significant augmented response in humoral and cellular immunity [P

[Determination and cloning of the gene encoding EG95 protein in Iranian isolate of Echinococcus granulosus]

Echinococcus granulosus is a cestode parasite that causes cystic hydatid disease in humans worldwide. The gene encoding EG95 protein may be a good candidate to design a DNA vaccine to prevent the disease. Considering the importance of EG95 gene and the scarceness of research on it in Iran, this study was carried out to determine and clone the gene encoding EG95 from Iranian isolate of E. granulosus.At the first stage, protoscoleces was isolated from hydatid cyst fluid and then RNA was extracted from protoscoleces and after performing RT-PCR, the amplified cDNA samples were detected by gel electrophoresis. In next stage, the obtained gene was cloned in pTZ57R/T vector. Two methods were used for conformation of cloning: colony PCR amplification and digestion with the EcoRI and XhoI restriction enzymes. Finally, the cloned EG95 gene in pTZ57R/T vector was sequenced. Homological comparison of sequences showed that cDNA of EG95 in Iranian isolate of E. granulosus had 492 bp and was different from the standard strain of EG95 reported from New Zealand and Australia [X90928.1]. Moreover, cloning of EG95 gene in pTZ57R/T plasmid was confirmed by digestion of this plasmid with the restriction enzymes. The EG95 gene was cloned in pTZ57R/T plasmid successfully and this plasmid can be used to design a DNA vaccine in further studies

[Cloning and sequencing the plasmid encoding Toxoplasma gondii Microneme 3 protein]

Toxoplasma gondii, an obligatory intracellular protozoan parasite, causing toxoplasmosis in human and animal with worldwide spread. Microneme 3 [MIC3] protein, a 90 kDa parasite factor attaching to the host cells in the beginning of the invasion, is secreted in all stages of parasite development [e.g. sporozoite, tachyzoite and bradyzoite] and also is considered as a potent antigen. Therefore, besides the immunogenicity and the candidacy for vaccine design, the protein is used for diagnostic purposes, as well. The aim of the present study was to transfer MIC3 gene into plasmid vector [PTZ57R/T] for subcloning in eukaryotic and prokaryotic plasmids. Toxoplasmia genomic DNA extracted using phenol-chloroform method and MIC3 gene was then amplified by PCR with specific primers. Electrophoresis was performed by using agarose gel and PCR product was purified by T4 DNA ligase enzyme into a cloning vector. Finally, recombinant plasmid was transformed into E.coli [Top10 strain]. The extracted clone was verified with PCR, digestion enzymes and sequencing. The PCR product was seen as a 1052bp band in agarose gel [1%]. The recombinant plasmids was restricted by HindIII and EcoRV enzymes and two obtained 2886 and 1052bp bands showed that the MIC3 gene was cloned in PTZ57R/T plasmid. The results revealed that the cloning and transformation of MIC3 gene in pTZ57R/T was done successfully

effect of vitamin D3 alone and mixed with IFN-gamma on tachyzoites of toxoplasma gondii [RH strain] proliferation and nitric oxide [NO] production in infected macrophages of BALB/C mice

Toxoplasma gondii is an obligatory interacelullar parasite that infects nucleated cells in its intermediate hosts. The aim of the present study was to determine the effect of vitamin D3 on the multiplication of T. gondii in peritoneal macrophage of Balb/c mice and nitric oxide production by macrophages. According to usage of vitamin D3 [one dose or seven doses] and INF gamma in vitro and in vivo, this study was divided into four experiments. In all experiments, the macrophages were collected from peritoneum and cultured in RPMI-1640. Then the supernatants were collected after 24 h and their nitric oxide was measure. After 96 h, the macrophages were collected and stained and the number of tachyzoites was measured. The first experiment [the mice were infected with tachyzoites and after 2 h, got one dose vita min D3 intraperitonealy] showed the best results. The mean of tachyzoites per macrophages was 2.37, and mean +/- SD of nitric oxide was 187.8 +/- 9. High-level production of nitric oxide may be related to the only one injection of vita min D3. The injection in long time might suppress the immune system

Determination of HBV genotypes among HBs Ag positive blood donors in Tehran, Iran using PCR-RFLP

Hepatitis B virus [HBV] is one of the major causative agents of acute and chronic liver disease worldwide and is believed to be responsible for a million deaths annually. On the basis of a comparison of complete genomic sequences, HBV has been classified into eight genotypes A-H which show a geographical distribution. Some genotypes are associated with different clinical outcomes. Identification of HBV genotypes is important to begin and follow up the treatment. In this cross-sectional study, the serum samples of blood donors were collected from Tehran Blood Transfusion Centers in period during "2005-2006". Sera of 55 blood donors who were positive for hepatitis B surface antigen were selected. DNA was extracted using commercial kit and the S gene sequence was amplified by nested-PCR. PCR products were then analyzed for restriction enzymes that would be genotype specific. Genotype D was found the only type in all HBV DNA positive serum samples, in Tehran. Genotype D is dominant among Tehran's blood donors, which is consistent with Iran and the Middle East dominant genotype

[Expression and neutralization of simian rotavirus recombinant glycoprotein [NSP4] with specific antibody in neonatal mice]

Rotaviruses are the most important cause of severe diarrhea in infants and young children in both developed and developing countries. Rotaviruses are members of the reoviridae family and contain 11 double- stranded RNA segments. Segment 10 encodes nonstructural glycoprotein NSP4, that can induce diarrhea in newborn mice. It has been suggested that NSP4 may be a key determinant for rotavirus pathogenesis and a target for vaccine development. Hence, the aim of this study was to examine the entire expression of mammalian rotavirus [sa11] NSP4 gene in E.coli and also its biological and immunogenesity properties in animal models. In this etarget for vaccine development. Hence, the aim of this study was to examine the entire expression of mammalian rotavirus [sa11] NSP4 gene in E.coli and also its biological and immunogenesity properties in animal models. In this experimental study, expression of NSP4 was demonstrated by Western blotting. The recombinant protein from pYS50 was purified by Preparative SDS polyacrylamid gel electrophoresis. Antibody against recombinant NSP4 was raised in rabbit. Intraperitoneal administration of full-length recombinant NSP4 caused diarrhea in 100% of the 4 to 5 days BALB/C neonatal mice. Intraperitoneal and oral inoculation of NSP4 antiserum significantly decreased diarrhea. Interaperitoneal administration of the full-length NSP4 induced diarrhea in 100% [5/5] of 4 to 5 days old BALB/C mice. Diarrhea in mice that received antibody against recombinant NSP4 was significantly decreased [p<0.000]. These results indicated successful expression of the biologically active full-length NSP4 in E.coli and confirmed that recombinant NSP4 was able to induced diarrhea in neonatal mice and also had enterotoxigenic activity. The recombinant NSP4 which produced in this study can be applied as antigen for detection of specific antibodies against NSP4 in human sera

[Prevalence of anti-HBc and anti-HBs in HBsAg negative blood donors in Tehran]

Current serological screening tests for blood-borne hepatitis viruses has reduced the risk of post-transfusion hepatitis dramatically. Occult hepatitis B virus [HBV] infection might allow the release of viremic units into the blood supply if blood is tested only for hepatitis B surface antigen [HBsAg]. Screening for anti-HBc has been shown as an alternative for detection of HBV infection. The aim of this study was to evaluate the prevalence of HBV infection markers in HBsAg negative blood donors. In this descriptive cross-sectional study, 2000 HBsAg negative samples were collected from blood centers in Tehran. All HBsAg negative samples were tested for anti-HBc using ELISA method. Then, all HBsAg negative and anti-HBc positive samples were tested for anti-HBs by the same method. All data were analyzed statistically using Chi-square test. Results One hundred ninety nine [9.95%] out of the 2000 HBsAg negative blood donors were anti-HBc positive [confidence interval of 7.66%-12.24%]. Out of the 199 anti-HBc-positive samples tested for anti-HBs, 149 [75%] were anti-HBs-positive [confidence interval of 65.5%-85.5%], and 102 [50.3%] had an antibody titer greater than 100 IU/ml. Conclusions In our study, the prevalence rate of anti-HBc in HBsAg negative blood donors was high. While anti-HBc-positive blood may be a potential source of HBV transmission, routine application of anti-HBc screening is not feasible in our country as it would seriously affect the blood supply adequacy. Therefore, more sensitive techniques such as minipool PCR testing after virus enrichment are essential for detecting HBV DNA in HBsAg-negative chronic HBV carriers

Cloning and sequencing of Leishmania major thiol-specific-antioxidant antigen [TSA] gene

Leishmaniasis is caused by parasitic protozoa of the genus Leishmania which, in the infected host are obligate intracellular parasite. TSA is the immuno-dominant antigen of Leishmania major which is considered as the most promising molecule for a recombinant or DNA vaccine against leishmaniasis. Genomic DNA of TSA protein was extracted and amplificated as a template. Then the PCR product was cloned into pTZ57R/T vector. Finally, the recombinant plasmid was extracted from transformed Escherichia coli [TG1 strain] and sequenced. MRHO/IR/75/ER [an Iranian strain] of L. major and TSA gene [Accession number LmjF15.1080] were used. Sequence analysis of cloned TSA gene into pTZ57R/T vector showed high homology of 90% with LmjF15.1080 [TSA gene] and strain "LV39" [Accession no. AF069386] and strain "Friedlin" [Accession no.AF044679]. We cloned TSA gene of L. major successfully. Recombinant plasmid was confirmed. It is ready to express recombinant protein for further studies

Expression of nonstructural glycoprotein NSP4 of SA11 Simian rotavirus in escherichia coli and production of antibody aginst it

Rotavirus non structural glycoprotein NSP4 can induce diarrhea in newborn mice. It has been suggested that NSP4 may be a key determinant for rotavirus pathogenesis and a target for vaccine development. In order to study the biological and morphological role of NSP4 a large amount of the purified protein and antibody against it are required. Simian rotavirus SA11 was propagated in BSCI cell, purified on cesium chloride gradients, and its genomic RNA was extracted. A cDNA from RNA segment 10 was synthesized and amplified by RT-PCR. cDNA fragment was cloned into plasmid vectors. The recombinant plasmid was characterized by restriction enzyme and sequencing. Construction of expression plasmid containing nsp4 gene was performed and expression of NSP4 was demonstrated by SDS-PAGE, Western blot, ELISA and IF using polyclonal antibody against NSP4 from SA11 infected BSC1 cells. A polyclonal antiserum against purified recombinant NSP4 was raised in rabbit; which was reacted with NSP4 in BSCI cells infected with SA11 rotavirus. These results indicated successful expression of the full-length NSP4 in E.coli to produce antibody against it and to study its biological activities