Background: With respect to the prevalence of leishmaniosis in our country and the problems occurred in the therapeutic approaches and also with respect to the previous successful reports of paromomycin efficacy in this regard, the present study was conducted to determine the effects of paromomycin and gentamicine sulphate on promastigotes of Leishmania major.

Materials and methods: It was an experimental study. The base of films composed of ethyl cellulose and HPMC (hydroxyl propyl methyl cellulose) containing paromomycin 15% and gentamicine 0.5%. To determine the rate and duration release from the films and its killing effects on leishmania promastigotes, a cloning system of parasite was established by using a set of modified NNN medium without liquid layer. The medium plates were divided in 3 groups, the media contain of drug films, placebo films, and media containing no film.

Results: results showed no colonies of Leishmania in a 30mm radius envelop the drug films. On the other hand, grown colonies of Leishmania promastigotes were found in placebo and control plates. Frequency of dead parasites were 98% and 5% in the placebo-control plates and drug plates, respectively (p<0.000).

Conclusion: Paromomycin and gentamicine drug films have good potential of drug releasing and suitable killing effects in vitro condition. It is suggested that the therapeutic effect of this new form of drug be achieved in human models.

Background: 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.

Materials and Methods: 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.

Results: 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.

Conclusion: The results revealed that the cloning and transformation of MIC3 gene in pTZ57R/T was done successfully.

Background: Toxoplasmosis, as a widespread and important disease, can cause severe complications in the congenital and HIV cases. Vaccination is one of the preventive approaches in humans and domestic animals. The GRA7, an excretory 29 kDa Toxoplasma gondii dense granule antigen released by the infected host cells , has been considered as a candidate to produce a vaccine .

Materials and Methods: In this experimental study, the inserted plasmid containing GRA7 gene was extracted from TOP10 bacteria and digested with the BamH1 and EcoR1 enzymes. The isolated gene was inserted into the pcDNA3 plasmid . The cloning was confirmed by the PCR and sequencing. The protein expression was confirmed by the SDS-PAGE and Western blotting.

Results: The results showed that the GRA7 gene was cloned into the pcDNA3 plasmid. The isolated gene cloned in pcDNA3 was confirmed by PCR and showed the 733bp band. The pcGRA7 plasmid expressed in the CHO cells showed the 29 kDa band using the SDS-PAGE and Western blotting.

Conclusion: Considering that cloning of GRA7 gene has been done in the pcDNA3 plasmid and transformed in the eukaryotic cells, it can be used for the DNA vaccine researches.

Background: Nef protein is one of the HIV regulatory proteins. This protein has various conserved epitopes inducing the efficient immune responses in HIV-1 infected individuals. Thus, Nef protein has been proposed as a suitable candidate for vaccine design. In current study, our goal was the cloning and expression of Nef protein in prokaryotic expression system and its purification using the reverse staining method. 

Materials and Methods: The coding sequence of Nef protein was amplified from pUC19-nef vector by PCR. Then, nef gene was inserted into the pGEX6p2 expression vector. This construct was transformed into the E.coli BL21 E.coli strain and subsequently protein expression was induced by IPTG anti-repressor. The protein expression was confirmed by SDS-PAGE and Western blot using anti-Nef antibody. Protein purification was performed by reverse staining method.

Results: The PCR and digestion analysis showed a clear band of 648 bp in agarose gel indicating the correct cloning of HIV-nef in pGEX6p2 expression vector. In addition, the detection of a clear 50 kDa band   in Western blotting using Anti-Nef antibody suggests the Nef protein expression induced by IPTG. Finally, the purified protein was obtained by reverse staining method.

Conclusion: The recombinant Nef protein expressed in E.coli was purified by reverse staining method. The Nef protein has the potential of antigenicity for vaccine designing against HIV infections.