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Introduction & Objective: Brucella is a facultative intracellular pathogen and one of the etiologic agents of brucellosis that can infect humans and domestic animals. Attenuated strains such as B. melitensis Rve1 and B. abortus S19 and Rb51 are being used to control brucellosis in domestic animals. However, no safe and effective vaccine is available for human use. This study was designed to evaluate the immunogenicity and the protective efficacy of a divalent fusion DNA vaccine encoding both the B. melitensis Omp31 protein and P39 protein, designated pCDNA3 recombinant vector. Materials & Methods: This experimental study was performed in Biotechnology Research Center of Islamic Azad University, Shahrekord branch in summer, 1386. Construction of pCDNA3 recombinant vector containing Omp31 and P39 genes of B. melitensis was completed. Then, 12 Balb/c mice were immunized intramuscularly with 100 mg per 50 micro liters of this DNA vaccine. Control mice, 12 Balb/c mice, were simultaneously injected with PBS. During the 1st, 7th, 15th and 30th days the mice received the injections. Afterwards, the ELISA cytokine assay was performed and data were analyzed by SPSS software. Results: Intramuscular injection of the divalent DNA vaccine elicited cellular immune responses in Balb/c mice. The ELISA cytokine assay with serum of vaccinated mice showed high level of IFN-γ and low changes of IL-4 in compare with control mice. Conclusion: Use of divalent genetic vaccine based on the Omp31 and P39 genes can elicit a strong cellular immune response against Brucellosis.

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ABSTRACT

Background & purpose: Hydatidosis is a zoonotic disease that caused by infection with the larvae of Echinococcus granulosus. Different antigens produced in larval stage of this parasite that recombinant vaccine base these antigens created significant immunity in infected animals. One of the important antigens is p14-3-3 that it's recombinant antigen created considerable immunity in mouse models. In this study according to the high immunity of antigen epitopes region the coding sequence of T-cell epitopes of P14-3-3 was cloned into pEGFP-N1vector in order to produce an effective DNA vaccine model to stimulate high level of Th1 immune response.

  Material and method: In this study bioinformatics tools were used to prediction of linear T-Cell epitopes of Echinococcus granulosus P14-3-3 &zeta antigen. The nucleotide coding sequence of these epitopes was synthesized by PCR. the ampliqon was digested with XhoI restriction enzyme and cloned into pEGFP–N1 vector That has been purificated by modified sambrook method with CaCl2 and PEG6000..Positive colony was selected by direct colony PCR and confirmed by the sequencing.and evaluation of it's expression in Eukaryotic cells was done by transformed to CHO cell line with electroporation.

Results: Linear T-cell epitopes of Echinococcus granulosus P14-3-3 after prediction,synthesis and amplification wae successfully cloned into pEGFP-N1 vector that purificated by new method with maximum vector and minimum RNA concentration.The expression of new constract in CHO cell line as a eukaryotic cells achivment by fluorescent microscope and will be used as a DNA vaccine model to evaluation immuno response in mouse models.

  Discussion: Successfully cloning of The linear T-cell epitppes coding sequence of Echinococcus granulosus P14-3-3&zeta antigen into pEGFP-N1 verificated by sequencing and fluorscent microscope images demonstrated expression of recombinant protein in CHO cell line