:: Volume 20, Issue 8 (11-2015) ::
__Armaghane Danesh__ 2015, 20(8): 666-676 Back to browse issues page
Prediction and cloning linear Tcell epitopes of P14-3-3 antigen into pEGFP–N1 as a DNA vaccine model to induse immuno response against hydatidosis and it's expression in CHO cell line
R Mesri1, M Esmaelizad 2, SA Angaji1, M Tahmaseb1, M Ahmadzadeh1, S Mohammadi1
1- 1Department of Cell and Molecular Biology, Kharazmi University, Tehran, Iran
2- 2Department of Central labratoary, Razi Vaccine and Serum Research Institute, Karaj, Alborz, Iran. , m.esmaelizad@rvsri.ac.ir
Abstract:   (3550 Views)


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

Keywords: Echinococcus granulosus, DNA vaccine, cloning, P14-3-3
Full-Text [PDF 341 kb]   (989 Downloads)    
Type of Study: Research | Subject: Parasitology
Received: 2015/06/6 | Accepted: 2015/08/31 | Published: 2015/10/12

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Volume 20, Issue 8 (11-2015) Back to browse issues page