Isolation, gene expression and PthA effector protein production of Xanthomonas citri subsp citri causal agent of citrus bacterial canker
Maryam Mokhtari | Mohmmad Reza Safar Nezhad | Seyyed Mahdi Alavi | Adam Torkman Zehi
The citrus bacterial canker is among the most important diseases in Mexican lime gardens in southern area of Iran. The disease is caused by <em>Xanthomonas citri</em> subsp. <em>citri (Xcc)</em>. The PthA bacterial protein, as an effector protein, is crucial in pathogenesis pathway. Inhibition of its functions through antibody could lead to suppression of disease in plant. The present study was performed for isolation and expression of <em>pthA</em> gene and purification of PthA recombinant protein. For this aim the specific primers were designed for isolation of 606 bp of hydroxyl terminal of this gene followed by PCR amplification and cloning into pTZ57R/T vector<em>.</em> The coding sequence of truncated <em>pthA</em> was inserted to pET28a bacterial expression vector as a C-terminal fusion to 6XHis tag. Production of recombinant protein was performed in BL21(DE3) strain of <em>E. coli. </em>The purification was done under native condition through affinity chromatography on nickel column. Total yield was assessed by SDS-PAGE and western blotting analysis. The results confirmed production of PthA recombinant protein with molecular weight around 26 kDa. The purified recombinant protein could be used as an antigen for production of recombinant monoclonal antibodies.
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