Cloning of unique immunogenic region of Brassica mitogen activated protein Kinase 3 (MAPK3) in expression vector & purification of MAPK3 protein
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Date
2017-07
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G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand)
Abstract
The productivity of rapeseed and mustard is severely affected by Alternaria blight disease which is caused by the fungal pathogen, Alternaria brassicae. So far there is no source of resistance gainst this disease. It is being thought that the disease resistant varieties of Brassica can be developed provided that molecular mechanism of pathogenesis / defense is delineated. MAP kinase signaling pathways are hypothesized to play important role in defense against Alternaria blight disease of Brassica. MAPK3 has recently been shown to play important role in defense response against necrotrophic fungal pathogens e.g. Botrytis cinerea. It was realized the regulation of MAPK3 protein at translational level during pathogenesis/defense response towards Alternaria blight disease will throw light on it’s role during pathogenesis process. However this objective can be fulfilled if monospecific polyclonal antibodies are available against MAPK3 protein. In order to achieve the above objective , the full length sequence of MAPK3 gene was retrieved from Brassica rapa genome and an immunogenic unique region was selected on the basis of BLAST and analysis of B and T cell epitopes. This unique immunogenic region of Brassica juncea MAPK3 was amplified and cloned in pGEMTeasy cloning vector to perform efficient restriction digestion of insert DNA for directional cloning into pRSETC expression vector. The sequence and correct reading frame of cloned MAPK3 gene was confirmed by sequencing. Then the recombinant pRSETC vector was introduced into C41 strain of E.coli for efficient synthesis of MAPK3 protein. The synthesis of 6X-His-MAPK3 recombinant protein of 20.6 kDa was induced by 0.7 mM IPTG and analyzed through SDS-PAGE. The cloned MAPK3 gene contained B and T cell epitopes which were different from epitopes present in other MAP kinases. This suggested the cloning of unique immunogenic region of MAPK3 in the pRSETC expression vector. In next step, MAPK3 protein can be purified by Nickel affinity chromatography and protein can be injected into rabbit for raising monospecific antibody against it.
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