CLONING AND CHARACTERIZATION OF rubisco PROMOTER FROM SUNN HEMP (Crotalaria juncea L.) FOR ITS ACTIVITY IN EXPRESSING FMDV GENES IN HOMOLOGOUS SYSTEM
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Date
2008-11-26
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University of Agricultural Sciences GKVK, Bangalore
Abstract
Genetically engineered microorganisms are important sources of industrial and medicinal proteins. Over the past decade Plant host system has been investigating as potential host system for expressing proteins of therapeutic and diagnostic use. However concerns regarding the stability and environmental safety need to be addressed for taking up the Plant system as potent host system. Chloroplast engineering however is expected to resolve some of the issue. Since, the FMD is a major concern in the world over and attempts have been directed towards development of alternative vaccines for conventional virus based immunoprofilactics, so present investigation used FMD immunogen for genetic engineering of chloroplast DNA and constructing transfer vector for transformation of Sunnhemp a fodder crop through Biolistic method. Using pBS-KS+ vector as a back bone rbcL promoter (1Kb) along with a 5’ end of gene with MCS (375bp) was amplified from genomic and chloroplast DNA and cloned in BamHI and HindIII, and BamH1 and SacI respectively, region that are highly conserved and flank the gene of interest and helps in homologous recombination. To the constructed transfer vector prbcL Trans, FMDV immunogenic gene P1-2A (2.3Kb) was cloned in the MCS BamHI and NotI. The plasmid was transferred into DH5α (E. coli) cells and the cultures were harvested at different time periods and the proteins from the lysates were analyzed for FMDV specific gene P1-2A. SDS-PAGE analysis showed an addition band of 90KDa indicating there was an expression of FMDV gene under cloned promoter. The specificity of the expression protein was confirmed by western blot. The constructed vector with immunogenic P1-2A gene (prbcL P12A) was used for transforming Sunnhemp calli standardized which induced a good callus from leaf explants in MS basal media with 2.0mg/L BAP + 0.2mg/L 2,4D through Biolistic method. The callus subject to bombardment with plasmid coated tungsten particles was co-cultivated for 15 days. The callus survived was further subjected to PCR for screening of the putative transgenic callus. The PCR was done using VP1 specific primer which amplify 700bp VP1-2A region. Two out of seven calli showed positive amplification resulting in a putative transgenic sunn hemp calli.
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