Identification and Molecular Cloning of Heat Shock Protein-70 (HSP-70) Gene of Trypanosoma evansi Isolated from Camel

Abstract

Present study was carried out to isolate the Heat Shock Protein-70 gene of Trypanosoma evansi using PCR. The desired amplicons of Heat Shock Protein-70 gene from genomic DNA of T. evansi were successfully amplified by PCR using gene specific primers at annealing temperature of 54°C. Amplified PCR product was identified on the basis of its size in agarose gel electrophoresis as 1956 bp. For cloning the purified DNA fragment was ligated to the pGEM-T Easy vector and ligated mixture was transformed into Escherichia coli JM109 strains. The cells containing recombinant plasmid were identified on the basis of white/blue colony selection on LB agar containing X-Gal, IPTG and ampicillin. Screening of recombinant was done by Restriction Enzyme digestion of plasmid DNAs using EcoRI and confirmed on the basis of gene size, i. e. 1956 bp for Heat Shock Protein-70 gene. Colony PCR was done for quick screening of plasmid inserts directly from E. coli colonies in the presence of insert specific primers.