Identification and molecular cloning of cysteine protease gene of Trypanosoma Evansi Isolated from Camel

Abstract

A molecular study was carried out to isolate cysteine protease gene of Trypanosoma evansi using PCR. The desired amplicons of cysteine protease gene from the genomic DNA of T. evansi were successfully amplified by PCR using gene specific primers at annealing temperature of 55°C. Amplified PCR product was identified on the basis of its size in agarose gel electrophoresis as 1533 bp. For cloning the purified DNA fragment was ligated to the pGEM-T Easy vector and the 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 recombinants was done by restriction enzyme digestion of plasmid DNA using EcoRI and confirmed on the basis of gene size, i. e. 1533 bp for cysteine protease gene. Colony PCR was done for quick screening of plasmid inserts directly from E. coli colonies in the presence of insert specific primers.