Camels and Camelids

IDENTIFICATION AND MOLECULAR CLONING OF CYSTEINE PROTEASE GENE OF Trypanosoma evansi ISOLATED FROM CAMEL

Journal Edition: August 2017
Article DOI: 10.5958/2277-8934.2017.00030.3
Published On: 03-09-2018 18:58

Hakim Manzer, S.K. Ghorui1, G.S. Manohar, S.K. Kashyap2, N. Kumar and Sashikant Kankar
Department of Veterinary Parasitology, 2Department of Veterinary Microbiology and Biotechnology,
College of Veterinary and Animal Science, Rajasthan University of Veterinary and Animal Sciences, Bikaner, India
1National Research Centre on Camel, Jorbeer, Bikaner, India

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.
Key words: Camel, cloning, cysteine protease, Trypanosoma evansi