Amplification, cloning and sequence analysis of paraflagellar rod 2 gene of trypanosoma evansi isolated from Indian dromedaries

Abstract

The present study was carried out to amplify the Paraflagellar Rod 2 gene of Trypanosoma evansi from camel by polymerase chain reaction, clone the amplicon in a suitable plasmid vector and characterization of pfr2 gene through sequencing. The desired amplicon of pfr2 gene of Trypanosoma evansi was amplified by PCR using gene specific primers and identified on the basis of size of the pfr2 gene. The amplicon of expected size was purified from the 1% low melting agarose gel. DNA fragment of interest was then ligated to the pGEM- T Easy vector and ligated mixture was transformed into Escherichia coli JM109 strains for cloning. After cloning, screening of recombinants was done by Restriction Enzyme digestion of plasmid DNA and by Colony PCR for quick screening of plasmid insert directly from E. coli colonies in the presence of insert specific primers. After confirmation of clone of pfr2 genes the plasmid DNA was sequenced and coding sequences of pfr2 gene according to the result obtained was of 1767 bp. Tree topology of pfr2 gene is based on the Neighbor-Joining method with 100% bootstrap values and identified pfr2 gene sequence showed a close homology with other Trypanosoma and Leishmania spp. gene sequences.