Molecular characterization of Paraflageller rod 1 gene of Trypanooma evansi from Indian Dromedaries

Abstract

The study was conducted for characterisation of Paraflagellar Rod 1 (pfr1) gene of Trypanosoma evansi from camel at molecular level. Genomic DNA of T. evansi from camel was used to amplify the pfr1 gene by polymerase chain reaction. Cloning of the amplicon was done in a suitable bacterial plasmid vector and characterisation of pfr1 gene was carried out through sequencing. The desired amplicon of pfr1 gene of T. evansi was amplified by PCR using gene specific primers and identified on the basis of size of the pfr1 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. 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 pfr1 genes the plasmid DNA was sequenced and coding sequences of pfr1 gene according to the result obtained was of 1769 bp. Tree topology of pfr1 gene is based on the Neighbor-Joining method and maximum parsimony method with 100% bootstrap values and identified pfr1 gene sequence showed a close homology with other Trypanosoma and Leishmania spp. gene sequences.