Amplification, cloning and sequence analysis of alternative oxidase gene of Trypanosoma evansi isolated from Indian dromedarius

Abstract

The present study was carried out to isolate the Alternative oxidase (aox) gene of Trypanosoma evansi by polymerase chain reaction, clone the amplicons in a suitable bacterial plasmid vector and characterisation of the gene through sequencing. The desired amplicon of aox gene of T. evansi was amplified by PCR using gene specific primers and identified on the basis of size of the gene. The amplicon of expected size was purified from the 1% low melting agarose gel. The 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. After confirmation of clone, the plasmid DNA was sequenced and coding sequence of aox gene according to the results obtained was of 990 bp. Tree topology of aox gene is based on the Neighbor-Joining method with 100% bootstrap values and identified aox gene sequence showed a close homology with other Trypanosoma spp. gene sequences.