Molecular Characterization of Alternative oxidase and Trans-sialidase genes of Trypanosoma evansi of Camel

Abstract

The present study was carried out to isolate the Alternative oxidase (aox) gene of Trypanosoma evansi using PCR, clone the amplicons in a suitable plasmid vector and then characterization of gene through sequencing. For this investigation, morphologically suspected T. evansi infected camel was confi rmed by examination of Giemsa stained blood smear of camel blood. Desired amplicons of aox gene was amplifi ed from genomic DNA of the parasite by PCR using gene specifi c primers. Amplifi ed PCR products were analyzed on 1.2% agarose gel stained with ethidium bromide and identifi ed on the basis of size of the aox gene. The amplicons of expected size were purifi ed from the 1% low melting agarose gel employing Illustra GFX PCR DNA and Gel Band Purifi cation Kit. The DNA fragment of interest was then ligated to the pGEM- T Easy vector and ligated mixture was transformed into Escherichia coli JM109 strains. The cells containing recombinant plasmid was identifi ed on the basis of blue/white colony selection on LB agar containing X-Gal, IPTG and ampicillin. Screening of recombinants was done by Restriction Enzyme digestion of plasmid DNAs using EcoRI and found that the release of DNA fragments around 990 bp for aox gene. Colony PCR was done for quick screening of plasmid inserts directly from E. coli colonies in the presence of insert specifi c primers. After confi rmation of clones of aox gene, the plasmid DNA was sequenced and coding sequence of aox gene according to the result obtained was of 990 bp. The phylogenetic and sequence analysis was done by use of Praline, Clustal X and MEGA5 softwares. Tree topology of aox gene is based on the Neighbor-Joining method and maximum parsimony with 100% bootstrap values. Multiple sequence alignment of obtained protein sequences of aox gene was performed with Praline sequence software. Identifi ed aox gene sequence showed a close homology with other Trypanosoma spp. gene sequences.