Amplification cloning and sequence analysis of alternative oxidase gene of Trypanosoma evansi isolated from indian dromedaries

Abstract

The present study was carried out to isolate the Alternative oxidase and Trans-sialidase genes of Trypanosoma evansi using PCR, clone the amplicons in a suitable plasmid vector and then characterization of above genes through sequencing. For this investigation, morphologically suspected T. evansi infected camel was confirmed by examination of Giemsa stained blood smear of camel blood. After confirming infection, DNA isolation from collected pellet of Trypanosoma evansi was done as per the protocols given by ready to use kit from Illustra blood genomic prep. mini kit with slight modifications. The desired amplicons of aox and ts genes were then amplified by PCR using gene specific primers. Amplified PCR products were analyzed on 1.2% agarose gel stained with ethidium bromide and identified on the basis of size of the aox and ts genes. The amplicons of expected size were purified from the 1% low melting agarose gel employing Illustra GFX PCR DNA and Gel Band Purification 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 could be identified 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 74 that the release of DNA fragments around 990 bp for aox and 2241 bp for ts gene. Colony PCR was done for quick screening of plasmid inserts directly from E. coli colonies in the presence of insert specific primers. After confirmation of clones of aox and ts genes, the plasmid DNAs were sequenced and coding sequences of aox and ts genes according to the results obtained were of 990 and 2241 bp respectively. The phylogenetic and sequence analysis was done by use of Praline, Clustal X and MEGA5 softwares. Tree topology of aox and ts gene is based on the Neighbor-Joining method and maximum parsimony with 100% bootstrap values. Multiple sequence alignment of obtained protein sequences of aox and ts genes was performed with Praline sequence software. Identified aox and ts gene sequences showed a close homology with other Trypanosoma spp. gene sequences.