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Advisor: Dr. G. S. Manohar
Title: Molecular characterization of Cysteine Protease gene and Heat Shock Protein 70 gene of Trypanosoma evansi isolated from camel
Publisher: Rajasthan University of Veterinary and Animal Sciences Bikaner-334001
Language: en
Type: Thesis
Agrotags: null
Abstract: The present study was carried out to isolate the Cysteine Protease gene and Heat Shock Protein 70 gene of Trypanosoma evansi using PCR. Amplicons were cloned in a suitable plasmid vector and then characterization of both the genes was done by sequencing. For this investigation, morphologically suspected Trypanosoma evansi infected camel was confirmed by examination of Giemsa stained blood smear of camel blood. After confirming infection, the T. evansi collected from camel blood were propagated in Swiss albino mice and the blood of mice was collected from heart region after dissecting the mice which had massive infection. DEAE cellulose chromatography was done for purification of trypanosomes from blood of mice. DNA extraction was done from collected pellets of Trypanosoma evansi using the phenol-chloroform extraction followed by ethanol precipitation. The desired amplicons of cysteine Protease and HSP-70 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 Cysteine Protease and HSP-70 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 126 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 that the release of DNA fragments around 1533 bp for Cysteine Protease and 1956 bp for HSP 70 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 Cysteine Protease and HSP-70 genes the plasmid DNAs were sequenced and coding sequences of Cysteine Protease and HSP-70 genes according to the results obtained were of 1533 bp and 1956 bp, respectively. The bioinformatic analysis of both Cysteine Protease and Heat Shock Protein-70 genes was done by using Bio Edit Sequence Alignment Editor, ExPASy: SIB Bioinformatics Resource Portal and NCBI open Reading Frame finder. Six Open Reading Frames were generated (3 sense and 3 Anti sense) for both Cysteine Protease and Heat Shock Protein-70 genes by ExPASy and NCBI ORF finder. The original nucleotide sequence from which primers were designed and the consensus nucleotide sequence which was formulated from forward and reverse primer sequences were aligned by Pairwise Optimal Global Alignment using BLOSUM62 similarity matrix and similarities and identities were evaluated.
Subject: Veterinary Parasitology
Theme: Molecular characterization of Cysteine Protease gene and Heat Shock Protein 70 gene of Trypanosoma evansi isolated from camel
These Type: M.V.Sc.
Issue Date: 2016
Appears in Collections:Thesis

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