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MOLECULAR CHARACTERIZATION OF TH2 CYTOKINE GENE (IL-10) OF CAMEL (Camelus dromedarius)

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2016
Dr. S S SONI
Rajasthan University of Veterinary and Animal Sciences, Bikaner - 334001
Rajasthan University of Veterinary and Animal Sciences, Bikaner - 334001

The present study was carried out to understand determinant of cell mediated immunity of Camelus dromedarius i.e. Interleukin-10 produced by Th2 cells mainly B cells, T cells, macrophages, Th2 subset of CD4+ cells. For this investigation peripheral blood mononuclear cells were separated from Camelus dromedarius using Histopaque® 1077 and were cultured in RPMI 1640 medium. PBMCs were stimulated with Concavalin-A to maximize the expression of genes. Total RNA was separated from stimulated cells using TRIZOL reagent. cDNA was synthesized from total RNA using reverse transcriptase (Superscript III). Using base sequence of Camelus bactrianus, primers were designed for specific amplification of interleukin-10 gene of Camelus dromedarius. The IL-10 gene was successfully amplified from cDNA using Taq polymerase and was identified on the basis of its size homology with that of Camelus bactrianus in agarose gel electrophoresis i.e. 600 bp. The IL-10 gene of Camelus dromedarius was cloned in pGEM-T easy vector at its 3’ T overhangs and recombinant plasmid was named as pGEM-TILT and transformed in Escherichia coli DH5α strains. The cells containing recombinant plasmid could be identified on the basis of blue/white colony selection on LB agar containing X-Gal and ampicillin. The interleukin-10 gene was isolated from recombinant plasmids on ligation with restriction enzyme EcoRI and identified on the basis of its size. Using the ideal Camel model, further study of IL-10 characterization and function may provide insight on the understanding of the cell mediated immune system.

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