MOLECULAR CHARACTERIZATION OF TH2 CYTOKINE GENE (IL-10) OF CAMEL (Camelus dromedarius)
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Date
2016
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Rajasthan University of Veterinary and Animal Sciences, Bikaner - 334001
Abstract
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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