Cloning, Sequencing And Expression Of Buffalo Cytokine Genes
Loading...
Date
2004
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Tamil Nadu Veterinary and Animal Sciences University
Abstract
To evaluate the buffalo lymphocyte transformation test the non-
radioactive DAPI-DNA microassay is method of choice found in the present
study. It was found that 5 x 10 5 spleen lymphocyte populations stimulated with
320ng of Con-A yielded higher mean OD values at 48 hrs. duration of
lymphocyte stimulation. The IL-2 cDNA amplification was found at 4, 8, 12 and
24 hrs. following RT-PCR amplification of RNA isolated from buffalo spleen
cells stimulated with Con-A (320 ng). The IL-8 cDNA amplification was
observed at 4, 8, 12, 24 and 48 hours following RT-PCR amplification of RNA
isolated from buffalo blood macrophages stimulated with PMA (10 ng/ml). The
assembled IL-2 cDNA revealed entire coding region of 464 bp and that of IL-8
cDNA revealed entire coding region of 240 bp. The buffalo shared 98.5 & 97
percent homology with cattle IL-2 and IL-8 at nucleic acid level and 97 & 96
percent homology with cattle at amino acid level, respectively. The purification of expressed rBuIL-2 and rBuIL-8 proteins were achieved
quickly and easily by affinity chromatography using Ni-NTA His-bind resin
columns. Yield of the pure proteins obtained in E.coli BL21 cells was
approximately 5 mg/lt. Both the proteins were functionally active in vitro through
maintenance of Con-A lymphoblast and in vitro chemotaxis of neutrophils by
rBuIL-2 and rBuIL-8 proteins respectively. In vivo activity of rBuIL-2 was shown
by its ability to maintain the SCC without influencing milk pH in cows at mid
lactation stage. Intradermal injections of rBuIL-8 attracted neutrophilic
infiltration in a dose dependent manner.
Description
Keywords
DAPI - DNA Microassay, rBuIL-2, rBuIL-8, Ni-NTA His bind resin SCC, Neutrophils Con-A lymphocyte blast, Macrophages