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Cloning, Sequencing And Expression Of Buffalo Cytokine Genes

KrishiKosh

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Title Cloning, Sequencing And Expression Of Buffalo Cytokine Genes
 
Creator Ramathilagam, G
 
Contributor Thiagarajan, V
 
Subject DAPI - DNA Microassay
rBuIL-2
rBuIL-8
Ni-NTA His bind resin SCC
Neutrophils Con-A lymphocyte blast
Macrophages
 
Description 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.
 
Date 2016-05-27T11:49:12Z
2016-05-27T11:49:12Z
2004
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/66366
 
Language en
 
Format application/pdf
 
Publisher Tamil Nadu Veterinary and Animal Sciences University