Thiagarajan, VRamathilagam, GTANUVAS2016-05-272016-05-272004http://krishikosh.egranth.ac.in/handle/1/66366To 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.enDAPI - DNA MicroassayrBuIL-2rBuIL-8Ni-NTA His bind resin SCCNeutrophils Con-A lymphocyte blastMacrophagesThesis