Protective immunity in mycobacterial infection is predominantly mediated by antibody independent cellular response involving recognition of mycobacterial antigens by T lymphocytes. In the present study secretary proteins of M. paratuberculosis (strain TEPS) was subjected to fractionation through DEAE Sephacel column chromatography and the fractionated protein evaluated for their T-cell reactivity. Crude culture filtrate protein was also resolved by SDS-PAGE followed by transfer to NCP for T-cell blotting. Fraction eluted through the column was subjected to SDS PAGE and the fractions having similar banding pattern were grouped together. According to such protein profile five groups were obtained out of them only one group was homogeneous having a 40 KD. MW peptide. All these five fractions were subjected to in-vitro lymphoproliferation test and cytotoxicity test on Black Bengal Indian goats. Young goats from Johne's disease free herd were housed in two groups, group-1 was sensitised with crude culture filtrate protein and group-11 was inoculated with live bacteria. In-vitro lymphocyte transformation test was done on day 80 and day 120 of post sensitisation. Highest thymidine uptake was recorded with 40 KD. MW protein fraction whereas other fractions did not show such a high response. A slight upward trend in Lymphoproliferation was observed on day 120 of post sensitisation as compared to day 80 in both the groups of animals. Animals sensitised with live bacteria have shown much higher stimulation index (SI-68.7) with same 40 KD. polypeptide, the proliferative response was considerably higher in comparison to animal sensitised with protein antigen. Cell mediated immune response was found to be persisting up to a period of 310 days as observed by lymphoproliferation. On T-cell blotting experiment using peripheral blood mononuclear cells from live bacteria sensitised goats, the crude antigen has shown major lymphoproliferative response around 30-50 KD MW region. T-cell response below and above this region was relatively poor. However on in-vitro cytotoxity assay, autologous monocycle targets pulsed with live bacteria have been found to be effectively lysed than that of target cells pulsed with fractionated protein antigens. Further study is in progress for in-vitro and in-vivo detection of CMI response in pre-clinical cases of paratuberculosis using the 40 KD MW protein antigen.