Abstract: Tumor necrosis factor alpha (TNF-) is a multi-functional cytokine that plays important role in immune response, the homeostasis of the immune system, apoptosis, cell proliferation, and differentiation. Whether pathogen and immune stimulation can enhance the protein level of TNF- is unclear. In this study, the cDNA of grass carp TNF- was cloned into the prokaryotic expression vector pET-32 for expression of His6-tagged TNF-. After purification through Ni2+-affinity chromotopraphy column, purified His6-TNF- was subjected to immunize mouse to get polyclonal antibody, anti- TNF-. Western blot analysis showed that the anti- TNF- could specifically recognize endogenic grass carp TNF- as well as His6- TNF-. Furthermore, our results indicated that the CIK TNF- level remain constant when challenged with GCRV or treated with immune stimulators in vitro. Since CIK cells seemed to be not a good cell line in investigating the response of TNF alpha signal pathway to stress, an in vivo experiment might be required to monitor the protein expression of TNF- in specific immune organs or cells.