Overexpressed CKLFSF2 (chemokine-like super family member 2) can be secreted into the supernatant of cultured cells, which exhibits the effects on cell-chemotaxis. To study the structural and functional characteristics of CKLFSF2 and make the polycolonal antibody, the recombinant plasmid pGEX-4T-3-CKLFSF C51 was constructed, following the expression in E. coli with high efficiency, purification using Glutathione Sepharose 4B and Sephacyl S-200, the recombinant CKLFSF2 C51 protein (typically >95% pure) was obtained. The N-terminal animo acid sequencing of CKLFSF2 C51 was performed and relative molecular mass is 5871.48 measured by ASIMA-CFR™ plus MALDI-TOF. ELISA assay detected the reactivity of the polycolonal antibody and Western blot show that CKLFSF2 was overexpressed in mammalian cells and the specific band was corresponding to the expectation. The recombinant CKLFSF2 C51 protein exhibits significantly chemotactic effect in PC-3 cells, which can be neutralised by anti-CKLFSF2 C51 polyantibody. The anti-CKLFSF2 C51 antibody can also be used for immunohistochemistry. Taken together, the recombinant CKLFSF2 C51 from E.coli has similar bioactivity as that from eukaryocytic expression system. The polycolonal antibodies of recombinant CKLFSF2 C51 can be used in immunohistochemistry, Western blot and to neutralize the chemotactic effect.