Abstract: Objective Expression for functional human DNA mismatch repair protein hMSH2 to use as a new candidate ligand for TCRγδ in researching of the function and antigen recognition pattern of γδT cells in immunity. Methods cDNA encoding hMSH2 was amplified with overlap-extension PCR and cloned into expression vector pAcGP67B and pET30a respectively. The recombinant vector was identified by restriction enzymes. The baculovirus DNA and pAcGP67B-hMSH2 were then cotransfected into sf9 cells. The recombinant hMSH2 protein was identified by western blotting. Meanwhile, the recombinant pET30a-hMSH2 was expressed in E.coli. The binding specificity of hMSH2 protein to OT3 peptide and TCRγδ was detected by ELISA and FCM. Functions of hMSH2 protein were further evaluated by MTT colorimetric assays and cytokines secret assay in vitro. Results The full length of hMSH2 gene was obtained by overlap-extension PCR and was cloned into the baculovirus expression vector and pET30a to construct the recombinant expression vectors, pAcGP67B-hMSH2 and pET30a-hMSH2, respectively. After 3 rounds of virus amplifying by re-infection, the soluble hMSH2 was detected in the supernatant. Purified hMSH2 not only binds specifically to OT3 peptide, but also to TCRγδ. Moreover, results indicated that the resultant protein can trigger the proliferation and IFN-γ secreting of γδT cells in vitro. Conclusion Functional hMSH2 recombinant protein was successfully expressed in baculovirus expression system holding more activity than that expressed by E.coli.