Abstract: The Tβ4 concatemer (2Tβ4) was synthesized by constructing 2Tβ4 prokaryotic expression system. 2Tβ4 was purified and recombined using Ni-NTA spin column, and its immunocompetence was measured by MTT. Under the induction of 0.05 mmol/L IPTG, 6.7 mg 2Tβ4 per gram bacteria was obtained by expressing PET-32a(+)-2Tβ4 in competent cell BL21 (DE3). No significant differences were observed between the effects of 2Tβ4 and standard Tβ4 on the multiplication of lymphocyte. This proves the validity of the established chicken 2Tβ4 prokaryotic expression system, and provides basic material for the application of Tβ4.