Abstract: The freshwater ciliate,Tetrahymena thermophila , has recently emerged as a novel manufacturing platform for recombinant subunit vaccines. We aim to further improve the screening efficiency of recombinant Tetrahymena to make it more widely used in the preparation of recombinant subunit vaccine. In this study, we constructed a new vector with the codon optimized puromycin resistance cassette replaced paromomycin resistance cassette to improve the screen efficiency and make the vector more optimum for expression. We also substitute thermo-inducible promoter HSP70 for MTT1 promoter which was induced by CdCl2. The expression of foreign proteins was triggered simply by increasing the cultural temperature. Construction of this vector would widen the application of T. thermophila expression system to preparation of more subunit vaccines.