关键词: 毕赤酵母, 枯草杆菌, 大肠杆菌, CD137L, T细胞增殖

Abstract: It was to construct a suitable expression system for CD137L by comparison of expression level of CD137L in Pichia yeast, Bacillus subtilis, and Escherichia coli and determine the effect of CD137L on T cell proliferation. CD137L was amplified by PCR with designed primers from yeast expression plasmid pPIC9K-CD137L and subcloned into vector pP43 or pET11a. Then recombinant plasmid pPIC9K-CD137L, pP43-CD137L and pET11a-CD137L were transformed into GS115, WB800 and BL21, respectively. Positive clones were screened and expression of CD137L was detected by SDS-PAGE. Then inclusion body of CD137L from Escherichia coli was refolded by dilute refolding and CD137L was purified by ion exchange chromatography. After that, bioactivity of CD137L was determined by T cell proliferation assay. CD137L was expressed by all the three expression system and further confirmed by Western blot assay. But expression level of CD137L in GS115 and WB800 was too weak for further study. In contrast, CD137L was efficiently expressed in BL21 as inclusion body. It was about 0.8 g inclusion body per 1 L cultured BL21 and 200 mg denatured protein was obtained. Then inclusion body was dissolved in 8 mol/L urea at 50℃or 60℃, and active protein CD137L was obtained after dilute refolding. Then refolded CD137L was purified by ion exchange chromatography, and purified CD137L demonstrated significantly costimulatory effect on T cell proliferation in dose-dependent manner. It was demonstrated that CD137L was efficiently expressed in Escherichia coli expression system compared with Pichia yeast and Bacillus subtilis, and purified CD137L kept costimulatory activity on T cell proliferation.

Key words: Pichia pastoris, Bacillus subtilis , Escherichia coli, CD137L , T cell proliferation