人源CD137L基因在不同表达系统中表达效率的比较

The Comparison of Expression Efficiency of Human CD137L in Different Expression System

比较分析人源CD137L在毕赤酵母、枯草杆菌及大肠杆菌中的表达差异,建立适合CD137L的表达系统并检测CD137L的生物学活性。设计PCR引物,以酵母表达质粒pPIC9K-CD137L为模板,扩增CD137L片段,分别构建枯草杆菌表达质粒及大肠杆菌表达质粒,再转化至相应的宿主菌并筛选阳性转化子;SDS PAGE电泳分析CD137L的表达情况并比较差异;稀释复性法复性CD137L包涵体,用离子交换层析纯化CD137L;T细胞激活试验检测CD137L的活性。CD137L在3个表达系统中均可以表达并且得到质谱鉴定,但是在毕赤酵母、枯草杆菌中的表达量微弱,而在大肠杆菌中CD137L以包涵体形式高效表达,平均1 L培养液能得到0.8 g包涵体沉淀,200 mg变性蛋白。经复性纯化后得到的CD137L能有效激活T细胞增殖并且其刺激活性呈现浓度依赖效应。与毕赤酵母、枯草杆菌相比,大肠杆菌表达系统能高效表达CD137L蛋白,并且经稀释复性后CD137L保持了刺激小鼠T细胞增殖的活性。

It was to construct a suitable expression system for CD137 L by comparison of expression level of CD137 L in Pichia yeast, Bacillus subtilis, and Escherichia coli and determine the effect of CD137 L on T cell proliferation. CD137 L was amplified by PCR with designed primers from yeast expression plasmid pPIC9K-CD137 L and subcloned into vector pP43 or pET11 a. Then recombinant plasmid pPIC9KCD137 L, pP43-CD137 L and pET11a-CD137 L were transformed into GS115, WB800 and BL21, respectively. Positive clones were screened and expression of CD137 L was detected by SDS-PAGE. Then inclusion body of CD137 L from Escherichia coli was refolded by dilute refolding and CD137 L was purified by ion exchange chromatography. After that, bioactivity of CD137 L was determined by T cell proliferation assay. CD137 L was expressed by all the three expression system and further confirmed by Western blot assay. But expression level of CD137 L in GS115 and WB800 was too weak for further study. In contrast, CD137 L 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 CD137 L was obtained after dilute refolding. Then refolded CD137 L was purified by ion exchange chromatography, and purified CD137 L demonstrated significantly costimulatory effect on T cell proliferation in dose-dependent manner. It was demonstrated that CD137 L was efficiently expressed in Escherichia coli expression system compared with Pichia yeast and Bacillus subtilis, and purified CD137 L kept costimulatory activity on T cell proliferation.