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目的表达、复性和纯化重组人白细胞介素7剪接变异体蛋白(interleukin-7 splice variants,IL-7splice variants),为下一步功能研究奠定基础。方法在IL-7剪接变异体(IL-7δ4/5以及IL-7δ3/4)cDNA克隆的基础上进行亚克隆,插入细菌表达载体pET-21b中,构建相应的原核表达载体,阳性克隆测序,证实其构建成功,转化入大肠杆菌BL21(DE3)中,对各蛋白表达条件进行优化、复性及纯化,利用Western blot鉴定重组蛋白。结果各表达蛋白与预期相对分子质量(10.3 ku、9.3 ku)相符,纯化后蛋白纯度高达95%以上,并经免疫印迹鉴定证实表达的蛋白为IL-7δ4/5和IL-7δ3/4。结论成功获得高纯度的IL-7剪接变异体复性蛋白。
Objective To express, renature and purify recombinant interleukin-7 splice variants (IL-7splice variants), which will lay the foundation for further functional studies. Methods The cDNA fragments of IL-7 splicing variants (IL-7δ4 / 5 and IL-7δ3 / 4) were subcloned and inserted into the bacterial expression vector pET-21b to construct the prokaryotic expression vector. The positive clones were sequenced, The recombinant protein was successfully transformed into E.coli BL21 (DE3). The protein expression conditions were optimized, renatured and purified. The recombinant protein was identified by Western blot. Results The expressed protein was in good agreement with the expected molecular weight of 10.3 ku and 9.3 ku. The purity of the expressed protein was over 95%. The expressed protein was confirmed to be IL-7δ4 / 5 and IL-7δ3 / 4 by Western blot. Conclusion High-purity IL-7 splice variant protein was successfully obtained.