重组人胸腺素β16的表达、纯化及其生物学活性研究

Study on the expression,purification and bioactivity of recombinant Tβ16

目的:人工合成人胸腺素β16(Thymosin β16,Tβ16)的DNA序列,克隆到大肠杆菌蛋白表达载体pET3c中,获得了高表达菌株。经发酵、破菌、层析纯化、酶切后获得了Tβ16表达产物,并研究其体内外生物学活性。方法:将构建的重组His-SUMO-Tβ16融合蛋白的表达载体转化大肠杆菌BL21(DE3)进行诱导表达。表达的融合蛋白经超声破碎、离子交换和金属亲和层析进行纯化后,用His-SUMO蛋白酶酶切,再经金属亲和层析和Superdex30凝胶层析,获得胸腺素β16。对胸腺素β16进行体内外生物学活性研究。结果:重组His-SUMO-Tβ16融合蛋白为可溶性表达,Tβ16比活性为5.3×105U/mg,在体外可促进BALB/c3T3细胞增殖,促进兔角膜细胞的增殖,促进血管内皮细胞的增殖和迁移以及促进鸡胚绒毛膜血管的增殖;在体内可促进家兔碱烧伤皮肤愈合。结论:成功构建了重组His-SUMO-Tβ16融合蛋白的大肠杆菌表达载体,获得了重组Tβ16蛋白,其具有很好的修复作用,为进一步Tβ16产业化开发奠定了基础。

Objective：To study the bioactivity of thymosin β16 （Tβ16） in vitro and in vivo. Methods： Recombinant His-SUMO-Tβ16 was constructed and transformed into E. coil BL21 （DE3） for induced expression. The product was treated by uhrasonication, ion-exchange chromatography and metal chelation chromatography respectively for purification. The fusion protein was cut by His-SUMO protease and then further purified by metal chelation chromatography and Superdex 30 gel chromatography. Results： Recombinant fusion protein His-SUMO-Tβ16 was soluble, whose specific activity was 5.3 × 10^5 U/mg. It could promote the proliferation of BALB/c 3T3 cells, rabbit corneal cells, and chicken embryo chorion vessels in vitro, and both the proliferation and migration of vascular endothelial cells in vitro were eubanced, and rabbit skin healing of alkali burns in vivo was accelerated. Conclusion： E. cnli expressing vector of recombinant His-SUMO-Tβ16 fusion protein is constructed successfully, and recombinant protein Tβ16 has significant repairing effects. The study established a good foundation for further industrialization of Tβ16.