蛇毒蛋白triflin功能区TFPR1在大肠杆菌中的表达及其功能初步研究

Production and Immunogenicity Analysis of Recombinant Snake Venom Protein TFPR1

目的:获得具有生物学活性的蛇毒蛋白triflin的致病相关蛋白1(PR-1)功能区TFPR1。方法:分析triflin空间结构及其保守结构域,获得TFPR1序列,并进行密码子优化、全基因合成,构建原核表达质粒p ET-TFPR1,在大肠杆菌BL21中以IPTG诱导表达,表达产物经Western印迹鉴定后,镍柱纯化、复性,对复性后的产物进行特性分析;以复性的TFPR1肌肉途径接种BALB/c雌性小鼠,分析其生物学活性。结果:TFPR1以包涵体形式表达,复性后蛋白纯度大于95%,无需佐剂免疫小鼠即可诱导机体产生效价为20万的抗TFPR1抗体。结论:制备了具有生物学活性的重组蛋白TFPR1,为后续研究TFPR1的生物学功能奠定了物质基础。

Objective：To find and express the functional region of triflin,one of the snake venom proteins from Trimeresurus flavoviridis.Methods： The optimized sequence named TFPR1 was obtained by analyzing the spatialstructure and conserved structural domain of triflin.The recombinant prokaryotic plasmid p ET- TFPR1 was con-structed by inserting the codon-optimized fragment to p ET-His,expression of protein was induced by IPTG andidentified by Western blot.The target protein was purified using high affinity Ni-charged resin,and then refoldedby gradient dialysis.The purified TFPR1 was intramuscularly immunized to BALB/c mice to evaluate its biologicalactivity.Results： TFPR1 was expressed in the form of inclusion bodies,and high purity of protein was obtained af-ter purification and refolding.The recombinant TFPR1 induced high titer of anti-TFPR1 antibody in the absenceof adjuvant.Conclusion： The recombinant protein TFPR1 with biological activity was successfully prepared,whichlays foundation for further work on evaluation of its biological functions.