【摘要】 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。更多还原

【Abstract】 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.更多还原