新型佐剂蛋白TFPR1在毕赤酵母系统中的表达及鉴定

Expression of a novel adjuvant TFPR1 in Pichia pastoris and its identification

目的：利用毕赤酵母系统表达一种基于蛇毒蛋白triflin的致病相关蛋白1（ PR-1）功能区蛋白 TFPR1。方法从质粒 pUC57-TFPR1中 PCR 扩增目的基因,插入毕赤酵母表达载体pPICZαA,构建重组表达质粒pPICZαA-TFPR1,转染入巴斯德毕赤酵母野生型X33菌株,经甲醇诱导表达重组蛋白TFPR1,采用ELISA法筛选阳性菌株后,SDS-PAGE及Western blot对表达产物进行鉴定。结果重组表达质粒pPICZαA-TFPR1经双酶切和测序鉴定证明构建正确；重组蛋白以分泌形式表达,相对分子质量约16＆#215;103。结论利用巴斯德毕赤酵母表达系统成功表达TFPR1蛋白,为进一步研究其功能和作为佐剂的应用提供参考资料。

Objective To express a recombinant protein TFPR1 （ the functional region of the snake venom proteins from Trimeresurus flavoviridis） in Pichia pastoris expression system. Methods The target gene was codon-optimized and synthesized according to the sequence of the conserved structural do-main of triflin and then cloned into the Pichia pastoris expression vector pPICZαA to construct the recombi-nant expression plasmid pPICZαA-TFPR1. The recombinant plasmid pPICZαA-TFPR1 was electroporated into the yeast strain X33. The transformed strains carrying expression plasmid were screened out with Zeocin and then induced by methanol to express the recombinant protein TFPR1. ELISA was performed for the screening of positive clones. SDS-PAGE and Western blot were used for further identification of the ex-pressed products. Results The recombinant plasmid pPICZαA-TFPR1 was successfully constructed. The recombinant protein TFPR1 was expressed in a secreted form at a molecular weight of 16＆#215;103. Conclusion The recombinant protein TFPR1 was successfully expressed in Pichia pastoris expression system, which laid a foundation for further researches on its biological function and application as an adjuvant.