PTD-NPY融合基因的克隆及其在毕赤酵母中的分泌表达.pdfVIP

生 物 工 程 学 报 Chin J Biotech 2008, September 25; 24(9): 1620-1624 Chinese Journal of Biotechnology ISSN 1000-3061 cjb@ © 2008 Institute of Microbiology, CAS CSM, All rights reserved 研究简报 PTD-NPY 融合基因的克隆及其在毕赤酵母中的 分泌表达 孙玉成, 周凤秋, 万家余, 高宏伟 军事医学科学院军事兽医研究所, 长春 130062 摘 要: 应用重叠延伸PCR 方法扩增HIV-1 TAT 蛋白转导结构域(PTD)与鼠源神经肽Y(NPY) 的融合基因, 克隆目的片 段并插入酵母表达载体 pPICZαA, 构建成重组表达质粒 pPICZα-PTD-NPY 。PCR 和酶切鉴定及测序正确后, 经限制性 内切酶Sac I 线性化重组表达质粒并通过电转化整合到巴斯德毕赤酵母菌GS115 的染色体基因组中。阳性重组酵母菌用 含 1%甲醇的培养基诱导其分泌表达。经过120 h 的诱导, 取上清浓缩除盐后进行 SDS电泳, 表明该系统成功表 达了PTD-NPY 融合蛋白, Western blotting 实验证实表达产物具有特异性。获得真核表达的PTD-NPY 融合蛋白, 为下一 步的应用研究提供了物质基础。 关键词: 蛋白转导, 神经肽 Y (NPY), 融合基因, 克隆, 分泌表达 Cloning of PTD-NPY Fusion Gene and Its Secretory Expression in Pichia pastoris Yucheng Sun, Fengqiu Zhou, Jiayu Wan, and Hongwei Gao Institute of Military Veterinary Medicine , Academy of Military Medical Sciences , Changchun 130062, China Abstract: The PTD-NPY fusion gene derived from HIV-1 TAT protein transduction domain and rat neuropeptide Y was amplified by overlap extension PCR, digested and subcloned into yeast expression vector pPICZαA to construct recombinant expression plasmid pPICZα-PTD-NPY. The cloned PTD-NPY fusion gene was identified by PCR and restriction enzyme digestion and sequenced. The exact recombinant plasmid was linearized by Sac I and integrated by electrotransformation into the genome of Pichia pastoris GS115 cells. Then, these positive recombinant yeast cells were induced by 10 mL/L methanol to express soluble PTD-NPY fusion protein. After 120 h of methanol induction, the SDSelectrophoresis result indicated PTD-NP