Autotaxin在sf9昆虫细胞的表达及纯化研究

Expression and purification of autotoxin in sf9 insect cells

目的 通过sf9昆虫细胞表达人Autotaxin（ATX）,获得具有生物学活性的目的蛋白。方法 采用TRIzol法提取人的黑色素瘤细胞总RNA,通过RT-PCR获得人ATX基因,构建p Fast BacTMHTA-ATX,Bacmid-ATX重组质粒,脂质体转染sf9昆虫细胞,收获重组ATX病毒;重组病毒侵染sf9昆虫细胞表达人ATX;通过His TrapTMHP和Hi Load 16/600 Superdex 200pg柱两部纯化获得目的蛋白并测定其磷脂酶D（lysophospholipase D,lyso PLD）活性。结果 用Bam H I和Xho I双酶切鉴定PCR产物及重组载体的正确性,重组病毒能够侵染sf9细胞表达人ATX,柱层析纯化目的蛋白,SDS-PAGE电泳纯度95%以上,每升表达液可得到纯化蛋白6mg,其明显具有溶血磷脂酶D（lysophospholipase D,lyso PLD）活性。结论 通过Bac-to-Bac表达系统构建的重组病毒杆粒能够感染sf9昆虫细胞,并正确表达具有生物学活性的人ATX,为开展ATX结晶与结构分析研究提供材料,为开发高效的ATX小分子药物抑制剂提供帮助。

Objective Production of autotoxin protein in sf9 insect cells with biological activity. Methods Autotaxin cDNA was cloned into pFastBac？HTA from melanoma cell by extraction of total RNA using TRIzol method and RT-PCR. Bacmid-ATX is isolated from transformed competent bacterial DH10 which carries Bac genomic sequences and transfected into sf9 using lipofectamine 2000. Recombinant ATX virus was amplified in sf9 and further used for infection and expression of ATX protein. Two step purification product using Histrap？HP and Hiload 16/600 Suerdex 200pg was determined for lysophospholipase D （lysoPLD） activity. Results Correct insertion o f PCR fragment is confirmed by BamH V Xho I digestion and sequencing. ATX virus can infect sf9 and induced enz^atic activity. Column purification and SDS-PAGE resulted 95% in purity and 6mg/liter in yield with significant lysoPLD activity. Conclusion ATX Baculovirus was successfully constructed that can infect sf9 cells and express active lysoPLD. Production of active ATX can be used for crystalography studies and screening for small pharmaceutical inhibitors.