草地贪夜蛾PP2A的克隆、序列分析及在大肠杆菌中的表达

Molecular cloning,sequence analysis and expression in Escherichia coli of the Spodoptera frugiperda protein phosphatase 2A

为建立斑蝥素等PP2A抑制剂的体外活性测定方法，用于以杀虫剂为目的的药物快速活性评价，本研究首次同源克隆了鳞翅目昆虫草地贪夜蛾Spodoptera frugiperda （J.E. Smith）的PP2A催化亚基cDNA全长序列，探索了该基因在大肠杆菌中的表达。结果显示，S. frugiperda的PP2A编码一条309个氨基酸的肽链，预测其蛋白质分子量为35.46 ku，等电点5.37。分析蛋白质氨基酸序列，没有发现信号肽和跨膜结构，推测该PP2A主要存在于胞质中。多重比对分析S. frugiperda及其它昆虫的PP2A，表明PP2A的保守性较高，可以用S. frugiperda PP2A作为研究斑蝥素等抑制剂的药物筛选靶标酶，用于杀虫剂的筛选。利用该基因构建pET30aPP2A原核表达载体转化至E. coli BL21（DE3）中，在16-30℃，0.2-0.8 mmol/L的IPTG下均能成功诱导PP2AHis表达，经Ni琼脂糖柱纯化后可以得到SDSPAGE呈现单一条带的纯化蛋白，电泳纯度大于90%，为进一步活性测定奠定了基础。

This study is aimed at establishing a method of PP2A activity assay for evaluating potential new pesticides in vitro. A Ser/Thr protein phosphatase 2A catalytic subunit gene was cloned from Spodoptera frugiperda （J.E. Smith） Sf9 cell culture and expressed in Escherichia coli. The fulllength cDNA sequence was 1 303 bp, encoding 309 amino acids with a predicted molecular weight of 35.46 ku and an isoelectric point of 5.37. This PP2A may mainly occur in cytoplasm without signal peptides and transmembrane structure. Multiple comparison analysis of S. frugiperda and other insects＇ PP2A indicate that PP2A is highly conserved. PP2A of S. frugiperda, the target enzyme for selection of inhibitors, can be used for screening insecticides in activity assays. A prokaryotic expression vector pET30aPP2A was constructed and transfected into the E. coli BL21 （DE3） stain, establishing a prokaryotic expression system. This expression system expressed PP2A well at conditions of 16-30℃ with a 0.2-0.8 mol/L IPTG inducer. We dissolved the inclusion in urea solution and purified it using a Ni／｜agarose column, obtaining purified PP2A which presented a single band after SDS／｜PAGE, showing the purity was over 90%. These results lay a foundation for research into PP2A activity assay.