[ Objective ] The predicted amino acid sequence of locus plu4437-plu4436 in the genome of Photorhabdus luminescens TT01 (referred to as pirA2B2) has a consistency of 50% and 45% with locus plu4093-plu4092( referred to as pirAlBl) , respectively. PirAlBl has been confirmed with oral insecticidal activity against Plutella xylostella and mosquito. The purpose of the present study was to examine whether pirA2B2 possesses insecticidal activity. [Methods] pirA2, pirB2 and pirA2B2 genes were PCR amplified and cloned. The recombinant expression vector pQE-pirA2, pQE-pirB2 and pQE-pirA2B2 were constructed and transferred into E. Coli M15, individually. The soluble PirA2, PirB2 and PirA2B2 proteins were detected from the supernatant of the recombinant M15 induced with IPTG by both SDS-PAGE and Western-blot. The proteins expressed in the three recombinant E. Coli M15 strains were purified by affinity chromatography combined with desalination technology and then quantified individually. The heamocoal and oral insecticidal activities of the expressed proteins were analyzed against the larvae of Galleria mellonella and Spodoptera litura. [Results] The results show; 1. PirA2B2 had heamocoal insecticidal activity against the fifth instar larvae of both G. Mellonella and S. Litura, with an LD50 of 4.0 and 2.8 (jug/larvae, respectively, 2. Neither PirA2 nor PirB2 alone had heamocoal insecticidal activity against the insects tested, while the mixture of PirA2 and PirB2 reconstituted full activity, and 3. PirA2B2 showed no oral insecticidal activity to the first instar larvae of either G. Mellonella or 5. Litura. [ Conclusion] pirA2B2 in the genome of P. Luminescens TT01 is a binary insecticidal toxin gene. [ Significance ] The successful expression and purifcation of PirA2B2 laid a foundation for further study on the function, insecticidal mechanism and expression regulation of the binary toxins.%[目的]Photorhabdus luminescens TT01基因组中的一对ORF plu4437-plu4436(简称pirA2 B2)的预测氨基酸序列与另一对已证明编码产物有口服杀虫活性的ORF plu4093-plu4092(简称pirA1B1)有50％和45％的一致性,本文旨在研究pirA2B2基因座的表达产物是否也有杀虫活性.[方法]PCR扩增并克隆了pirA2,pirB2和pirA2B2基因,构建了重组表达载体pQE-pirA2,pQE-pirB2和pQE-pirA2B2并分别转入M15菌株表达,经SDSPAGE和Western blot检测证明,3个重组菌株经IPTG诱导后,分别成功表达了可溶的PirA2,PirB2和PirA2B2 蛋白.用亲和层析结合脱盐技术对3个重组菌株表达的外源蛋白分别进行纯化,并通过生物测定确定纯化蛋白的杀虫活性.[结果]生物测定结果显示联合表达的PirA2B2对大蜡螟和斜纹夜蛾五龄幼虫均有明显的血腔杀虫活性,LD50分别为每虫4.0和2.8 μg,单独表达的PirA2或PirB2对上述2种害虫没有血腔杀虫活性,但两者的混合物具有与两者联合表达相似的杀虫活性；PirA2B2对大蜡螟和斜纹夜蛾初孵幼虫均无口服杀虫活性.[结论]pirA2B2是P.luminescens TT01菌株基因组中的另一个二元杀虫毒素基因.[意义]pirA2B2的成功克隆表达和杀虫功能的确定为进一步研究其与pirAlB1的关系以及该基因的表达调控等打下了基础.
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