美国白蛾hcapn3基因的克隆及HcAPN3蛋白与Cry1Ac结合特性分析

Cloning of midgut aminopeptidase hcapn3 gene from Hyphantria cunea and binding to Cry1Ac toxin of HcAPN3

通过比对分析已知昆虫氨肽酶N(aminopeptidase N,APN)氨基酸序列并结合本实验室的美国白蛾(Hyphantria cunea)中肠iTRAQ结果,设计了hcapn3基因特异引物,获得hcapn3基因片段。通过RACE-PCR技术获得美国白蛾中肠氨肽酶N基因(hcapn3)全长序列(GenBank登录号为KJ013598)。Blastp分析表明,获得的氨肽酶属于氨肽酶N3家族,命名为HcAPN3。序列分析显示HcAPN3包括952个氨基酸残基,具有典型的谷氨酸锌化氨肽酶(Gluzincin)结构域和羧基端(ERAP1_C)结构域。利用Bac to Bac表达系统在昆虫细胞中表达108kDa的HcAPN3蛋白。在原核表达系统中表达58kDa的Gluzincin结构域和49kDa ERAP1_C的结构域蛋白。Ligand Blot分析结果显示,HcAPN3蛋白及Gluzincin结构域可与Cry1Ac蛋白特异性结合,但ERAP1_C结构域未能与Cry1Ac结合。本研究首次克隆了美国白蛾氨肽酶基因并分析了HcAPN3与Cry1Ac的结合特性,为下一步功能研究提供基础。

The specific primers of aminopeptidase N gene were designed,and a fragment of hcapn3 was obtained by PCR method.A full-length gene（GenBank accession no.KJ013598）encoding APN of Hyphantria cunea was obtained by RACE-PCR method based on the sequences of published APNs from insects and iTRAQ of Hyphantria cunea midgut.Analysis of the protein sequences by NCBI Blastp revealed that it belonged to aminopeptidase N gene family 3,named HcAPN3.The primary structure of HcAPN3 contained 952 amino acids residues,a typical Gluzincin aminopeptidase family domain and ERAP1_C domain.The hcapn3 gene was successfully expressed in insect cells as secreted proteins（108 kDa）using recombinant baculoviruses（Bac to Bac system）.Further,the Gluzincin aminopeptidase family domain and ERAP1_C-like C-terminal domain were expressed in E.coli as58 kDa and 49 kDa proteins by SDS-PAGE analysis,respectively.In vivo Ligand-Blotting study demonstrated binding of Cry1 Ac toxin to both HcAPN3 and peptidase Gluzincin family domain,not to ERAP1_C domain.The first identified aminopeptidase N from H.cunea may be a candidate receptor for Cry1 Ac toxin.