家蚕半胱氨酸蛋白酶抑制剂BmCPI40鉴定及时空表达特征

摘要

【目的】鉴定家蚕（Bombyx mori）半胱氨酸蛋白酶抑制剂，并对其表达特征及调控进行分析，为深入研究半胱氨酸蛋白酶抑制剂在家蚕变态发育中的作用提供数据支持。【方法】根据家蚕基因组数据库和Primer 5.0软件设计BmCPI40去信号肽引物，对家蚕半胱氨酸蛋白酶抑制剂BmCPI40进行克隆，利用Clustal X和MEGA4.0软件对BmCPI40进行氨基酸序列和进化关系分析。采用原核表达系统对重组的BmCPI40进行表达、纯化和抗体的制备。运用 RT-PCR 和 qPCR 技术在核酸水平分析BmCPI40时空表达特征，利用 Western 和免疫荧光技术分别对BmCPI40进行蛋白水平检测和组织定位。qPCR方法检测20E诱导条件下BmCPI40在家蚕体壁中的表达量变化。用20E处理细胞，双荧光素酶报告系统对BmCPI40启动子序列活性进行检测，探究BmCPI40调控方式。【结果】鉴定、克隆、表达了家蚕的半胱氨酸蛋白酶抑制剂，并命名为BmCPI40，其ORF全长366 bp,编码121个氨基酸，其中前18氨基酸构成其信号肽，编码蛋白质大小约为12249.21 Da，等电点为4.43；进化树分析显示BmCPI40与一些病原微生物半胱氨酸蛋白酶的抑制剂结构域进化关系较近；BmCPI40的原核表达结果表明，重组的 BmCPI40以可溶蛋白的形式表达；BmCPI405龄第3天各组织Western blot结果显示，其主要存在于家蚕表皮中，头部也有微量存在；时期检测结果表明，BmCPI40幼虫期表达量明显高于蛹期，眠期及变态期表达量下调明显，这与核酸水平的qPCR结果相一致，进一步的体壁免疫荧光定位证实BmCPI40主要存在于体壁的皮细胞周围；qPCR表明，20E诱导5龄第2天家蚕幼虫后，BmCPI40表达量减少；双荧光素酶检测结果显示，20E诱导细胞后，BmCPI40启动子序列荧光素酶活性下降，证实BmCPI40受到了蜕皮激素的抑制作用。【结论】 BmCPI40的表达受到蜕皮激素的诱导下调，同时其可能参与了家蚕的蜕皮及变态发育过程。%[Objective] The objective of this study is to identify a newly discovered cysteine protease inhibitor of silkworm (Bombyx mori) and to investigate the expression characteristic and regulation mechanism, thereby providing data for further investigation on the inhibitor functions in the development process of molting and metamorphosis in the silkworm.[Method] Primers were designed using Primer Premier 5.0 software according to the Silkworm Genome Database, and were used to amplify the sequence ofBmCPI40 that the putative signal peptide was eliminated. The multiple sequence alignment and phylogenetic analysis of BmCPI40 was performed by bioinformatics software Clustal X and MEGA 4.0. The recombinant BmCPI40 protein was expressed in prokaryotic expression system, and then was used to produce the polyclonal antibody after purification. The spatial and temporal expression pattern ofBmCPI40 was investigated using RT-PCR and quantitative real-time PCR (qPCR), and the protein distribution and localization in different tissues and different developmental stages was analyzed using Western blot and immunofluorescence. In addition, the 20E-induced expression ofBmCPI40 was also studied after injection using qPCR. Furthermore, the promoter activity ofBmCPI40 was analyzed in silkworm cells with Dual Glo Luciferase Assay System.[Result]A new cysteine protease inhibitor of silkworm, named BmCPI40, was cloned and expressed. The open reading frame ofBmCPI40 was 366 bp that encoded a protein with 121 amino acid residues, the first 18 residues of which was predicted as signal peptides, and the isoelectric point was 4.43. Phylogenetic analysis indicated that this inhibitor showed high sequences identity with the propeptides of several microbes cysteine proteinases. Soluble protein was produced from recombinantE.coli cell. The spatial expression results suggested thatBmCPI40 was expressed high in the epidermis and weakly in the head. The temporal expression indicated that the expression of BmCPI40 in the larvae stage was significantly higher than that in the pupal stage, while the expression was significantly reduced during molting and metamorphosis. Immunofluorescence experiments showed that BmCPI40 was synthesized in the epithelial cells. Moreover, it was found thatBmCPI40 could be inhibited by ecdysone, because that its expression level decreased after injection with 20E in the silkworm and promoter activity also decreased after induction with 20E in theBmE cell.[Conclusion]Ecdysone induced the decrease of expression level ofBmCPI40, which may be involved in the molting and metamorphosis in the silkworm.