BmSPI38同型串联多聚体在大肠杆菌中的表达和抗真菌活性

Expression of BmSPI38 tandem multimers in Escherichia coli and its antifungal activity

摘要:

本研究旨在通过蛋白质工程手段获得结构均一性更好、活性更高、抗真菌能力更强的家蚕蛋白酶抑制剂BmSPI38的串联多聚体蛋白。利用原核表达技术获得BmSPI38串联多聚体蛋白，并通过蛋白酶抑制剂胶内活性染色、蛋白酶抑制实验和真菌生长抑制实验等探讨串联多聚体化对BmSPI38的结构均一性、抑制活性和抗真菌能力的影响。活性染色结果表明，基于多肽柔性接头的串联表达能够极大提高BmSPI38蛋白的结构均一性。蛋白酶抑制实验表明，基于接头的串联三聚体化和四聚体化能提高BmSPI38对微生物蛋白酶的抑制能力。孢子萌发实验表明，His₆-SPI38L-tetramer对球孢白僵菌（Beauveria bassiana）分生孢子萌发的抑制能力显著强于His₆-SPI38-monomer。真菌生长抑制实验显示，能够通过串联多聚体化来增强BmSPI38对酿酒酵母（Saccharomyces cerevisiae）和白色念珠菌（Candida albicans）的抑制能力。本研究成功实现BmSPI38的串联多聚体在大肠杆菌中的异源活性表达，并证实可通过串联多聚体化来增强BmSPI38的结构均一性和抗真菌能力，不仅可为培育抗真菌转基因家蚕提供重要的理论依据和新策略，还将推动BmSPI38的外源生产及在医疗领域的应用。

关键词:家蚕;蛋白酶抑制剂;串联多聚体;异源表达;结构均一性;抗真菌活性

Abstract:

The aim of this study was to prepare tandem multimeric proteins of BmSPI38, a silkworm protease inhibitor, with better structural homogeneity, higher activity and stronger antifungal ability by protein engineering. The tandem multimeric proteins of BmSPI38 were prepared by prokaryotic expression technology. The effects of tandem multimerization on the structural homogeneity, inhibitory activity and antifungal ability of BmSPI38 were explored by in-gel activity staining of protease inhibitor, protease inhibition assays and fungal growth inhibition experiments. Activity staining showed that the tandem expression based on the peptide flexible linker greatly improved the structural homogeneity of BmSPI38 protein. Protease inhibition experiments showed that the tandem trimerization and tetramerization based on the linker improved the inhibitory ability of BmSPI38 to microbial proteases. Conidial germination assays showed that His₆-SPI38L-tetramer had stronger inhibition on conidial germination of Beauveria bassiana than that of His₆-SPI38-monomer. Fungal growth inhibition assay showed that the inhibitory ability of BmSPI38 against Saccharomyces cerevisiae and Candida albicans could be enhanced by tandem multimerization. The present study successfully achieved the heterologous active expression of the silkworm protease inhibitor BmSPI38 in Escherichia coli, and confirmed that the structural homogeneity and antifungal ability of BmSPI38 could be enhanced by tandem multimerization. This study provides important theoretical basis and new strategies for cultivating antifungal transgenic silkworm. Moreover, it may promote the exogenous production of BmSPI38 and its application in the medical field.

Key words:Bombyx mori;protease inhibitor;tandem multimer;heterologous expression;structural homogeneity;antifungal activity