柞蚕ApMLEC的重组表达和功能分析

Analysis of recombinant expression and function of Antheraea pernyi ApMLEC

对柞蚕Malectin(ApMLEC)进行克隆、蛋白表达纯化和初步功能分析。基于已知的柞蚕cDNA数据库,通过PCR克隆ApMLEC基因,生物信息学分析序列信息;将基因连接至pET-28a原核表达载体,通过大肠杆菌BL21(DE3)表达系统和亲和层析法纯化重组蛋白,探究该蛋白结合多糖、凝集细菌和抑菌能力;通过将基因连接至转移载体pApBacDual-egfp探究其抗病毒作用。结果表明:基因由798个碱基构成,编码266个氨基酸,纯化得到分子质量约为33 ku、带有组氨酸标签的重组蛋白;ApMLEC能够与麦芽糖、脂多糖、肽聚糖结合;凝集细菌;抑制大肠杆菌和金黄色葡萄球菌的生长,且最小抑菌质量浓度均为250μg/mL;并抑制ApNPV病毒的复制。ApMLEC初步功能分析结果表明其可能在柞蚕天然免疫中发挥重要作用,为今后开展柞蚕免疫系统的研究奠定基础。

The study cloned,expressed,purified and performed preliminary functional analysis of Malectin(ApMLEC)from Antheraea pernyi.Based on the known cDNA database of Antheraea pernyi,the ApMLEC gene was cloned by PCR and the sequence information was analyzed by bioinformatics.The gene was ligated to the pET-28a prokaryotic expression vector,and the recombinant protein was purified by Escherichia coli BL21(DE3)expression system and affinity chromatography.The ability of the protein to bind polysaccharides,agglutinate bacteria and inhibit bacterial growth was investigated.The antiviral activity of the protein was explored by ligating it to the transfer vector pApBacDual-egfp.The results showed that the gene consisted of 798 bases encoding 266 amino acids and the purified recombinant protein had a molecular weight of about 33 ku with the His-tag.The ApMLEC could bind to maltose,lipopolysaccharide and peptidoglycan.It could agglutinate bacteria and inhibit the growth of Escherichia coli and Staphylococcus aereus with a minimum inhibitory mass concentration of 250μg/mL for both.It also inhibited the replication of Antheraea pernyi nuclear polyhedron viruses.The preliminary functional analysis of ApMLEC suggested that it might play an important role in the natural immunity of Antheraea pernyi,and laid the foundation for future research on the immune system of this species.