生物学杂志

生物学杂志 ›› 2023, Vol. 40 ›› Issue (3): 22-.doi: 10.3969/j.issn.2095-1736.2023.03.022

• 研究报告 • 上一篇    下一篇

柞蚕ApMLEC的重组表达和功能分析

刘智超1， 古苏云1， 刘丹梅2， 范　琦3， 刘宇博1， 张嘉宁1， 李文利1   

  1. 1. 大连理工大学 生命科学与药学学院， 盘锦 124221； 2. 辽东学院， 丹东 118003；
    3. 辽宁省海洋水产科学研究院， 大连 116023
  • 出版日期:2023-06-18 发布日期:2023-06-19
  • 通讯作者: 李文利，博士，副教授，研究方向为昆虫免疫，E-mail:biolwl@dlut.edu.cn
  • 作者简介:刘智超，硕士，研究方向为柞蚕免疫，E-mail:zhichaoliu@mail.dlut.edu.cn
  • 基金资助:
    辽宁省教育厅项目（LJKZ1124）

Analysis of recombinant expression and function of Antheraea pernyi ApMLEC

LIU Zhichao1, GU Suyun1, LIU Danmei2, FAN Qi3, LIU Yubo1, ZHANG Jianing1, LI Wenli1   

  1. 1. School of Life and Pharmaceutical Sciences, Dalian University of Technology, Panjin 124221, China;
    2. Liaodong University, Dandong 118003, China;
    3. Liaoning Ocean Fisheries Science Research Institute, Dalian 116023, China
  • Online:2023-06-18 Published:2023-06-19

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摘要： 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250 μg/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。

关键词: 柞蚕, ApMLEC, 抗病毒, 抑菌, 凝菌

Abstract: 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 250 μg/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.

Key words: Antheraea pernyi, ApMLEC, antiviral, antibacterial, agglutinating bacteria

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