Ramos Luís Felipe Costa, Rangel João Henrique de Oliveira, Andrade Guilherme Caldas, Lixa Carolina, de Castilho Livia Vieira Araujo, Nogueira Fábio César Sousa, Pinheiro Anderson S, Gomes Fabio Mendonça, AnoBom Cristiane Dinis, Almeida Rodrigo Volcan, de Oliveira Danielle Maria Perpétua
Department of Biochemistry, Institute of Chemistry, Center of Mathematical and Natural Sciences, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro, RJ, Brazil.
Alberto Luiz Coimbra Institute of Graduate Studies and Research (COPPE), Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro, RJ, Brazil.
J Venom Anim Toxins Incl Trop Dis. 2021 Mar 12;27:e20200127. doi: 10.1590/1678-9199-JVATITD-2020-0127.
Insects can be found in numerous diverse environments, being exposed to pathogenic organisms like fungi and bacteria. Once these pathogens cross insect physical barriers, the innate immune system operates through cellular and humoral responses. Antimicrobial peptides are small molecules produced by immune signaling cascades that develop an important and generalist role in insect defenses against a variety of microorganisms. In the present work, a cecropin B-like peptide (AgCecropB) sequence was identified in the velvetbean caterpillar and cloned in a bacterial plasmid vector for further heterologous expression and antimicrobial tests.
AgCecropB sequence (without the signal peptide) was cloned in the plasmid vector pET-M30-MBP and expressed in the BL21(DE3) expression host. Expression was induced with IPTG and a recombinant peptide was purified using two affinity chromatography steps with Histrap column. The purified peptide was submitted to high-resolution mass spectrometry (HRMS) and structural analyses. Antimicrobial tests were performed using gram-positive () and gram-negative ( and ) bacteria.
AgCecropB was expressed in BL21 (DE3) at 28°C with IPTG 0.5 mM. The recombinant peptide was purified and enriched after purification steps. HRMS confirmed AgCrecropB molecular mass (4.6 kDa) and circular dichroism assay showed α-helix structure in the presence of SDS. AgCrecropB inhibited almost 50% of gram-positive bacteria growth.
The first cecropin B-like peptide was described in and a recombinant peptide was expressed using a bacterial platform. Data confirmed tertiary structure as predicted for the cecropin peptide family. AgCecropB was capable to inhibit growth .
昆虫存在于众多不同的环境中，会接触到诸如真菌和细菌等致病生物。一旦这些病原体突破昆虫的物理屏障，先天免疫系统就会通过细胞和体液反应发挥作用。抗菌肽是由免疫信号级联反应产生的小分子，在昆虫抵御多种微生物的防御中发挥着重要且广泛的作用。在本研究中，在豆天蛾中鉴定出一种天蚕素B样肽（AgCecropB）序列，并将其克隆到细菌质粒载体中，用于进一步的异源表达和抗菌测试。
将AgCecropB序列（无信号肽）克隆到质粒载体pET-M30-MBP中，并在BL21(DE3)表达宿主中表达。用异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达，并用HisTrap柱通过两步亲和层析纯化重组肽。对纯化后的肽进行高分辨率质谱（HRMS）和结构分析。使用革兰氏阳性菌（ ）和革兰氏阴性菌（ 和 ）进行抗菌测试。
在28°C、0.5 mM IPTG条件下，AgCecropB在BL21(DE3)中表达。经过纯化步骤后，重组肽得到纯化和富集。HRMS证实了AgCrecropB的分子量（4.6 kDa），圆二色性分析表明在十二烷基硫酸钠（SDS）存在下其具有α-螺旋结构。AgCrecropB抑制了近50%的革兰氏阳性菌生长。
首次在 中描述了天蚕素B样肽，并利用细菌平台表达了重组肽。数据证实了天蚕素肽家族预测的三级结构。AgCecropB能够抑制 生长 。
