摘要
家蚕防御素(defensin)是家蚕抗菌肽家族的主要成员之一,为家蚕先天性免疫的重要效应因子。采用生物信息学方法分析家蚕防御素基因BmdefA、BmdefB的序列中各有2个外显子,2个基因分别定位于家蚕第4号和第13号染色体上;等电点预测BmdefA带有负电荷,属于罕见的阴离子型抗菌肽,而BmdefB带有正电荷,属于常见的阳离子型抗菌肽;预测2个基因编码的成熟肽分子质量均在4 kD左右。用RT-PCR方法检测BmdefA在家蚕整个发育过程中有表达,且在检测的幼虫和成虫各组织中均有表达;BmdefB从幼虫5龄第3天到成虫期表达,雄性表达量高于雌性,且在5龄第3天幼虫的生殖腺、脂肪体和血细胞中高水平表达。家蚕防御素BmdefA、BmdefB具有不同的分子特性和表达特征,暗示它们在家蚕先天免疫过程中可能行使不同的功能,甚至可能具有不同的抗菌机理。构建融合GST标签的家蚕防御素基因原核表达载体,通过在大肠杆菌细胞内诱导表达,获得可溶性的目的蛋白,使用GST亲和层析柱初步纯化表达的融合蛋白并进行Western blotting鉴定,为进一步研究BmdefA、BmdefB的体外活性和抑菌机制奠定基础。
Defensins of silkworm（Bombyx mori） are major members of silkworm antimicrobial peptide（AMP） family and important effectors of silkworm innate immune system.Bioinformatic analysis showed that two silkworm defensin genes,termed BmdefA and BmdefB,are located on chromosome 4 and chromosome 13 respectively.Each of them has two exons.Prediction on isoelectric point showed that BmdefA brings negative charges,belonging to the unusual anionic peptide family,whereas BmdefB brings positive charges,belonging to the usual cationic peptide family.Molecular masses of mature peptides encoded by both genes are predicted to be around 4 kD.RT-PCR assay revealed that BmdefA was expressed during all developmental stages of silkworm and in all observed tissues of silkworm larvae and adults;BmdefB was expressed from day 3 of the 5th instar to adult stage with higher expression in male individuals over female ones and high expression levels in gonad,fat body and hemocyte of day 3 larvae of the 5th instar.Silkworm defensins BmdefA and BmdefB had different molecular characteristics and expression patterns,suggesting that they play different roles in the innate immune process of silkworm and even function via different antimicrobial mechanisms.After prokaryotic expression vector for fusion expression of silkworm defensin genes with GST tag was constructed,induced expression in E.coli yielded soluble target proteins,which were further purified using GST affinity chromatography and identified by Western blotting.The present study establishes a good basis for further investigations on in vitro activity and antimicrobial mechanism of BmdefA and BmdefB.
出处
《蚕业科学》
CAS
CSCD
北大核心
2012年第2期249-256,共8页
ACTA SERICOLOGICA SINICA
基金
中央高校基本科研业务费专项(No.XDJK2009C158)
重庆市自然科学基金重点项目(No.CSTC2008BA1008)
西南大学研究生科技创新基金项目(No.kb2010004)
