中华蜜蜂半乳糖凝集素AcGalectin的表达、纯化及性质分析

Expression,purification and characterization of galectin AcGalectin in Apis cerana cerana( Hymenoptera: Apidae)

【目的】本研究旨在明确中华蜜蜂Apis cerana cerana半乳糖凝集素(galectin)的功能及其与病毒的相互作用。【方法】提取中华蜜蜂的总RNA,利用RT-PCR技术获得Galectin基因,然后利用生物信息学软件对基因序列及其推导氨基酸序列和结构特征进行分析;再依据大肠杆菌Escherichia coli密码子的偏嗜性对该基因密码子进行优化、原核表达,并制备重组蛋白;最后,通过Far-western blotting技术检测纯化的重组蛋白与纯化的中蜂囊状幼虫病毒(Chinese sacbrood virus,CSBV)、慢性蜜蜂麻痹病毒(Chronic bee paralysis virus,CBPV)和残翅病毒(Deformed wing virus,DWV)的结合情况。【结果】从中华蜜蜂中克隆到Galectin基因,并将其命名为Ac Galectin(Gen Bank登录号:MG557559),其cDNA全长为1 473 bp。Ac Galectin空间结构比较稳定,包含一个半乳糖识别结构域。系统进化树表明,Galectin明显分为两簇,膜翅目蜜蜂科昆虫的Galectin形成一个簇,而其他昆虫Galectin形成另一簇。经密码子优化后的Ac Galectin基因能够在大肠杆菌感受态细胞BL21(DE3)中高效表达。纯化的重组Ac Galectin蛋白可与CSBV和CBPV结合,但不与DWV结合。【结论】结果表明Ac Galectin与蜂病毒CSBV和CBPV有结合作用。本研究为Ac Galectin在CSBV和CBPV感染过程中的功能研究奠定了基础。

【Aim 】 This study aims to explore the function of galectin in Apis cerana cerana and its interaction with the honeybee virus. 【Methods】Galectin gene was amplified from A. cerana ceranas by RT-PCR using the extracted total RNA as the template, and then the sequence and structural characteristics of deduced amino acids were analyzed by using bioinformatics tools. The codons of the galectin gene were optimized based on the codon bias of Escherichia coli,and the recombinant protein wasproduced in E. coli and purified by affinity chromatography. The binding activities of the recombinant protein with Chinese sacbrood virus（ CSBV）,Chronic bee paralysis virus（ CBPV） and Deformed wing virus（ DWV） were analyzed by far-western blotting. 【Results】A galectin gene was cloned from A.cerana cerana and named Ac Galectin（ Gen Bank accession no： MG557559）,and its full-length cDNA is1 473 bp in length. Ac Galectin has a stable spatial structure containing a Gal-bind_lectin domain. The phylogenetic tree constructed based on the amino acid sequences from Ac Galectin and the previously reported galectins indicated that galectins form two clusters, one related to galectins from the Hymenoptera,and the other related to galectins from other insects. The codon-optimized Ac Galectin gene could be highly expressed in host bacteria BL21（ DE3）,and the purified recombinant Ac Galectin protein could bind with CSBV and CBPV,but not with DWV. 【Conclusion】The results indicate that Ac Galectin can bind with CSBV and CBPV,and this lays a foundation for the further study on the function of Ac Galectin in the process of CSBV and CBPV infections.