中华蜜蜂GABARAP基因克隆、生物信息学分析及原核表达载体的构建

Cloning,Bioinformatics Analysis and Prokaryotic Expression of GABARAP Gene in Apis cerana cerana

【目的】对中华蜜蜂(Apis cerana cerana)中自噬相关蛋白Atg8家族成员γ-氨基丁酸相关受体蛋白(gamma-aminobutyric acid receptor type associated protein,GABARAP)基因进行克隆鉴定和生物信息学分析,并在大肠杆菌BL21(DE3)感受态细胞中进行原核表达,以期为后续探究该基因的功能奠定基础。【方法】根据GenBank中西方蜜蜂(Apis mellifera)GABARAP基因序列(登录号:XM_001120069.5)设计引物,采用巢式PCR对中华蜜蜂GABARAP基因进行扩增、克隆;运用生物信息学软件对其氨基酸序列相似性、二级结构、三级结构进行比对和预测分析;构建GABARAP基因原核表达载体,利用Western blotting对GABARAP蛋白进行鉴定后并用IPTG诱导纯化。【结果】经巢式PCR扩增得到中华蜜蜂GABARAP基因序列;使用在线BLAST工具对氨基酸相似性进行分析显示,中华蜜蜂GABARAP与西方蜜蜂、大蜜蜂、小蜜蜂、欧洲熊蜂、东方熊蜂的氨基酸序列相似性为100%;氨基酸序列系统进化树显示,中华蜜蜂与西方蜜蜂亲缘关系最近,与秀丽隐杆线虫亲缘关系最远。生物信息学分析结果显示,GABARAP基因编码区全长354 bp,编码117个氨基酸,蛋白分子质量为13.99 ku,理论等电点为9.48;GABARAP蛋白二级结构主要由3个α-螺旋、4个β-折叠和6个多肽结合位点构成,二、三级结构预测结果一致;SDS-PAGE分析结果显示,IPTG诱导的GABARAP蛋白分子质量为35 ku;Western blotting鉴定结果证明了His单克隆抗体可以特异性识别GABARAP蛋白。【结论】本研究成功克隆了中华蜜蜂GABARAP基因,获得了GABARAP蛋白并进行了生物信息学分析,为进一步研究GABARAP基因及其蛋白在自噬发生中的作用提供了参考。

【Objective】The purpose of this study was to perform cloning,identification and bioinformatics analysis of gamma-aminobutyric acid receptor type associated protein(GABARAP)gene,a member of the Atg8 family of autophagy-related proteins in Apis cerana cerana.Prokaryotic expression was carried out in E.coli BL21(DE3)competent cell,hoping to lay the foundation for the subsequent exploration of the function of this gene.【Method】Primers were designed with reference to Apis mellifera GABARAP gene sequence in GenBank(accession number:XM_001120069.5).The nested PCR was used to amplify and clone the Apis cerana cerana GABARAP gene.The amino acid sequence similarity,secondary structure and tertiary structure were compared and predicted by bioinformatics software.The prokaryotic expression vector of GABARAP gene was constructed.The GABARAP protein was identified by Western blotting and induced by IPTG and then purified.【Result】The Apis cerana cerana GABARAP gene was amplified by nested PCR.The comparison of amino acid sequence similarity analysis using online BLAST tool showed that the GABARAP of Apis cerana cerana was 100%with that of Apis mellifera,Apis dorsata,Apis florea,Bombus terrestris and Bombus impatiens.The phylogenetic tree analysis of amino acid sequence showed that GABARAP of Apis cerana cerana was most closely related to Apis mellifera and farthest from Caenorhabditis elegans.The results of bioinformatics analysis showed that the CDS region of GABARAP gene fragment size was 354 bp,encoding 117 amino acids,the molecular weight of the protein was 13.99 ku and the theoretical isoelectric point was 9.48.The secondary structure of GABARAP protein was mainly composed of 3 alpha helices,4 beta sheets and 6 peptide binding sites,the predicted results of secondary and tertiary structures were the same.SDS-PAGE analysis showed that the molecular weight of GABARAP protein induced by IPTG was 35 ku.The results of Western blotting showed that His monoclonal antibody could specifically recognize GABARAP protein.【Conclusion】In this study,the GABARAP gene of Apis cerana cerana was cloned successfully,and the GABARAP protein was obtained and analyzed by bioinformatics,which provided a reference for further study on the role of GABARAP gene and its protein in autophagy.