家蚕新基因Bmbzj的表达、抗体制备及亚细胞定位  

Cloning,Expression and Subcellular Localization of a Novel Gene Bmbzjfrom Silkworm Pupae(Bombyx mori)

作　　者：高珍[1] 卜远虹[1] 金邦泽[1] 于威[1] 刘悦[2] 陈健[1] 张耀洲[1] 

机构地区：[1]浙江理工大学生物化学研究所,杭州310018 [2]浙江经贸职业技术学院应用工程系,杭州310018

出　　处：《浙江理工大学学报（自然科学版）》2012年第3期414-417,共4页Journal of Zhejiang Sci-Tech University(Natural Sciences)

基　　金：国家高技术研究发展计划(863计划;2011AA100603);浙江省自然科学基金项目(Y3090304;Y3110354)

摘　　要：在自建的家蚕蛹cDNA文库中发现了一条新基因序列,GenBank登录号为DY231426。该基因是一个同源性很低的基因,未发现保守结构域,将其命名为Bmbzj。Bmbzj基因全长661bp,由99bp的5′端非翻译区序列(5′UTR)、486bp的开放读码框(ORF)和76bp的3′端非翻译区序列(3′UTR)组成。生物信息学分析结果表明蛋白Bmbzj可能存在跨膜区和信号肽。将该基因插入到载体pGEX-4T-3中,构建了该基因编码的重组质粒pGEX-4T-3-Bmbzj,转化大肠杆菌Rosetta后,IPTG诱导表达了该融合蛋白,并利用亲和层析的方法纯化该重组蛋白,免疫新西兰兔制备多克隆抗体。亚细胞定位实验结果表明该蛋白只存在于细胞质中。A novel gene is identified in the process of analyzing the cDNA sequences from the Bombyx mori pupa cDNA library constructed by our laboratory（GenBank Accession： DY231426）.The gene shares low homologous with other genes and has no conserved sequence,it is named as Bmbzj.The full length of gene Bmbzj is 661 bp,which contains 5′UTR（99 bp）,ORF（486 bp） and 3′UTR（76 bp）.The gene Bmbzj is subcloned from Bombyx mori pupa cDNA library and inserted into prokaryotic expression vector pGEX4T-3,Escherichia coli Rosetta are transformed with the recombinant plasmid,the recombinant protein expression is induced with IPTG.The recombinant fusion protein GST-Bmbzj is purified using GST Trap FF column chromatography.Purified GST-Bmbzj is used to produce anti-Bmbzj polyclonal antibody.The subcellular immunofluorescence localization results indicate that the Bmbzj is distributed in cytoplasm.

关 键 词：家蚕 Bmbzj基因 原核表达 纯化 亚细胞定位 

分 类 号：Q74[生物学—分子生物学]