家蚕新基因Bmpseg的克隆、特征分析及亚细胞定位
lone, characteristics analysis and subcellular localization of a novel gene Bmpseg from silkworm pupae (Bombyx mori )
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。Unigene分析预测该基因只在蛹期表达，因此将其命名为pupa-sepecific expresstion gene (Bmpseg)。该基因是一个同源性很低的基因，未发现保守结构域。Bmpseg基因全长661bp，由 99 bp的 5′端非翻译区序列（5′UTR）、486 bp的开放读码框（ORF）和76 bp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Bmpseg可能存在跨膜区和信号肽。我们将该基因插入到载体pGEX-4T-3中，构建了重组质粒pGEX-4T-3-Bmpseg，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。
novel gene was identified in the process of analyzing the cDNA sequences from the Bombyx mori pupa cDNA library constructed by our laboratory (GenBankAccession: DY231426) The gene shares low homologous with other genes and has no conserved sequence. Unigene analysis predicts that this gene was only expressed in pupa stage, so it was named as pupa-sepecific expression gene(Bmpseg). The full length of gene Bmpseg is 661 bp, which contains 5′UTR (99 bp ), ORF (486 bp) and 3′UTR (76 bp).The gene Bmpseg was subcloned from Bombyx mori pupa cDNA library and inserted into prokaryotic expression vector pGEX4T-3, Escherichia coli Rosetta were transformed with the recombinant plasmid, the recombinant protein expression was induced with IPTG. The recombinant fusion protein GST-Bmpseg was purified using GST Trap FF column chromatography. Purified GST-Bmpseg was used to produce anti-Bmpseg polyclonal antibody. The subcellular immunofluorescence localization results indicated that the Bmpseg was distributed in cytoplasm.
陈健、张耀洲、陈剑清、王丹、于威、金邦泽、盛清、舒建洪、吴祥甫、刘立丽、聂作明、吕正兵、沈红丹
分子生物学昆虫学细胞生物学
家蚕新基因原核表达纯化亚细胞定位
Bombyx morinovel geneprokaryotic expressionpurificationsubcellular localization
陈健,张耀洲,陈剑清,王丹,于威,金邦泽,盛清,舒建洪,吴祥甫,刘立丽,聂作明,吕正兵,沈红丹.家蚕新基因Bmpseg的克隆、特征分析及亚细胞定位[EB/OL].(2009-02-12)[2025-08-02].http://www.paper.edu.cn/releasepaper/content/200902-582.点此复制
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