家蚕BmYippee蛋白的表达和亚细胞定位分析
Expression analysis and subcellular localization of BmYippee from silkworm,Bombyx mori
在对家蚕蛹期cDNA文库测序分析中，我们获得一条cDNA序列长度为1272 bp，该基因的开放阅读框（ORF）全长366 bp，编码121个氨基酸残基，预测分子量为13.72 kD，等电点为5.91并且具有一个保守的Yippee结构域。我们将它命名为家蚕Yippee蛋白BmYippee（Yippee from Bombyx mori），Genbank登录号为DN236885。，编码的蛋白有121个氨基酸残基，。我们将其ORF克隆到原核表达载体pET-28a中，构建重组质粒，双酶切鉴定及序列测定正确后转化大肠杆菌BL21(DE3)，经IPTG诱导表达的重组蛋白以包涵体形式存在，经过镍柱亲和层析纯化后获得高纯度的目的蛋白His-BmYippee，以该重组蛋白作为抗原免疫新西兰大白兔制备多克隆抗体，经ELISA检测，抗体效价可达1:8000。提取家蚕各个时期和五龄幼虫各组织的总RNA分别进行荧光定量PCR分析结果表明，该基因mRNA在蛹期转录水平最高，卵中最低，在生殖腺中转录水平最高，丝腺和脂肪最低。亚细胞定位结果表明，在Bm5细胞的细胞间期BmYippee在细胞核和细胞质中均有分布，而细胞分裂期时在细胞质中的某些区域有相对集中的分布。
pupae cDNA library of silkworm has been constructed in our Lab. We identified a gene with an open reading frame(ORF) of 366bp which encodes 121 amino acids containing a conservative domain Yippee. We nominated this gene as BmYippee(Yippee gene from Bombyx mori), and its accession number in Genbank is DN236885. The ORF of the gene was cloned into the prokaryotic expression vector pET-28a and the recombinant plasmid was transformed into E.coli BL21(DE3). The fusion protein was successfully expressed,purified and then immuned New Zealand rabbit to get antiserum. The titer of the polyclonal antibodies reaches 1:8000 measured by ELISA. We compared the quantity of BmYippee mRNA in different tissues of the fifth instar larva and different stages of silkworm using RT-PCR. The subcellular localization of BmYippee shows that it was localized in both cytoplasm and nucleus during interphase and at several regions of cytoplasm during mitotic phase of Bm5 cells.
陈健、毛洁、王鑫、吕正兵、舒特俊、张耀洲
分子生物学细胞生物学昆虫学
Bombyx moriYippeeRT-PCR亚细胞定位
Bombyx moriYippeesubcellular localizationRT-PCR
陈健,毛洁,王鑫,吕正兵,舒特俊,张耀洲.家蚕BmYippee蛋白的表达和亚细胞定位分析[EB/OL].(2009-02-11)[2025-05-25].http://www.paper.edu.cn/releasepaper/content/200902-505.点此复制
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