家蚕CIB基因的表达分析及其亚细胞定位

李晓璐; 杨光; 郑青亮; 张耀洲

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家蚕CIB基因的表达分析及其亚细胞定位

首发时间：2009-11-24

李晓璐 ¹ 杨光 ¹ 郑青亮 ¹ 张耀洲 ¹

1、浙江理工大学生物化学研究所

摘要：钙整合素结合蛋白一般含有4个EF-hand结构域，可与包括血小板αⅡbβ3 integrin 等许多效应蛋白结合并能调节他们的功能。我们从本实验室构建的家蚕蛹cDNA文库中获得一条cDNA序列，通过同源序列比对后发现其为家蚕钙整合素结合蛋白 (Bombyx mori calcium and integrin binding protein，BmCIB)基因, 全长为939 bp，ORF为558 bp，编码165个氨基酸。我们构建了重组表达载体pET28a- BmCIB，在E. coli BL21(DE3)中诱导表达了含有His标签的融合蛋白BmCIB。通过包涵体纯化与亲和层析法进一步纯化融合蛋白并免疫雄性新西兰兔，制备并获得了多克隆抗体。通过实时定量PCR和Western blot分析，我们发现BmCIB 的mRNA和表达的蛋白在五龄家蚕各组织及发育时期广泛分布。其mRNA转录水平以生殖腺和蛹期中最高，蛋白表达在各发育时期均可检测到，各组织中以生殖腺中最多。通过免疫荧光法分析BmCIB蛋白在家蚕Bm5细胞的亚细胞定位，发现其定位于细胞质中。以上工作为进一步研究BmCIB功能奠定了基础。

关键词：钙整合素结合蛋白 CIB 原核表达实时定量PCR 亚细胞定位

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Expression analysis and subcellular localization of the CIB from silkworm, Bombyx mori

Li Xiaolu ¹ Yang Guang ¹ Zheng Qingliang ¹ Zhang Yaozhou ¹

1、Institute of Biochemistry, College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, PRC

Abstract：The calcium and integrin binding protein (CIB), is an EF-hand-containing protein that binds many effector proteins including the platelet alphaIIbbeta3 integrin and potentially regulates their functions. Here we screened a cDNA sequence from silkworm pupa cDNA database constructed by our laboratory. After blasting, we found that it is Bombyx mori calcium and integrin binding protein gene(BmCIB). The CIB cDNA is 939-bp in size, containing a 558-bp open reading frame that encodes 165 amino acids. We obtained His-tagged fusion protein by transforming pET28a-CIB recombinant plasmid into E. coli BL21 (DE3). To obtain the anti-BmCIB polyclonal antibody,we immunized a male New Zealand rabbit with fusion protein which was purified with liquid solution of inclusion bodies and metal-chelating affinity chromatography. According to real-time Quantitative PCR and Western blot analyses,we found that BmCIB mRNA and protein are widely distributed in a variety of tissues and different developmental stages of silkworm. The transcriptional level of BmCIB mRNA is highest in gonad and pupa. The expression of protein can be detected in all the developmental stages and is highest in gonad among different tissues. Moreover,after being stained with immunofluorescence, the subcellular localization of the BmCIB was analyzed. The result show that BmCIB proteins are all accumulated in cytoplasm. The protein of BmCIB has been cloned and expressed successfully. The specific polyclonal antibody against the protein of BmCIB has been obtained which can be used for further research. Additionally we have completed the related expression and subcellular localization analysis of BmCIB which can be used for further research into the function of CIB．

Keywords： calcium and integrin binding protein CIB prokaryotic Expression Real-time Quantitative PCR Subcellular localization

基金：

1. 国家自然科学基金,国家自然科学基金（2007AA021703；2007AA120402,2005CB121006）

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李晓璐，杨光，郑青亮，等. 家蚕CIB基因的表达分析及其亚细胞定位[EB/OL]. 北京：中国科技论文在线 [2009-11-24]. https://www.paper.edu.cn/releasepaper/content/200911-686.

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论文编号	200911-686
论文题目	家蚕CIB基因的表达分析及其亚细胞定位
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