家蚕整合素基因Bmintegrin αPS3的鉴定及亚细胞定位

Identification and Subcelluar Localization of Bmintegrin αPS3 from Silkworm(Bombyx mori)

【目的】克隆家蚕整合素基因BmintegrinαPS3,分析其mRNA表达及亚细胞定位特征,并进行原核表达及蛋白纯化,为进一步探究BmintegrinαPS3在家蚕中的功能奠定基础。【方法】利用RACE方法克隆获得BmintegrinαPS3的cDNA全长序列;采用RT-PCR方法检测BmintegrinαPS3的时空表达;在原核表达系统中表达获得BmintegrinαPS3重组蛋白;并构建BmintegrinαPS3真核表达载体,转染家蚕胚胎细胞系分析其蛋白亚细胞定位。【结果】家蚕整合素基因BmintegrinαPS3编码框全长为2 895 bp,编码965个氨基酸,含有整合素家族蛋白典型的integrin alpha结构域,预测其为跨膜蛋白。构建了BmintegrinαPS3的原核表达系统,并利用亲和层析纯化获得高纯度的BmintegrinαPS3原核表达蛋白,为进一步制备抗体获得足够的抗原。在家蚕细胞系中外源表达BmintegrinαPS3蛋白,显示其主要定位于细胞质和细胞膜中。另外,RT-PCR结果显示BmintegrinαPS3 mRNA在血细胞中为特异持续表达。【结论】获得了BmintegrinαPS3的完整cDNA序列及表达特征,经原核表达、纯化获得其融合蛋白,在细胞水平上初步分析了其亚细胞定位情况。

[Objective] Bmintegrin αPS3 was cloned, analyzed, and overexpressed either in Escherichia coli or silkworm cell line to provide a theoretical basis for further studying the function ofBmintegrin aPS3 in silkworm. [Method] The Bmintegrin αPS3 eDNA including the complete ORF sequence was cloned by RACE method. The spatial-temporal expression profile of Bmintegrin αPS3 was investigated by RT-PCR. Bmintegrin aPS3 protein was overexpressed using prokaryotic expression. In addition, Bmintegrin αPS3 was constructed into a transit expression vector, and transfected into silkworm embryonic cell line to analyze the subcellular location of Bmintegrin aPS3. [Result] Bmintegrin αPS3 eDNA consisted of 2 895 bp, and encoded 965 amino acids containing the conserved integrin alpha domains, which was predicted to be a transmembrane protein. Bmintegrin aPS3 protein fxom E. coli was overexpressed and purified using affinity chromatography, and the purity reached more than 99%. In addition, the overexpressed Bmintegrin αPS3 protein in embryonic cell line was located in cytoplasm and membrane. RT-PCR results showed that Brnintegrin αPS3 mRNA was specifically and continually expressed in silkworm hemocytes. [Conclusion] The Bmintegrin aPS3 eDNA was cloned and its expression patterns in silkworm were analyzed. The subcelluar localization of recombinant Bmintegrin αPS3 protein was investigated in silkworm embryonic cell line. The Bmintegrin αPS3 protein was overexpressed in E. coli and purified for further antibody preparation.