家蚕整合素Bmintegrin β1基因的克隆及表达

Cloning and expression of Bmintegrin β1 in silkworm Bombyx mori

摘要:

整合素作为一种跨膜糖蛋白，与生物体的生理和病理等多个过程密切相关。为了探究其在家蚕中扮演的角色，通过PCR及RACE技术克隆得到了家蚕整合素β家族成员Bmintegrin β1，通过结构域预测网站对其结构域进行了预测，并构建进化树对其进化关系进行分析，此外通过原核表达系统及蛋白纯化方法获得其重组蛋白，使用重组蛋白免疫小鼠获得其多克隆抗体，并采用半定量PCR及Western blotting方法检测Bmintegrin β1的时空表达。文中得到家蚕整合素Bmintegrin β1的3种剪切形式，且这3种剪切体具有长为2 502 bp的开放阅读框架，编码833个氨基酸。根据预测该蛋白含有Integrin-B-tail、跨膜区等一系列整合素家族保守的结构域。进化树结果表明该蛋白与同为鳞翅目成员的烟夜蛾和黑脉金斑蝶的整合素蛋白最为接近。此外我们通过原核表达系统及亲和层析技术获得了高纯度的Bmintegrin β1蛋白，进一步制得多克隆抗体。并通过免疫印迹反应证明该抗血清能特异识别Bmintegrin β1重组蛋白。之后通过半定量PCR及免疫印迹检测，结果显示该蛋白在家蚕的各组织以及血细胞各时期均有较高表达且在血液中表达量最高。总之，这项研究为家蚕整合素家族的研究提供了基础。

Abstract:

Integrins are transmembrane glycoproteins, closely related to many physiological and pathological processes. In order to explore its role in silkworm, by PCR and Rapid-amplification of cDNA ends (RACE) technology, the full-length cDNA of Bmintegrin β1 in silkworm was acquired. The domain was predicted by domain prediction website. Phylogenetic tree was constructed to analyze its evolutionary relationship. By prokaryotic expression system, protein purification method and immunizing mouse, the antibody against Bmintegrin β1 recombinant protein was obtained. The spatial-temporal expression profile of Bmintegrin β1 was investigated by semi quantitative PCR and Western blotting. Then we identified all 3 different spliceosomes, and they shared a common open reading frame of 2 502 bp, encoding 833 amino acids. Bmintegrin β1 contained all the classic domains of the integrin family, such as Integrin-B-tail, transmembrane domain etc. Phylogenetic analysis indicated that Bmintegrin β1 was close to the homologous proteins from Heliothis assulta and Danaus plexippus. In order to understand the function of Bmintegrin β1 further, we generated the antibody. In addition, Western blotting demonstrated that the antibody recognized the Bmintegrin β1 recombinant protein. Then, semi quantitative PCR and Western blotting results showed that Bmintegrin β1 was widely expressed in most of tissues, among of them, it’s exhibited the highest expression level in hemacyte. Overall, this study provides a foundation for the study of silkworm integrin family.