家蚕整合素Bmintegrin β1基因的克隆及表达

发布时间：2018-04-10 02:05

本文选题：Bmintegrin　切入点：β　出处：《生物工程学报》2017年12期

【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Bmintegrinβ1,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]:Integrin as a transmembrane glycoprotein, is closely related with the organism's physiology and pathology and other processes. In order to explore the role played in the silkworm, through PCR and RACE technology cloned Bombyx mori family member Bmintegrin integrin beta 1, predict the site to predict the structure of domain by domain, and construct evolution the evolutionary relationships were analyzed by prokaryotic expression system and method for protein purification to obtain the recombinant protein using recombinant protein immunized mice polyclonal antibody, and using semi quantitative PCR and Western blotting method to detect the expression of temporal Bmintegrin beta 1. 3 splicing forms of silkworm Bmintegrin beta 1 integrin in this paper and, the 3 shear body with length of 2502 BP open reading frame encoding 833 amino acids. According to the prediction of the protein containing Integrin-B-tail, transmembrane region and a series of integration Domain family conservative. Phylogenetic tree analysis shows that the protein with Lepidoptera members of Helicoverpa assulta and the monarch of the integrin is closest. In addition we through the prokaryotic expression system and affinity chromatography to obtain high purity Bmintegrin beta 1 protein to prepare polyclonal antibody. And through the blot proved that the antiserum could specifically recognize the recombinant protein Bmintegrin beta 1. By semi quantitative PCR and Western blotting showed that the protein, in different tissues of silkworm and the period of blood cells have high expression in the blood and scale up the highest results. In conclusion, this study provides a basis for the study of silkworm integrin family.

【作者单位】：西南大学家蚕基因组生物学国家重点实验室;
【基金】：国家自然科学基金(No.31672496) 重庆自然科学基金(No.cstc2016jcyj A0425) 重庆高校创新团队建设计划(No.CXTDX201601010)资助~~
【分类号】：Q78;Q966

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