摘要
克隆获得了棉铃虫β-actin基因,生物信息学分析表明,基因序列长1 131 bp,编码376个氨基酸,理论分子量为41.77 k D,理论等电点5.16。氨基酸序列与其他物种肌动蛋白比较有98%-99%的一致性,与鳞翅目的家蚕具有99%的一致性。分析棉铃虫β-actin和小家鼠β-actin的抗原决定簇位点,发现两者抗原表位有70.63%的一致性。同时原核表达、纯化了棉铃虫β-actin蛋白,Western-blot结果表明表达正确。将纯化后的蛋白免疫ICR小鼠4次后,小鼠抗血清效价最高可达388 800,并且能与天然棉铃虫蛋白特异性结合。
As one of the important internal controls, β-actin plays an crucial role in maintaining cell structure, cell movement, and cell division of physiological activity. It is one of the widely used internal controls in the detection of gene transcription and protein level. In this study, β-actin gene was cloned from cotton bollworm. Bioinformatics analysis showed that the open reading frame of β-actin was 1 131 bp, encoding 376 amino acids, the theoretical molecular weight was 41.77 k D, and isoelectric point was 5.16. The similarity of amino acid between the β-actin of cotton bollworm and one of other species was 98%-99%, while 99% similarity with Bombyx mori. Analysis of β-actin antigenic determinant sites from cotton bollworm and Mus musculus indicated that their homology was 70.63%. Concurrently, β-actin protein from prokaryotic expression of the gene was purified, and Western blot analysis showed that the expression was correct. The purified protein was used to immunize ICR mouse 4 times, the titer of mouse antiserum β-actin reached 388 800 by ELISA assay. Moreover, the antiserum was able to specifically bind with the total protein of the cotton bollworm.
出处
《生物技术通报》
CAS
CSCD
北大核心
2015年第12期138-145,共8页
Biotechnology Bulletin
基金
国家自然科学基金项目(31471781)
国家级大学生实训项目(201410755025)
关键词
棉铃虫
Β-ACTIN
克隆
原核表达
多克隆抗体
Helicoverpa armigera
β-actin
cloning
prokaryotic expression
polyclonal antibody
作者简介
黄丽娜，女，硕士研究生，研究方向：昆虫分子毒理学；E—mail：huanglinaxju@163．com
通讯作者：刘小宁，女，博士，副教授，研究方向：农业昆虫与害虫防治；E-mail：liuxn0103@sina．com
