摘要
目的对广州管圆线虫中间纤维蛋白(intermediate filament,IF)基因进行体外表达、纯化、鉴定和抗原性质分析。方法将IF基因的重组表达载体pET-IF在大肠杆菌中表达,表达产物经组氨酸亲和层析纯化,SDS-PAGE鉴定蛋白质分子量,质谱分析氨基酸序列,生物信息学分析蛋白质结构,Western blot鉴定与人、大鼠和小鼠感染血清及人IgG4亚类的抗原反应性。结果SDS-PAGE、质谱分析和Western blot证明本研究获得的纯化蛋白为预期蛋白IF,其分子量为47.8kDa,肽指纹图谱与秀丽杆线虫IF蛋白家族具有高度同源性,并具有典型的IF结构特征,且IF结构的尾部存在抗原决定簇,融合蛋白IF能与大鼠感染血清、小鼠感染血清和患者血清IgG结合,也能与患者IgG4亚类结合。结论体外获得了抗原IF纯化蛋白,研究显示其可用于人类外周血检测并有助于检测现症感染和疗效考核。
To analyze intermediate filament （IF） protein of Angiostrongylus cantonensis, IF cDNA was expressed in vitro and its expression products were purified and identified. Recombinant pET-IF was irdused-expressed in E. coli and expression products were purified by His-binding resin affinity column. Protein molecular weight were identified by SDS-PAGE. Protein property was analyzed by mass spectrum and its structure feature was analyzed by bioinformatics. Western blot was used to detect its antigenic reaction to rat, mouse and human infected-serum and IgG4 antibody of human. As a result, SDS- PAGE, mass spectumn and western blot identified that the purified expression products just were the aim protein IF （47. 6 kDa） whose mass spectrometry fingerprint has higher homology with IF protein family of Caenorhabditis elegans , it has the specific features of IF protein and the tail domain perhaps has antigenic determinant. IF protein could response to IgG antibody of rat, mouse and human infected-serum, it could be reaction to IgG4 antibody of patient. In this research, the purified IF protein was obtained in vitro and it maybe benefit for testing current infection and analyzing curative effect.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2007年第6期590-594,共5页
Chinese Journal of Zoonoses
基金
国家"十五"科技攻关重大项目(2003BA712A0307)
国家自然科学基金广东省政府联合基金(U0632003)
广州市科技三项(2005Z37561
2006Z3C7191)
广东省医学科研基金(B2006003)
广东省人民医院科学技术研究基金(Y200532)资助
关键词
广州管圆线虫
抗原
中间纤维蛋白
基因
Angiostrongylus cantonensis
antigen
intcrmediate filament
cDNA library
