天津科技大学学报2016，Vol.31Issue(6)：27-33,7.DOI:10.13364/j.issn.1672-6510.20160029
莱茵衣藻IFT139蛋白抗原的原核表达、纯化及多克隆抗体的制备
Prokaryotic Expression,Purification and Polyclonal Antibody Preparation ofChlamydomonas reinhardtiiIFT139 Protein Antigen
摘要
Abstract
IFT139 is a key component of the intraflagellar transport(IFT)complex A in Chlamydomonas reinhard-tii.Mutations,deletions or silence ofift139 gene can affect cilia assembly and depolymerization,and thus lead to ciliopa-thy.To further investigate the role of IFT139 in ciliogenesis,ift139 5′-458 bp EST cDNA fragment was firstly amplified and the recombined plasmids pMAL-c2-ift139 and pET-28a(+)-ift139 were constructed and transferred intoEscherichia coli BL21(DE3).Under IPTG induction,fusion proteins MBP-IFT139 and HIS-IFT139 with a molecular weight of 6.0×104and 2.5×104,respectively,were obtained.The fusion protein MBP-IFT139 was purified by affinity adsorption purification(more than 95%, purity)and then used for immunizing New Zealand white rabbits to produce antiserum.When the titer of antiserum reached 256,000,it was purified by Protein A,and then affinity adsorption purification was followed by nitrocellulose mem-brane purification procedure.The specificity of polyclonal antibodies was evaluated with Western blot.The result showed that the polyclonal antibody can specifically recognize IFT139.The research has provided an important tool for further clari-fying the role of IFT139 in ciliogenesis and cilipathy.关键词
莱茵衣藻/纤毛/IFT139/表达纯化/多克隆抗体Key words
Chlamydomonas reinhardtii/cilia/IFT139/expression and purification/polyclonal antibody分类
生物科学引用本文复制引用
田伟,董彬,李镇芳,孟德梅,樊振川..莱茵衣藻IFT139蛋白抗原的原核表达、纯化及多克隆抗体的制备[J].天津科技大学学报,2016,31(6):27-33,7.基金项目
国际遗传工程与生物技术中心(ICGEB)研究资助项目(CRP/CHN15-01) （ICGEB）
