【摘要】 过氧化物氧还酶6(peroxiredoxin6,Prdx6)是一种双功能蛋白质,具有GSH过氧化物酶和磷脂酶A2活性。前期研究构建布鲁氏菌强弱毒株感染绵羊白细胞层SSH c DNA文库,发现Prdx6在不同毒力布鲁氏菌感染时存在一定的差异性表达,推测Prdx6可能在布鲁氏菌感染中发挥一定的作用。为了更好地研究其在生物体中的作用,以RT-PCR技术,提取小鼠巨噬细胞系RAW264.7总RNA,反转录制备c DNA,设计特异引物克隆Prdx6开放阅读框核酸序列,并构建原核表达载体,进行原核表达,以镍柱纯化获得纯度较高的Prdx6蛋白,免疫家兔制备高特异性、高灵敏度的鼠Prdx6兔源多克隆抗体,为后续进一步研究Prdx6蛋白功能提供科学依据和应用工具。更多还原

【Abstract】 Peroxiredoxin6( Prdx6) is a bifunctional protein that includes glutathione peroxidase and calcium-independent phospholipase A2. A SSH c DNA library of buffy coat from Ovis aries was constructed through infecting with virulent and avirulent strains of Brucella,and a differentially expressed Prdx6 was cloned.It indicated that Prdx6 might take part in the infection of brucella. In order to study the Prdx6 protein function,here total RNAs from the mice macrophage cell line Raw 264. 7 were extracted,c DNAs were reverse transcribed,and the open read frame( ORF) of mouse Prdx6 gene was cloned using the specific primes by means of RT-PCR.Then,the prokaryotic expression vector of Prdx6 was constructed,and the recombinant Prdx6 protein with a His tag was expressed by induction of IPTG, and purified by affinity chromatography of Ni-NTA. A specific polyclonal antibody against mouse Prdx6 was obtained by immunizing a rabbit with the purified Prdx6,which would benefit the further study on the functions of Prdx6 protein in the future.更多还原