猪生长分化因子11的原核表达及抗体制备  

Expression of Growth/Differentiation Factor 11 Gene in E.coli and Its Antibody Preparation

作　　者：孙琳琳[1] 孙亿[1] 臧丽[1] 姜运良[1] 

机构地区：[1]山东农业大学动物科技学院,泰安271018

出　　处：《畜牧兽医学报》2009年第11期1577-1581,共5页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金："863"高新技术项目(2006AA10Z1E1)

摘　　要：本研究旨在获得猪GDF-11的特异性抗体,以便于进一步研究猪GDF-11的功能。对猪GDF-11的编码区进行了克隆、表达载体的构建和转化、原核诱导表达和纯化以及将GDF-11注射到小鼠中并分离抗体。从猪胚肾中分离总RNA,经RT-PCR扩增得到猪GDF-11基因的编码区,插入pMD18-T载体中。再经限制性内切酶BamHⅠ和EcoRⅠ双酶切,将回收的GDF-11酶切片段插入原核表达载体pGEX-4T-2中,获得重组原核表达质粒pGEX-4T-2-GDF-11。重组质粒经测序鉴定后转化大肠杆菌BL21(DE3),并经IPTG诱导产生了65 ku的GST-GDF-11融合蛋白。融合蛋白经凝血酶酶切,分离纯化出42 ku左右的GDF-11蛋白;然后注射小鼠,制备了多克隆抗体,其滴度最高可达1∶25 600,而且特异性好,表明得到了猪GDF-11的多克隆抗体。The objective of the present study is to obtain specific antibody against porcine GDF-11 protein to further reveal its function in pigs.The coding region of porcine GDF-11 was cloned,expression vector was constructed and transformed,inducible expression and purification were carried out and antibody was isolated after GDF-11 injected into mice.A GDF-11 coding region sequence was amplified from total RNA isolated from porcine embryo kidney tissues by RT-PCR and inserted into pMD18-T.After being digested with BamHⅠand EcoRⅠ,the GDF-11 fragment was inserted into the prokaryotic expression vector pGEX-4T-2 and sequenced,and the recombinant plasmid pGEX-4T-2-GDF-11 was obtained.The competent E.coli cell strain BL21（DE3） were transformed by the recombinant plasmid pGEX-4T-2-GDF11 and induced by IPTG to produce fusion protein GST-GDF-11 of 65 ku.The expressed fusion protein GST-GDF-11 was digested with thrombin to release GDF-11 protein of 42 ku which was subsequently used to immunize mouse.The anti-GDF-11 polyclonal antibody with high sensitivity（1：25 600） and specificity was obtained.

关 键 词：猪 生长分化因子11 E.COLI 表达 抗体 

分 类 号：S813.3[农业科学—畜牧学]