| 猪生长分化因子11的原核表达及抗体制备 |
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| 引用本文： | 孙琳琳,孙亿,臧丽,姜运良.猪生长分化因子11的原核表达及抗体制备[J].畜牧兽医学报,2009,40(11). |
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| 作者姓名： | 孙琳琳 孙亿 臧丽 姜运良 |
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| 作者单位： | 山东农业大学动物科技学院,泰安,271018 |
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| 基金项目： | "863"高新技术项目 |
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| 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BarnH Ⅰ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.
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| 关 键 词： | 猪 生长分化因子11 表达 抗体 |
Expression of Growth/Differentiation Factor 11 Gene in E. coli and Its Antibody Preparation |
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| SUN Lin-lin,SUN Yi,ZANG Li,JIANG Yun-liang.Expression of Growth/Differentiation Factor 11 Gene in E. coli and Its Antibody Preparation[J].Acta Veterinaria et Zootechnica Sinica,2009,40(11). |
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| Authors: | SUN Lin-lin SUN Yi ZANG Li JIANG Yun-liang |
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| Abstract: | 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 BamH Ⅰ and EcoR Ⅰ, the GDF-11 frag-ment was inserted into the prokaryotic expression vector pGEX-4T-2 and sequenced, and the re-combinant 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 di-gested with thrombin to release GDF-11 protein of 42 ku which was subsequently used to immu-nize mouse. The anti-GDF-11 polyclonal antibody with high sensitivity (1 : 25 600) and specifici-ty was obtained. |
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| Keywords: | E coli pig GDF-11 E coli expression antibody |
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