SUA41蛋白表达和纯化及其多克隆抗体制备

Expression,Purification and Polyclonal Antibody Preparation of SUA41 Protein

旨在制备特异性SUA41多克隆抗体,为深入研究其在植物生长发育中的功能提供有力的分子生物学和生物化学的工具。PCR扩增拟南芥SUA41基因中编码280个氨基酸(401-680位氨基酸)的特异片段,经过GATEWAY的DNA重组技术构建了原核表达载体pDEST17-SUA41,用热休克法转化到E.coliBL21(DE3)star感受态细胞,以异丙基β-D-硫代半乳糖苷(IPTG)诱导表达出6×His-SUA41融合蛋白,用8 mol/L尿素缓冲液溶解包涵体并且经过水逐级去除尿素获得提纯的融合蛋白,并利用Western blotting鉴定确认。融合蛋白经Ni金属螯合柱亲和层析得以纯化,用SDS-PAGE进一步纯化。纯化的融合蛋白经过SDS-PAGE后切胶回收,免疫小白兔,制备多抗血清,然后用Western blotting进行检测,鉴定血清特异性和效价。结果显示,融合蛋白6×His-SUA41免疫兔,产生特异性的SUA41兔抗血清,可以检测到细菌和拟南芥组织中SUA41蛋白。用水提纯变性剂尿素溶解的包涵体蛋白具有可行性。制备的特异性SUA41兔抗血清效价高,能够有效地识别大肠杆菌表达的和拟南芥的SUA41蛋白。在有合适的对照情况下,该兔抗血清可以用于分析植物中SUA41蛋白的功能。

It was to prepare rabbit antiserum against SUA41 for further study of the function of SUA41 protein and the relationship between SUA41 protein and plant development.We cloned a specific fragment（1 021-2 040 bp）from Arabidopsis SUA41 gene,constructed a fusion expression vector pDEST17-SUA41,and transferred it into E.coli BL21（DE3）Star.Cells were indued by IPTG to express fusion polypeptide containing a 6×His-tag at the N-terminal of SUA41 fragment in inclusion bodies.Incluson bodies were dissolved in 8 mol/L urea buffer,cleared progressively by water,and purified through Ni-NTA affinity chromatography,and confirmed by Western blot.The fusion protein was further purified by SDS-PAGE.Polyacrylamide gel containing fusion proteins 6×His-SUA41 was used to immunize rabbits to develop a polyclonal antiserum.The rabbit antiserum was evaluated by western blot and showed its effectiveness and specility.Results showed a prokaryotic expression vector pDEST17-SUA41 was successfully constructed.The fusion protein 6×His-SUA41 was efficiently expressed in inclusion bodies in BL21 Star,cleared with water,and purified by Ni-NTA and SDS-PAGE.The rabbit was immunized with this purified fusion proteins and prepared a polyclonal antiserum,which could react effectively with SUA41 from E.coli and Arabidopsis tissues.Water could be used to purify inclusion body proteins dissolved in urea buffer,which could promote affinity purification of proteins with Ni-NTA Resin.The prepared SUA41 antiserum had high affinity to react SUA41 protein,and it may be used to analyse the function of SUA41 protein in the presence of suitable controls.