拟南芥蛋白磷酸酶TOPP4的原核表达和多克隆抗体纯化
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国家自然科学基金(31070247)


Prokaryotic Expression and Polyclonal Antibodies Purification of Protein Phosphatase TOPP4 in Arabidopsis
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    摘要:

    以拟南芥野生型Col-0为材料,对其I型蛋白磷酸酶(TOPP)家族进行序列分析,对家族成员之一的TOPP4进行原核表达及多克隆抗体的制备和纯化。结果显示:(1)该研究构建出原核表达载体pEGM-4T-3-TOPP4和pET-28a-GFP-N150并转入大肠杆菌BL21(DE3)中。(2)经IPTG诱导,表达出分子量约为62 kD的GST-TOPP4和分子量约为34 kD的His-GFP-N150可溶性重组蛋白。(3)纯化的重组蛋白GST-TOPP4作为抗原免疫新西兰兔后,获得了效价大于1∶400 000的多克隆抗体血清。(4)抗体血清经连接了His-GFP-N150蛋白的溴化氢活化的树脂纯化,得到特异性较高的anti-TOPP4多克隆抗体。研究认为,该研究纯化出了特异的TOPP4蛋白多克隆抗体。

    Abstract:

    Arabidopsis wild-type Col-0 was chosen as material to analyze the amino acid sequence comparison of type one protein phosphatase (TOPP) family.The TOPP4,a member of TOPP family,was prokaryotic expression to prepare and purify polyclonal antibodies.The results indicated as follow:(1)The prokaryotic expression plasmids of pEGM-4T-3-TOPP4 and pET-28a-GFP-N150 were constructed and transformed into Escherichia coli BL21 (DE3) strain.(2)The soluble GST-TOPP4 protein of 62 kD and His-GFP-N150 protein of 34 kD were expression by IPTG induction.(3)The recombinant protein GST-TOPP4 was purified,and then used as the antigen to immune the rabbits.The prepared polyclonal antibodies serum,whose titer was over 1∶400 000,was achieved.(4)The serum was purified by CNBr-activated Sepharose,which was linked with His-GFP-N150 protein,to obtain anti-TOPP4 polyclonal antibodies with great specificity.Our studies suggested that the specific TOPP4 polyclonal antibodies have been purified.

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王 玮,管利萍,张 静,等.拟南芥蛋白磷酸酶TOPP4的原核表达和多克隆抗体纯化[J].西北植物学报,2014,34(10):1937-1943

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  • 在线发布日期: 2014-10-31
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