抗人KIAAO100蛋白多克隆抗体的制备

摘要

目的:制备抗人KIAA0100蛋白多克隆抗体,为今后研究人KIAA0100蛋白的结构和功能奠定基础.方法:采用生物信息学软件对人KIAA0100蛋白第1 557～2 234位氨基酸序列进行原核密码子优化,将优化后的核苷酸序列经人工合成后克隆入原核表达载体pET-30a中,构建重组质粒;采用限制性酶切分析对重组质粒进行鉴定;将正确的重组质粒转化人大肠杆菌BL21(DE3)细胞,以表达羧基末端携带6×组氨酸标签的重组蛋白;使用重组蛋白作为免疫原免疫新西兰大白兔制备抗人KIAA0100蛋白多克隆抗体;使用蛋白质印迹分析检测该多克隆抗体对人KIAA0100蛋白的识别能力.结果:限制性酶切分析显示:重组质粒被成功构建;SDS-PAGE分析结果显示:主要以包涵体形式存在的重组蛋白被成功表达;蛋白质印迹分析证实:该多克隆抗体能够高效地识别人KIAA0100蛋白.结论:成功制备了抗人KIAA0100蛋白多克隆抗体,以上研究结果为我们将来研究人KIAA0100蛋白的结构和功能奠定了基础.%Objective:To preparation of polyclonal antibody(pAb) against human KIAA0100 protein and lay the foundation for the study of the structure and function of human KIAA0100 protein.Methods:Bioinformatics software was used to optimize Escherichia coli (E.coli) rare codons into E.coli normal codons of human KIAA0100 protein segment(1 557 ～ 2 234),and then the synthetic optimal nucleotide sequence were cloned into prokaryotic expression vector pET-30a to construct the recombinant plasmid.After being identified by restriction enzyme digestion analysis,the recombinant plasmid were transformed into E.coli BL21 (DE3) cells,and then the recombinant human KIAA0100 protein segment(1 557 ～2 234) were expressed as a fusion protein with C-terminal 6 × histidine (His)-tag.The New Zealand white rabbits were next immunized by the recombinant protein.The pAb was finally identificated by Western blot analysis.Results:Restriction enzyme digestion analysis showed that the recombinant plasmid was successfully constructed.SDS-PAGE analysis attested that the recombinant protein was successfully expressed as inclusion body.Western blot analysis proved that the pAb could sensitively identified human KIAA0100 protein.Conclusion:In this study,the pAb against human KIAA0100 protein is successfully prepared,which will be of great help on the study of the structure and function of human KIAA0100 protein.