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p ET-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入大肠杆菌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（ p Ab） 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 p ET-30 a 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 p Ab 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 p Ab could sensitively identified human KIAA0100 protein. Conclusion： In this study,the p Ab 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.
出处
《现代肿瘤医学》
CAS
2017年第5期681-686,共6页
Journal of Modern Oncology
基金
国家自然科学基金资助项目(编号:0817 18110232)
