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目的制备本课题组报告的肝癌相关抗原Kinectin的多克隆抗体,并鉴定其免疫学特性。方法以本课题组前期构建的人MBP-Kinectin融合片段重组质粒为基础,原核表达和亲和层析大量纯化MBP-Kinectin融合蛋白,并免疫新西兰兔制备多克隆抗体;用间接ELISA法测定抗体效价,用Western blot鉴定抗体的特异性。同时用Kinectin多克隆抗体检测Kinectin在正常成人肝细胞HL-7702和人肝癌细胞株SMMC-7721内的表达。结果通过免疫新西兰兔获得抗Kinectin抗体,ELISA测定纯化的Kinectin抗体效价最高为1∶12 800,Western blot结果显示Kinectin抗体具有较好的特异性。免疫细胞化学和western blot检测发现Kinectin蛋白在肝癌细胞株内的表达显著高于正常成人肝细胞株。结论制备的抗Kinectin抗体具有较高的效价和特异性,能满足Western blot和免疫细胞化学检测Kinectin蛋白的要求,为进一步深入研究Kinectin在肝癌发生发展中的作用奠定基础。
Objective To prepare the polyclonal antibody of Kinectin which is reported by our group and identify its immunological characteristics. Methods The MBP-Kinectin fusion fragment was constructed based on the prokaryotic expression vector of human MBP-Kinectin fusion protein. The purified MBP-Kinectin fusion protein was purified by prokaryotic expression and affinity chromatography and immunized New Zealand rabbit to prepare polyclonal antibody. The antibody titer , The specificity of the antibody was identified by Western blot. At the same time, the Kinectin polyclonal antibody was used to detect the expression of Kinectin in normal adult hepatocytes HL-7702 and human hepatoma SMMC-7721 cells. Results The anti-Kinectin antibody was obtained by immunizing New Zealand rabbits. The highest titer of the purified Kinectin antibody was 1:12 800 by ELISA. Western blot results showed that the Kinectin antibody had good specificity. Immunocytochemistry and western blot showed that the expression of Kinectin in hepatocellular carcinoma cells was significantly higher than that in normal adult hepatocytes. Conclusion The prepared anti-Kinectin antibody has high titer and specificity, which can meet the requirement of Western blot and immunocytochemistry detection of Kinectin protein, which lays the foundation for further study on the role of Kinectin in the occurrence and development of hepatocellular carcinoma.