肝癌相关抗原Kinectin的多克隆抗体制备、鉴定及初步应用

Preparation and identification of a polyclonal antibody to hepatocellular carcinoma associated antigen Kinectin

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。

This paper aimed to prepare the polyclonal antibodies against hepatocellular carcinoma associated antigen Kinectin,and identify the immune characteristic of anti-kinectin polyclonal antibody.We purified the MBP fusion protein MBP-kinectin from prokaryotic expression system constructed in our previous work.Then the polyclonal antibodies to MBP-kinectin were prepared by immunizing New Zealand rabbits with the purified fusion protein MBP-kinectin.The titer and specificity of the antibody were determined by ELISA and Western blot respectively.ELISA detection results showed that the titer of anti-kinectin polyclonal antibody was about 1∶128 000,while Western blot results showed that the polyclonal antibody to kinectin could bind to MBP-kinectin fusion protein specifically.Furthermore,the expression.of kinectin in hepatic cell HL-7702 and hepatocellular carcinoma cell SMMC-7721 were assayed both by immunohistochemistry technique and Western blot.We found that the protein expression of kinectin,which mainly present in the cytoplasmic area,was higher in hepatocellular carcinoma cell SMMC-7721 than in normal hepatic cell HL-7702.All results showed that polyclonal antibodies to kinectin are successfully preparation and can be applied to Western blot and immunohistochemistry.This work also lays a foundation for further exploring of the role of kinectin in the development of hepatocellular carcinoma.