人肝癌相关抗原Cdc25C特异性多肽抗体的制备及其应用

Preparation and application of human HCC-associated antigen Cdc25C specific peptide antibody

目的制备抗人肝癌相关抗原Cdc25C特异性多肽抗体,为进一步研究Cdc25C的功能奠定基础。方法通过生物信息学软件对人Cdc25C蛋白的抗原属性进行分析,综合考虑多肽的纯度、氨基酸组成、长度、亲水性和二级结构等,得到若干个同源性低、抗原性较高的候选多肽。多肽合成后与KLH佐剂偶联,免疫新西兰大白兔,制备相应的多克隆抗体。经ELISA和Western blotting方法鉴定其活性,并以免疫组化法检测多肽抗体的效果。结果多肽抗体的效价较高,特异性强。以多肽抗体对重组表达的Cdc25C融合蛋白进行Western blotting鉴定,于73k D附近有明显反应条带。以全蛋白的单克隆抗体和多肽抗体检测同一肝细胞癌癌旁组织,购买的Cdc25C单克隆抗体与制备的Cdc25C多肽多克隆抗体平均光密度分别为0.538±0.192、0.410±0.122,两者比较差异无统计学意义(t=0.109,P>0.05)。结论成功制备抗人肝癌相关抗原Cdc25C特异性多肽抗体;筛选获得的候选多肽具有Cdc25C全蛋白一样的抗原性,制备的多肽抗体具有和针对Cdc25C全蛋白抗体一样的效果。

Objective To prepare polyclonal antibody against human hepatocellular carcinoma（ HCC）-associated antigen Cdc25 C specific polypeptide,and to lay a foundation for further study of the function of Cdc25 C. Methods The properties of Cdc25 C protein antigen were analyzed by bioinformatics software. Several candidate partial peptides of low homology and high antigenicity were designed based on the purity,amino acid composition,length,hydrophilic and secondary structure. Three partial peptides of Cdc25 C were synthesized. The synthesized peptide was then used to immunize after coupling with KLH. The activity of anti-Cdc25 C antibody was analyzed by ELISA and Western blotting,and its effect was detected by immunohistochemistry. Results The polypeptide antibody had high titer and specificity. The antibody identified against recombinant Cdc25 C fusion protein by Western blotting,and the reaction bands appeared at 73 k D. We used the whole protein monoclonal antibody and peptide antibody to detect the same HCC cancer adjacent tissue,and its average optical density was 0. 538 ± 0. 192 and 0. 410 ± 0. 122,respectively,no significant difference was found between them（ t =0. 109,P〈0. 05）. Conclusion Polyclonal antibody against human HCC-associated antigen Cdc25 C specific polypeptide is successfully prepared. The candidate peptides obtained from the screening have the same antigenicity as Cdc25 C whole protein,and the prepared polypeptide antibody has the same effect as the antibody against the Cdc25 C whole protein.