人肿瘤转移相关基因mag-2的多克隆抗体的制备和鉴定

Polyclonal antibody specific to human tumor metastasis-associated mag-2 gene: preparation and identification

目的:制备和鉴定针对人肿瘤转移正相关基因mag-2蛋白产物的特异性多克隆抗体。方法:采用生物软件分析mag-2蛋白序列特性,预测特异性抗原表位,人工合成17肽抗原表位片段,将之与钥孔碱血蓝蛋白(KLH)耦联,采用背部皮下及皮内多点注射法免疫日本大耳白兔,收集分离血清,间接酶联免疫吸附试验(ELISA)测定其效价,Western印迹试验鉴定其特异性及其在多种肿瘤细胞中的表达。结果:成功免疫日本大耳白兔,ELISA结果表明得到的抗血清与免疫多肽有良好的结合,获得了高效价的多克隆抗体;Western印迹试验结果显示抗血清检测到的条带大小约为55×103,与mag-2理论蛋白产物相对分子质量相符;发现mag-2在多种肿瘤细胞中均有表达。结论:成功制备了mag-2特异性多克隆抗体,为深入研究mag-2基因在肿瘤转移中的作用提供了一个有用的工具。

Objective.To prepare and identify a polyclonal antibody specific to human tumor metastasis-associated mag- 2. Methods：Based on the data of antigen anticipating of mag-2 produced by a hio-software, a mag-2-specific peptide of 17 amino acids was synthesized and conjugated with key hole limpet hemocyanin （KLH） to immunize Japanese rabbit subcutaneously and intrademlally on its back. The rabbit serum was collected. Its titer was determined by indirect enzyme linked immunosorbent assay （ ELISA）, Western blotting assay was performed to test the serum＇s specificity and the expression of mag-2 in several tumor cell lines. Results： The Japanese rabbit was successfully immunized. The ELISA revealed that the serum obtained could well combine with the immunogen peptide and that the polyclonal antibody＇s titer was high. A band was detected by the polyclonal antibody in Western blotting assay, and its molecular mass was 55 x 103 , which was the very molecular mass of mag-2 gene＇s theoretically ceded protein. The mag-2 protein was expressed in several tumor cell lines. Conclusion：A polyclonal antibody specific to human tumor metastasis-associated mag-2 was successfully prepared, which would provide a useful tool for the further study of the functions of mag-2 in tumor metastasis process.