肿瘤睾丸抗原OY-TES-1氨基端截短蛋白及其多克隆抗体的制备

Preparation of cancer-testis antigen OY-TES-1 truncated N-terminal half protein and its polyclonal antibody

目的获得原核表达的OY-TES-1氨基端截短蛋白(OY-TES-1-N),并制备其多克隆抗体。方法扩增编码OY-TES-1-N 268个氨基酸(A6-R273)的cDNA序列;将PCR产物插入原核表达载体pMAL-C2,构建重组质粒,并转化DH5α菌;通过蓝白斑筛选、DNA测序筛出阳性菌;在优化条件下用IPTG对阳性菌进行诱导表达MBP/OY-TES-1-N融合蛋白;上Amyloseresin亲和层析柱纯化,行Western blot鉴定。以融合蛋白作为抗原免疫新西兰白兔制备抗血清,经活化琼脂糖微球纯化后,采用ELISA法及Western blot法分别检测抗血清的效价和多克隆抗体的特异性。结果成功诱导表达出MBP/OY-TES-1-N融合蛋白。以该蛋白免疫新西兰兔制备抗血清,抗体效价为1∶1 000,Western blot检测证实该抗体能与目的蛋白发生特异性结合。结论成功地表达并纯化了MBP/OY-TES-1-N融合蛋白,并制备了特异性多克隆抗体。

This study is aimed to obtain the prokaryotic expression of OY-TES-1 truncated N-terminal half protein and prepare its polyclonal antibody,which will lay a foundation for the research of its function.The fragments of OY-TES-1-N（containing 268 amino acids of N-terminal half） gene were amplified by Polymerase Chain Reaction（PCR）;PCR product was inserted into the expression vector pMAL-C2 to construct the recombinant plasmid,and then it was transformed into E.coli DH5α;the positive bacteria was selected by blue-white selected test,and DNA sequencing;the expression of the MBP/OY-TES-1-N fusion protein was induced by IPTG under the optimum conditions in positive bacteria;purification of fusion protein of MBP/OY-TES-1-N was performed by the amylose resin column,then tested by the Western blot.The MBP/OY-TES-1-N fusion protein was successfully expressed.New Zealand rabbits were immunized with the fusion protein to prepare the antisera.The titers of prepared antibody were 1∶ 1 000.Western blot revealed specific reactions of the polyclonal antibody with target protein.In this study,the fusion protein MBP/OY-TES-1-N was successfully expressed and purified,and its specific polyclonal antibody was also prepared.