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

作　　者：陈玉升[1] 韦连登[2] 范蓉[3] 罗彬[2] 何少健[2] 林永达[2] 陈芳[2] 谢小薰[2] 肖绍文[1] 

机构地区：[1]广西医科大学第一附属医院神经外科,南宁530021 [2]广西医科大学第一附属医院基础医学院,南宁530021 [3]广西中医学院,南宁530001

出　　处：《免疫学杂志》2011年第3期254-258,共5页Immunological Journal

基　　金：国家自然科学基金(30760055、81060207);广西自然科学基金(桂科自0832144、0728148);广西大型仪器协作网资助项目(666-2008-079、691-2008-104);广西高发疾病研究创新性团队基金(桂教人[2007-59])

摘　　要：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.

关 键 词：OY-TES-1 融合蛋白 克隆 纯化 多克隆抗体 

分 类 号：R737.21[医药卫生—肿瘤]