抗CMTM4多克隆抗体的制备、纯化和鉴定  

Preparation,purification and characterization of the polyclonal antibody against human CMTM4

作　　者：李婷[1] 郭晓欢[1] 王彧[1] Markus Plate 邵陆宁[1] 宋泉声[1] 马大龙[1] 韩文玲[1] 

机构地区：[1]北京大学人类疾病基因研究中心北京大学基础医学院免疫学系,北京100083

出　　处：《细胞与分子免疫学杂志》2008年第1期41-44,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：高等学校全国优秀博士论文作者专项资金资助(200257);国家自然科学基金资助项目(30571691);国家高技术研究发展计划(863)资助项目(2006AA02A305)

摘　　要：目的:制备抗CMTM4的多肽抗体并鉴定其性能。方法:通过TMHMM、DNAStar等软件对CMTM4的3种剪接体,CMTM4-v1,v2和v3的蛋白序列进行分析,选取了4段序列合成多肽并与钥孔戚血蓝素(KLH)偶联。其中第1、2、3条多肽为3种剪接体共有,混合后免疫家兔,制备Ab1;而第4条多肽为CMTM4-v1所特有,单独免疫家兔,制备Ab2。ELISA法检测抗体效价。免疫亲和层析法纯化后,进行West-ern blot和免疫组织化学检测,鉴定这2种抗体的特异性与适用范围。结果:得到兔抗人CMTM4多肽抗体Ab1和Ab2,效价分别达1∶105和1∶106。纯化后的Ab1用于Western blot可特异识别超表达的CMTM4-v1和v2,并可用于免疫组织化学实验;而Ab2仅可用于Western blot,特异性识别超表达的CMTM4-v1。Ab1还可特异识别小鼠内源性Cmtm4。结论:用多肽免疫家兔制备的2种抗CMTM4抗体不仅具有较高的效价以及特异性,并且Ab1还可以特异识别小鼠Cmtm4,为CMTM4的功能研究提供了有力的支持。AIM： To prepare, purify and characterize the polyclonal antibodies against CMTM4. METHODS： Four polypeptides named peptide 1, 2, 3, and 4 were synthesized based on the bioinformatics analysis of the three isoforms of CMTM4, CMTM4-v1, -v2, and-v3, and coupled with keyhole limpet hemocyanin （KLH） for immunization. Among them, peptide 1, 2, and 3, common for CMTM4-v1, v2, and v3, were mixed for immunization to prepare the antibody which can recognize all of the three isoforms （ Abl ） ; while peptide 4, which is specific for CMTM4-v1, was injected separately into New Zealand rabbits to prepare the antibody specifically targeting CMTM4-v1 （Ab2）. ELISA assay was used to detect the titers of the antibodies. After purification by immunoaffinify chromatography, Ab1 and Ab2 were identified by Western blot and immunohistochemistry assays. RESULTS： The titers of Ab1 and Ab2 were 1： 10^5 and 1：106, respectively. Western blot confirmed their high specificity. Ab1 could also be used for immunohistochemistry analysis, but Ab2 could not. Immunohistochemistry analysis with Abl demonstrated the high expression level of CMTM4 in human testis, which was consistent with the previous resuit of Northem blot. Moreover, Western blot and immunohistochemistry analysis verified that Ab1 can also recognize mouse Cmtm4. CONCLUSION： Two specific antibodies against human CMTM4 were obtained, which will be helpful for further study.

关 键 词：CMTM4 多肽抗体 免疫亲和纯化 

分 类 号：Q78[生物学—分子生物学] R392-33[医药卫生—免疫学]