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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。
OBJECTIVE: To prepare anti-CMTM4 polypeptide antibodies and identify their properties. Methods: The protein sequences of three CMTM4 spliceosomes, CMTM4-v1, v2 and v3 were analyzed by software TMHMM and DNAStar. The 4-mer peptide was synthesized and coupled with keyhole limpet hemocyanin (KLH). The first, second and third polypeptides were shared by three kinds of spliceosomes, and the mixed rabbits were immunized to prepare Ab1. The fourth polypeptide was unique to CMTM4-v1 and was used to immunize rabbits separately to prepare Ab2. Antibody titers were measured by ELISA. Immuno-affinity chromatography purification, West-ern blot and immunohistochemistry detection, identification of the specificity of these two antibodies and the scope of application. RESULTS: Rabbit anti-human CMTM4 polypeptide antibodies Ab1 and Ab2 were obtained at titers of 1:105 and 1:106, respectively. The purified Ab1 was used to detect the over-expression of CMTM4-v1 and v2 by Western blot, and could be used in immunohistochemistry. Ab2 was only used in Western blot to specifically recognize CMTM4-v1 overexpression. Abl also specifically recognizes murine endogenous Cmtm4. CONCLUSION: The two anti-CMTM4 antibodies raised by rabbit immunized with the polypeptide not only have high potency and specificity, but also Ab1 can specifically recognize mouse Cmtm4, which provides strong support for the functional study of CMTM4.