基于表位多肽的抗TRIM22抗体的制备及其初步应用

 目的 制备抗TRIM22多肽抗体及鉴定其性能。 方法 将TRIM22 N端15个氨基酸的多肽（MDFSVKVDIEKEVTC）与钥孔嘁血蓝蛋白（KLH）偶联，免疫新西兰白兔制备抗血清；将TRIM22多肽与Affi-Gel 10偶联制备免疫亲和层析柱, 兔抗血清经亲和层析纯化后得到TRIM22多肽特异性抗体；采用ELISA测定抗体效价、Western blot来鉴定抗体特异性，并利用该抗体通过间接免疫荧光实验来检测TRIM22蛋白的细胞内定位。 结果 获得了抗TRIM22特异性抗体。Western blot结果显示该抗体能特异识别真核及原核表达的TRIM22蛋白。利用该抗体进行的间接免疫荧光实验发现TRIM22蛋白主要定位在HepG2细胞核中。 结论 该抗体的制备为TRIM22分子的功能研究提供了有力的工具。

The preparation and preliminary application of the epitope peptide-based antibody against TRIM22

Objective To prepare,purify and characterize the polyclonal anti-peptide antibody against TRIM22. Methods A synthetic peptide(MASGILVNVKEEVTC) corresponding to the amino-terminal amino acid sequence of TRIM22 was conjugated to keyhole limpet hemocyanin carrier protein and used to immunize New Zealand white rabbits.The specific antibody was purified from immune sera using columns of TRIM22 peptide coupled to Affi-gel 10.ELISA was used to determine the antibody titer,and Western blot was used to determine the specificity of the antibody.The intracellular localization of TRIM22 was investigated by indirect immunofluorescence staining with the anti-TRIM22 antibody. Results A peptide polyclonal antibody against TRIM22 was obtained.Western blot analysis showed that this antibody could specifically recognize TRIM22 proteins expressed by both prokaryotic and eukaryotic systems,and indirect immunofluorescence staining showed that TRIM22 was mainly localized to the nuclei of HepG2 cells. Conclusions The preparation of TRIM22 antibody provided an useful tool for the further functional study of TRIM22.