VSTM1多克隆抗体的制备和鉴定  

Preparation and identification of the polyclonal antibody against human VSTM1

作　　者：李婷[1] 郭晓欢[1] 王平章[1] 宋泉声[1] 马大龙[1] 韩文玲[1] 

机构地区：[1]北京大学基础医学院免疫学系,卫生部医学免疫学重点实验室,北京大学人类疾病基因研究中心,北京100191

出　　处：《细胞与分子免疫学杂志》2012年第12期1291-1294,1299,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：高等学校博士学科点专项科研基金资助课题(20110001110016)

摘　　要：目的:制备VSTM抗体,鉴定其性能。方法:VSTM1有两种主要的剪接体VSTM-v1和VSTM-v2。前者为膜分子,后者为分泌蛋白,与前者相比后者仅缺少穿膜区。构建VSTM-v2的原核表达载体,诱导表达并纯化带两种不同标签的VSTM-v2重组蛋白,Trx-His-S-VSTM1-v2和GST-VSTM1-v2。用前者免疫家兔制备VSTM1抗血清,ELISA检测效价;后者偶联Sepharose 4B制备免疫亲和层析柱对抗血清进行纯化。通过Western blot法和免疫荧光细胞化学技术,鉴定该抗体的特异性与适用范围。结果:免疫得到兔抗人VSTM1抗血清,效价达到1∶106。纯化后的抗体用于Western blot可特异识别过表达和内源性的VSTM1,并可用于免疫荧光细胞化学技术检测细胞膜表面过表达的VSTM-v1。结论:成功制备了兔抗人VSTM1抗体,具有较高的效价及特异性,可以识别内源性VSTM1并可用于免疫荧光细胞化学技术。AIM： To prepare and characterize the polydonal antibody against human VSTMI. METHODS： VSTM1 has two main isoforms, VSTMI-v1, a type I transmembrane protein, and VSTMI-v2, a classical secretory protein, lacking only the transmembrane domain compared with VSTMI-v1. Two recombinant prokaryotic proteins of VSTMI-v2, Trx-His-S-VSTMI-v2 and GST-VSTMI-v2, were constructed, expressed, purified, and then used for immunization of New Zealand rabbits to prepare anti-VSTM1 antibody and coupling with CNBr-activated Sepharose 4B to purify the antibody by immunoaffinity chromatography, respectively. ELISA was performed to detect the titers of the antiserums. After purification, the antibody was identified by Western blotting and immunofluorescence cytochemistry. RESULTS： The titers of the antiserums from the two immunized rabbits were both 1：106. Western blotting confirmed that the purified antibody could recognize both the overexpressed and endogenous VSTMI specifically. Immunofluorescence cytochemistry verified that the antibody could also recognize the overexpressed VSTMI-vl on the surface of HEK293T cells. CONCLUSION： The rabbit antibody against human VSTM1 of a high titer has been obtained, which can be used for recognizing endogenous VSTM1 in immunofluorescence cytochemistry.

关 键 词：免疫基因组学 细胞因子 白细胞分化抗原 VSTM1 多克隆抗体 

分 类 号：R392.11[医药卫生—免疫学] R392-33[医药卫生—基础医学]