CD26多克隆抗体的制备与鉴定  

Preparation and characterization of polyclonal antibodies against CD26

作　　者：宋敏[1] 范娅涵[1] 胡川闽[2] 赵树铭[1] 

机构地区：[1]第三军医大学西南医院输血科,重庆400038 [2]第三军医大学医学检验系暨药学院临床生物化学教研室,重庆400038

出　　处：《中国输血杂志》2009年第7期535-538,共4页Chinese Journal of Blood Transfusion

基　　金：重庆市自然科学基金(编号:2007-23);第三军医大学回国人员启动基金(编号:2007XG51);第三军医大学第一附属医院临床创新基金B类(编号:SWH2006B020)资助目

摘　　要：目的针对CD26酶催化结构域制备多克隆抗体。方法应用RT-PCR技术以人白细胞mRNA为模板,扩增获取编码CD26催化结构域的基因序列,克隆入原核表达载体PET32a后,转化BL21感受态细菌,经IPTG诱导表达得到his-CD26融合蛋白;亲和层析柱纯化并经Western-blot鉴定后,用此重组蛋白免疫2只新西兰纯种大白兔,获取免疫血清共180 ml,经蛋白A柱纯化及抗原抗体亲和纯化后,采用间接ELISA法检测抗体效价,Western-blot及免疫细胞化学染色进行抗体效价及特异性鉴定。结果构建出PET32a/CD26原核表达质粒,并在大肠杆菌BL21中获得高效表达,经his亲合层析纯化后蛋白量达2.3 mg/ml;制备的多抗血清纯化后效价达256 000,经Western-blot证明能特异性的识别CD26重组蛋白,免疫细胞化学染色显示能特异的结合于H9细胞。结论成功制备了能特异性识别CD26的多克隆抗体。Objective To prepare polyclonal antibody against the catalytic center of CD26. Methods The coding gene of CD26 amplified using RT-PCR technology with mRNA template from human leucocyte was inserted into prokaryotie expressive vector PET-32a, and then the recombinant plasmids were transformed into BL21 and induced by IPTG. The fusion protein was purified by Ni^＋ affinity column chromatography, and then was used to immune two rabbits after being con- formed by Western-blot. The 180 ml of the polyclonal antibody serum was then purified by protein A column and antibody affinity column. The titer and specificity were determined by ELISA, Western-blot and Immunocytocbemistry. Results PE332a/CD26 prokaryotic expression plasmids were constructed successfully and highly expressed in BI21. Antigen concentration of the fusion protein was up to 2. 3 mg/ml after being purified by Ni^＋ affinity column chromatography. The polyclonal antibody against CD26 was obtained with titer up to 1： 256000. Western-blot and Immunocytochemistry revealed that the final purified antibody was specific to the recombinant and native CD26. Conclusion The highly specific polyclonal antibody against the catalytic center of CD26 was obtained successfully.

关 键 词：CIY26 原核表达 包涵体纯化 多克隆抗体 制备 鉴定 

分 类 号：R446.62[医药卫生—诊断学] Q513.2[医药卫生—临床医学]