树鼩CD4蛋白多克隆抗体的制备及检测  

Preparation and Determination of CD4 Polyclonal Antibodies of Tree Shrew

作　　者：卢孔杰[1] 徐婧雯[1] 张雪梅[1] 吴忠香[1] 任芳芳[1] 马娜[1] 巩蔚[1] 严丽蔚 朱文兵[1] 董少忠[1] LU Kong-jie XU Jing-wen ZHANG Xue-mei WU Zhong-xiang REN Fang-fang MA Na GONG Wei YAN Li-wei ZHU Wen-bing DONG Shao-zhong(Institute of Medical Biology, Chinese Academy of Medical Science and Peking Union Medical College, Yunnan Key Laboratory of Vaccine Research ＆ Development on Severe Infectious Disease, Kunming 650118, China)

机构地区：[1]中国医学科学院/北京协和医学院医学生物学研究所,云南省重大传染病疫苗研发重点实验室,昆明650118

出　　处：《实验动物与比较医学》2016年第4期243-249,共7页Laboratory Animal and Comparative Medicine

基　　金：国家科技支撑计划项目（2014BAI01B01）

摘　　要：目的 原核表达、纯化树鼩CD4蛋白并制备多克隆抗体,检测树鼩体内的CD4蛋白.方法 克隆树鼩CD4基因并连接到表达载体,构建重组质粒pET30a（＋）-CD4和pGEX-5X-1-CD4,表达并纯化重组蛋白His-CD4和GST-CD4,用不同佐剂[弗氏佐剂、MF59和A1（OH）3]制备多抗,用Western blot法检测血清抗体特异性.结果 蛋白表达产物His-CD4和GST-CD4的相对分子质量分别约为53.5×103和70.0×103,纯化后蛋白浓度分别为600 μg/mL和1 mg/mL;三组佐剂均能诱导产生抗体,抗体几何平均滴度（Geometric Mean Titer,GMT）的比较结果为：弗氏佐剂组（GMT：97 420）＞Al（OH）3佐剂组（GMT：67 202）＞MF59佐剂组（GMT：55 128）;其中弗氏佐剂组显著高于原蛋白组（P＜0.001）;Al（OH）3组显著高于原蛋白组（P＜0.01）;而MF59组显著高于原蛋白组（P＜0.05）.结论 制备了特异性良好的小鼠抗血清,能够特异性地结合树鼩的CD4蛋白.为下一步本实验室制备CD4单克隆抗体、CD4蛋白分子的功能研究以及树鼩的病毒感染模型等免疫相关实验的进行提供了基础.Objective To express tree shrew （Tupaia belangeri） CD4 protein in prokaryotic cells, purify the expressed product and prepare its polyclonal antibodies,and determinate CD4 protein in vivo. Methods CD4 gene was cloned and inserted into express vectors. And then recombinant plasmids pET30a （＋）-CD4 and pGEX-5X-1-CD4 were constructed. The expressed recombinant proteins His- CD4 and GST-CD4 were expressed and purified. The polyclonal antibodies of CD4 protein were pre- pared with three different adjuvants including A1 （OH）a, MF59 and Freund＇s. The serum antibody speci- ficity was determined by Western blot. Results The expressed recombinant proteins His-CD4 and GST-CD4, with relative molecular masses of about 53.5 ~ 103 and 70.0 ~ 103 respectively. And the concentrations of proteins His-CD4 and GST-CD4 were 600 ktg/ml and lmg/ml respectively. Three groups of adjuvants all can induce antibodies. The comparison results of Geometric Mean Titer （GMT） is that the Freund＇s adjuvant group （GMT： 97 420） showed the highest geometric mean of antiserum, followed by A1 （OH）3 adjuvant group （GMT： 67 202） and MF59 adjuvant group （GMT： 55128）. The Freund＇s adjuvant group was significantly higher than the original protein group （P〈0.001）. The AI（OH）3 adjuvant group was significantly higher than the original protein group （P〈0.01）. The MF59 adjuvant group was higher than the original protein group （P〈0.05）. Conclusions Mouse antiserum with high specificity was prepared,and it could bind to CD4 protein of tree shrew in vivo, which laid a foundation of further study on the preparation of monoclonal antibody against CD4 and the research of CD4 function and immune related experiments of tree shrews as viral infection models.

关 键 词：树鼩 CD4蛋白 表达 纯化 多克隆抗体 

分 类 号：R392-33[医药卫生—免疫学]