1株抗鸡CD8α链单克隆抗体的鉴定  

Identification of A Monoclonal Antibody against Chicken CD8α Chain

作　　者：朱静文[1] 余为一[1] 

机构地区：[1]安徽农业大学安徽省人兽共患病重点实验室,安徽合肥230036

出　　处：《安徽农业科学》2011年第20期12221-12222,共2页Journal of Anhui Agricultural Sciences

基　　金：国家自然基金项目(30671537)资助

摘　　要：[目的]获得研究鸡CD8分子的单克隆抗体。[方法]用自行设计的引物PCR扩增鸡CD8分子α链部分基因片段,构建2个原核重组质粒,该片段经原核表达和纯化后免疫BALb/c小鼠,最后将免疫小鼠脾细胞与骨髓瘤细胞融合,细胞上清液用ELISA检测。[结果]PCR扩增获得大小约为510 bp的基因片段。构建的pET-32a-CD8α导入大肠杆菌,诱导表达获得了大小为39 kD的融合蛋白H is-CD8α。融合细胞经亚克隆,其上清液用ELISA检测和筛选,获得1株稳定传代和分泌抗CD8α的抗体的杂交瘤细胞株。该株细胞被命名为C11。上清液的ELISA抗体效价达1:600。[结论]该研究以原核表达的CD8α为抗原制备了1株单克隆抗体。[Objective] The aim of the study was to obtain monoclonal antibody against chicken CD8 molecule.[Method] A fragment of chicken CD8α gene was amplified by PCR with a pair of designed primers.Then two recombinant plasmids containing the amplified fragment were constructed.After prokaryotic expression and purification,the obtained recombinant protein was used to immunize BALb/c mice.Finally,the spleen cells were fused with myeloma cells（SP2/0）,and antibody titer of culture supernatant was detected by ELISA.[Result] A 510-bp gene fragment was amplified by PCR.The recombinant plasmid pET-32a-CD8α was transformed into E.coli,and 39 kDa His-CD8α fusion protein was induced to expression.After subcloning,the culture supernatant was detected by ELISA.A hybridoma cell strain,which could stably excrete antibody against CD8α protein,was obtained and named C11.The ELISA titer of cell supernatant was higher than 1：640.[Conclusion] A hybridoma cell strain has been established using CD8α expressed in prokaryotic system as immunogen.

关 键 词：鸡 CD8Α 原核表达 单克隆抗体 

分 类 号：S858.31[农业科学—临床兽医学]