Syncytin蛋白的原核表达及其多克隆抗体的制备和鉴定  

Preparation and characterization of polyclonal antibodies against syncytin

作　　者：孙鹥[1,2,3] 庞伟[1] 欧阳东云[1,2] 郑永唐[1] 

机构地区：[1]中国科学院昆明动物研究所中国科学院和云南省动物模型与人类疾病机理重点实验室,650223 [2]中国科学院研究生院,100049 [3]云南省第一人民医院检验科,650011

出　　处：《免疫学杂志》2010年第2期124-127,131,共5页Immunological Journal

基　　金：中国科学院"西部之光"人才计划在职博士生资助项目

摘　　要：目的克隆人syncytin基因并在大肠杆菌中表达,并用表达后的融合蛋白制备syncytin蛋白多克隆抗体。方法 PCR扩增人syncytin基因编码区的DNA片段,将其克隆入原核表达质粒pET30a(+),转化大肠杆菌BL21,诱导产生syncytin-His融合蛋白。采用割胶回收的方法纯化目的蛋白,免疫新西兰白兔,制备多克隆抗体。通过ELISA、Western-Blot和免疫组织化学等方法来检测抗体的灵敏度和特异性。结果成功表达并纯化了syncytin-His融合蛋白,SDS-PAGE分析表明融合蛋白主要以包涵体形式存在;ELISA法测定抗体效价为1:10 000;Western-Blot和免疫组织化学结果显示所制备的抗体能特异性识别syncytin蛋白。结论成功制备和鉴定了人syncytin多克隆抗体,多克隆抗体特异性强和效价高,为下一步研究Syncytin的生物学功能奠定了基础。Syncytiu plays a crucial role in syneytiotrophoblast formation. This study is aimed to generate and identify the rabbit polyclonal antibodies against human syneytin. We amplified the syncytin gene by PCR, and then inserted it into cloning vector. After digestion by BamH I/Not I, the recombinants were cloned into expression vector pET30a（＋） and transferred into E. coli BL21 （DE3）; fusion protein His/syncytin was induced by IPTG, and then analyzed by SDS-PAGE; the protein straps about 42 000 were cut, grinded, and then used to immunize New Zealand rabbits. Sera of the immunized rabbits were collected 10 days after the second enhanced immunization; the antiserum was detected by ELISA, Western blot, and immunohistochemistry staining. In this study, the prokaryotic expression vector pET30a （＋）/syneytin was successfully constructed and fusion protein Syncytin-His was expressed efficiently. The levels of polyclonal antibodies were raised in the immunized rabbits. The syncytin polyclonal antibodies reveal high titer and specificity, which may be applied in syncytin function research.

关 键 词：SYNCYTIN 原核表达 多克隆抗体 

分 类 号：R392.11[医药卫生—免疫学]