摘要
旨在制作TGF-β二型受体结合蛋白TRIP-1的抗体,以便推进对该蛋白在TGF-β信号通路以及癌症发生发展中的功能研究。首先克隆出人的TRIP-1基因,构建其原核重组表达载体pET-His-TRIP-1,将其转化至大肠杆菌BL21(DE3)中,诱导获得TRIP-1蛋白,纯化后将其分别免疫新西兰大耳兔和昆明小鼠(KM),免疫4次后取血清,并对抗血清进行效价和亲和力测定。通过Western blot和免疫荧光试验鉴定抗血清特异性。最后,利用proteinG初步纯化抗血清。结果表明,通过重组表达获得抗原蛋白,纯化后免疫新西兰大耳兔和昆明鼠可得到效价较高的抗TRIP-1的多克隆抗体,在稀释倍数较高的情况下仍可以用于Westernblot和免疫荧光试验,且有非常好的特异性。
To produce antibody against the TGF-β type Ⅱ receptor binding protein, TRIP-1, contributing to study TRIP-1 ＇s function in TGF-β signaling pathway and cancer development. Human＇s TRIP-1 gene was cloned into the prokaryotic expression vector of pET-His, and then transformed into E. coli BL21 to induce TRIP-1 protein expression. Purified TRIP-1 protein was used to immunize New Zealand rabbits and Kunming mice （ KM ） . The serum was removed and the titer and affinity was determined after immunization 4 times. The antiserum was highly purified by proteinG. Results indicated that the titer of the anti-serum from murine and rabbit are high enough for Western blot and immunofluorescence assay.
出处
《生物技术通报》
CAS
CSCD
北大核心
2013年第3期139-143,共5页
Biotechnology Bulletin
基金
国家自然科学基金项目(30570960
30900749)
深圳市重点实验室建设与提升计划
关键词
原核表达纯化
TRIP-1蛋白
动物免疫
抗血清
抗体鉴定
Prokaryotic expression and purification
TRIP-1
Animal immunization
Anti-serum
ntibody identification
