可溶性肿瘤坏死因子-α的高效表达及其抗体的制备

High Expression of Soluble Tumor Necrosis Factor-α (TNF-α) in E. coli and Preparation of Specific Antibody against TNF-α

为了制备 TNF-α特异性抗体 ,用 PCR的方法克隆出 TNF-α片段的编码基因并将之插入 PET- 2 8a (+)原核表达载体 ,成功地在大肠杆菌中获得 5 0 %～ 6 0 %的高效表达。经 SDS- PAGE电泳 ,切下 TNF-α特异条带 ,直接免疫大白兔 ,制备出 TNF-α特异性抗体 ,抗体的效价可达 1∶ 6 40 0。利用该抗体成功地检测出 m TNF- NIH3T3细胞膜上的跨膜型 TNF-α的表达情况。结果表明 :制备出的抗体具有较高的特异性 ,对

In order to prepare the specific antibody against soluble tumor necrosis factor α (sTNF α), a gene fragment coding sTNF α was cloned by using PCR and inserted into PET 28a(+) expression vector. Recombinant protein was expressed effectively in E.coli and separated by SDS PAGE. By using the TNF α specific band in SDS PAGE agrose as antigen, the rabbits were immunized and the antibody against TNF α was obtained with the titer being 1∶6400. The antibody was used to successfully detect the expression of transmembrane TNF α on the cellular membrane of mTNF NIH3T3. It was suggested that the antibody was high specific and was useful for immunological detection of TNF α.