TNF受体(P55)胞外区基因的克隆及其在大肠杆菌中的表达

Cloning and Expression of Extra-cellular Domains of Tumor Necrosis Factor Receptor P55 in E.coli

TNF与多种疾病密切相关。为了获得大量具有生物学活性的可溶性TNF受体用以拮抗TNF的毒性作用 ,在原核表达系统中表达了TNFR(P5 5 )的胞外区与TrxA的融合蛋白。将TNFR(P5 5 )胞外区去信号肽的前三个结构域基因克隆入融合蛋白表达载体 pET 32a ,在大肠杆菌BL2 1(DE3)中高效表达了TrxA TNFR融合蛋白。表达产物以包涵体形式存在 ,经过变性和复性 ,并经镍金属鳌和柱亲和层析纯化 ,得到了纯度较高的可溶性受体蛋白的初纯品。免疫学实验及L92 9细胞体外实验均表明 :该蛋白具有TNFR(P5 5 )特异的抗原性、与TNF结合的活性以及良好的抑制TNF的生物学活性。

TNF (tumor necrosis factor)is one of the most important cytokines In addition to its outstanding anti tumor function and other very important functions,it is a key factor in the pathophysiology of many diseases Soluble TNF receptor can inhibit TNF’s activity by binding to TNF but fails to transduct the signals into cells We successfully amplified the first three domains in the extra cellular region of TNF receptor P55 by PCR and cloned into prokaryotic expression vector pET 32a The expressed protein was fused with TrxA and produced in the form of inclusion body After denaturation and renaturation,we got biologically active receptor protein,which has good antigenicity and binding ability to TNF It can also inhibit the cytotoxic activity of TNF on L929 cell culture