抗CEA单链抗体与人hTNF-α融合基因的构建、表达和纯化

采用基因重组技术将已获得的抗癌胚抗原（ＣＥＡ） 单链抗体基因（ＳｃＦｖ） 与人肿瘤坏死因子α（ｈＴＮＦ α） 基因拼接成抗ＣＥＡＳｃＦｖ ｈＴＮＦ α（免疫毒素） 融合基因， 并克隆进大肠杆菌分泌性表达载体ｐＥＴ ２２ｂ （ ＋ ） 中， 在大肠杆菌中表达了６ ×Ｈｉｓ免疫毒素融合蛋白， 其６ ×Ｈｉｓ 位于ＳｃＦｖ 的Ｎ 端， 在胞内以可溶性存在， 其表达量占菌体总蛋白的６％ ， ＳＤＳ ＰＡＧＥ 和Ｗｅｓｔｅｒｎ ｂｌｏｔ 显示表达产物分子量为４３ｋＤ， 与其基因编码蛋白的理论推算值相符。经Ｎｉ２＋ ＩＤＡＳｅｐｈａｒｏｓｅ６Ｂ亲和柱一步纯化的６×Ｈｉｓ 免疫毒素纯度可达９０％ 以上， 得率为０－２ｍｇ／１００ｍｌ。表达产物除了具有与ＣＥＡ 结合的活性， 也具有对Ｌ９２９细胞杀伤的功能。

Construction,Expression and Purification of the Fusion Gene of Anti CEA Single Chain Antibody and Human Tumor Necrosis Factor

By using genetic recombination techniques,the gene encoding single chain antibody(ScFv)to CEA and human necrosis factor α(hTNF α) were combined to form anti CEA ScFv hTNF α (immunotoxin) fusion gene The construct was expressed in E coli with high level as soluble protein,accounting for 6% of the total bacterial proteins This gene product,characterized by SDS PAGE and Western blot appeared to be a protein with molecular mass of 43 kD The purity of 6×His immunotoxin purified by affinity chromatography through Ni 2+ IDA Sepharose 6B reached up to more than 90% Besides its binding activity with CEA,the expressed product also exhibited the activity to kill the L 929 cell in vitro