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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（ｈＴＮＦα） 基因拼接成抗ＣＥＡＳｃＦｖｈＴＮＦα（免疫毒素） 融合基因， 并克隆进大肠杆菌分泌性表达载体ｐＥＴ２２ｂ （ ＋ ） 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％ ， ＳＤＳＰＡＧＥ 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。
The anti-CEAScFvhTNFα (immunotoxin) fusion gene was spliced with the anti-carcinoembryonic antigen (CEA) single-chain antibody gene (ScFv) and human tumor necrosis factor α (hTNFα) gene obtained by gene recombination technology. It was cloned into the E. coli secretory expression vector pET-22b (+) and the 6×His immunotoxin fusion protein was expressed in E. coli. The 6×His was located at the N-terminus of ScFv and was found to be soluble in the cell. The 6% of the bacterial total protein, SDS-PAGE and Western blot showed that the molecular weight of the expressed product was 43 kD, which was consistent with the theoretically calculated value of the protein encoded by the gene. The purity of 6×His-immunotoxin purified by Ni2+IDASepharose6B affinity column can reach 90% or more, and the yield is 0-2mg/100ml. In addition to the activity of binding to CEA, the expression product also has the function of killing L929 cells.