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淋巴毒素(lymphotoxin,LT) 通过TNFR1受体传递凋亡信号,从而发挥抗肿瘤活性. 对LT的受体结合区域进行多点随机突变,利用噬菌体展示重组人淋巴毒素(rhLT) R46,S106,L130三点随机突变组合文库和S106～F110区域随机突变体库. 噬菌体库与固相化TNFR1受体进行亲和筛选,富集了能与TNFR1受体结合的rhLT突变体. 随机挑选20个单克隆噬菌体进行ELISA受体结合鉴定,其中80%的克隆与TNFR1受体特异性结合,有4个克隆与TNFR1受体的结合能力高于野生型序列的rhLT. 将这4个结合力高的突变体克隆于pET32a(+)载体,经过大肠杆菌表达和纯化操作后,检测这些突变体蛋白与TNFR1受体的结合活性和对L929细胞的杀伤活性,发现3个克隆的受体结合性质与其展示于噬菌体上时基本吻合,其中C199克隆与TNFR1的结合活性比野生型序列的rhLT提高了近30%,且其对L929细胞的杀伤活性提高了近90%. 应用噬菌体展示技术对淋巴毒素进行体外进化研究的尝试,为下一步的蛋白质结构和功能研究提供了思路,可成为大分子药物开发的有效工具.
Lymphotoxin (LT) exerts anti-tumor activity by transmitting apoptotic signals through the TNFR1 receptor.Multiple random mutagenesis of the receptor binding domain of LT is performed, and phage display recombinant human lymphotoxin (rhLT) R46, S106, L130 Three-point random mutagenesis library and a random mutants library from S106 to F110, phage library and immobilized TNFR1 receptor were screened and enriched rhLT mutants capable of binding TNFR1 receptor were randomly selected 20 monoclonal The phages were identified by ELISA receptor binding, of which 80% of the clones specifically bound to the TNFR1 receptor and 4 clones had higher binding capacity to the TNFR1 receptor than the wild-type sequence of rhLT.The four high binding mutants Were cloned into pET32a (+) vector. After E. coli expression and purification, the binding activity of these mutant proteins to TNFR1 receptor and the cytotoxic activity against L929 cells were tested. The binding properties of the three clones were found to be similar to that of the phage displayed in phage The results showed that the binding activity of C199 clone with TNFR1 was increased by nearly 30% compared with that of wild-type sequence, and its cytotoxicity to L929 cells increased by nearly 90% .Using phage display technology, Attempts to study the in vitro evolution of baptism of toxins provide ideas for the next step in the study of protein structure and function and can be an effective tool for the development of macromolecular drugs.