抗CD_3抗体(HIT3a)基因突变及其表达研究

目的　提高可溶性抗CD3 scFv片段的表达量 ,并测定其生物学活性。方法　用PCR法致抗CD3 scFv基因突变 ;用DNA限制性酶切指纹图谱以及Westernblot法筛选突变克隆 ;用间接免疫荧光法测定抗体的特异性结合活性 ;用12 5I标记抗体 ,进行竞争抑制试验 ;采用51Cr释放试验 ,进行抗CD3scFv片段介导的T淋巴细胞细胞毒性测定。结果　DNA序列测定结果表明 ,抗CD3 scFv抗体突变克隆菌株m2为重链第六位氨基酸突变 ,即E(GAG)→Q(CAG)。突变后的可溶性抗CD3 scFv片段表达量(1μg/ml)比突变前 (0 .0 1μg/ml)高 10 0倍。突变前、后的抗CD3 scFv片段与Jurkat细胞 (CD3 )的结合特异性未改变。m2能竞争性封闭亲代鼠源性抗体HIT3a与CD3 阳性的Jurkat细胞的结合位点。体外杀伤实验结果显示 ,由m2与IL 2共刺激产生的CD3 AK细胞的细胞毒活性比单用IL 2刺激产生的LAK细胞强。结论　通过对抗CD3 scFv抗体基因进行定点突变 ,实现了该抗体片段的高表达 ;m2能与CD3 的Jurkat细胞结合 ;也能激活人外周血中的T淋巴细胞产生CD3 AK细胞毒活性

The mutation of anti-CD_3 antibody (HIT3a) gene and its expression

Objective To improve the expression of anti CD 3 single chain Fv (scFv) by site mutation and identify its biological activity. Methods Anti CD 3 scFv gene was mutated by PCR,the target clones were screened by both the fingerprints of DNA restriction endonuclease digestion and Western blot, the antigen binding activity of scFv was examined by FACS, competitive inhibition was performed with 125 I labeled HIT3a and the cytotoxic effect mediated by the anti CD 3 scFv activated T lymphocytes was analyzed by 51 ?Cr released assays. Results The DNA sequencing showed that the 6th amino acid of the anti CD 3 antibody (HIT3a) heavy chain gene was mutated from E(GAG) to Q(CAG). The expression of mutated anti CD 3 scFv (m2) was increased by 100 times higher than that of the parent scFv, and there was no difference in the Jurkat cell (CD 3 ) binding activity between the (m2) and parent scFv. The preliminary results of competitive assays showed that m2 could partially block the sites of CD 3 Jurkat cells where the parent antibody bound to. Cytotoxicity assays demonstrated that CD 3AK cells induced by IL 2 and m2 showed stronger cytotoxic effect than that of LAK cells induced by IL 2 alone in vitro.Conclusion By site mutation, a high expression fragment m2 of anti CD 3 scFv antibody was obtained. The results of some experiments indicated that m2 could bind to CD 3 Jurkat cells, furthermore, by co stimulated with IL 2, it could activate peripheral T lymphocytes and induce CD 3AK cytotoxic effect.