论文部分内容阅读
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
Objective To improve the expression of soluble anti-CD3 scFv fragment and determine its biological activity. Methods The mutation of CD3 scFv gene was induced by PCR. The mutant clones were screened by restriction endonuclease digestion and Western blotting. The specific binding activity of the antibody was determined by indirect immunofluorescence. The competitive inhibition assay was performed with 125I labeled antibody. The 51Cr release assay was used to determine the cytotoxicity of T lymphocytes mediated by anti-CD3 scFv fragments. Results The results of DNA sequencing showed that mutant M2 of anti-CD3 scFv antibody was the sixth amino acid mutation of heavy chain, ie E (GAG) → Q (CAG). The expression level of the mutated soluble anti-CD3 scFv fragment (1 μg / ml) was 10 times higher than that of the pre-mutation (0.1 μg / ml). The binding specificity of the anti-CD3 scFv fragment before and after the mutation to Jurkat cells (CD3 +) was unchanged. m2 competitively blocks the binding site of the parent murine antibody HIT3a to CD3-positive Jurkat cells. In vitro cytotoxicity assays showed that the cytotoxic activity of CD3 AK cells co-stimulated with m2 and IL2 was stronger than that of LAK cells stimulated with IL2 alone. Conclusion The site-directed mutagenesis of the anti-CD3 scFv antibody results in the high expression of this antibody fragment; m2 binds to CD3 + Jurkat cells; it also activates T lymphocytes in human peripheral blood to produce CD3 AK cytotoxicity