抗CD3 scFv基因的真核表达及其生物学活性鉴定

目的：真核表达抗CD3单链抗体(scFv)，并研究其生物学活性。方法：将编码抗CD3 scFv的DNA片段插入真核表达载体pDisplay中。对所构建重组表达载体进行测序确认后，以电穿孔法将重组质粒导入Hela细胞，以原位杂交法检测抗CD3 scFv的表达，以3H TdR掺入法检测其在体外对T细胞的活化作用，以MTT比色法观察转染的。Hela细胞与T细胞混合培养后，诱发的细胞毒性T细胞的杀伤作用。结果：成功地构建了抗CD3 scFv的真核表达载体，并在Hela细胞中获得表达。所分泌的抗CD3 scFv在抗CD28mAb存在的条件下，能够刺激T细胞活化。将转染的Hela细胞与T细胞混合培养能够诱发CTL的杀伤作用。结论：真核表达的抗CD3 scFv具有刺激T细胞活化的活性，可用于构建对肿瘤进行免疫治疗的双功能分子。

Eukaryotic expression of anti-CD3 single chain Fv antibody gene and the characterization of its bioactivities

AIM: To express anti-CD3 scFv in Hela cells and investigate its biological activity. METHODS: DNA fragment encoding anti-CD3 scFv was inserted into eukaryotic expression vector pDisplay. The recombinant expression vector was sequenced and then transfected into Hela cells by electroporation method. The expression of anti-CD3 scFv was identified by in situ hybridization. In vitro T lymphocyte activation was then detected by 3H-TdR incoporation method. Anti-CD3 scFv gene-transfected Hela cells were co-cultured with and T cells cytotoxicity was measured by MTT colorimetry. RESULTS: Anti-CD3 scFv gene was correctly inserted into pDisplay and expressed in Hela cells. The secreted anti-CD3 scFv was able to activate T lymphocytes in the presence of anti-CD28 mAb. Cytotoxicity could be observed when anti-CD3 scFv gene-transfected Hela cells were mixed and co-cultured with T lymphocytes. CONCLUSION: Anti-CD3 scFv expressed by Hela cells can activate T lymphocytes.