OX40-IgG1融合基因重组表达载体的构建及鉴定

Construction and identification of a recombinant expression vector encoding OX40-IgG1 fusion gene

目的:构建含人OX40-IgG1融合基因的真核表达载体pDC315-OX40Ig,表达具有生物学活性的OX40Ig融合蛋白,为诱导异体复合组织移植免疫耐受的基因治疗作准备。方法:全基因合成目的基因OX40Ig,即人OX40胞外段序列及人IgG1 Fc段序列,将该片段插入到pUC57(+)载体后,亚克隆至真核表达载体pDC315(+),构建pDC315-OX40Ig重组质粒。构建的重组质粒经PCR、酶切及测序鉴定后,用脂质体法转染于NI H/3T3细胞。以SDS-PAGE与Western Blotting检测细胞培养上清中OX40I g融合蛋白的表达情况;MTT法观察OX40Ig对混合淋巴细胞反应(MLR)的抑制作用。结果:合成的目的基因全长1320bp,构建的重组质粒经PCR、双酶切及测序鉴定正确。SDS-PAGE与West ern Blotting证实OX40Ig融合蛋白在3T3细胞中实现表达,表达产物相对分子量为48000左右,与理论预期值相符。体外实验证实OX40Ig蛋白能够抑制异基因淋巴细胞的MLR。结论:成功构建了人OX40-IgG1融合基因真核表达载体pDC315-OX40Ig,体外实验证实表达的OX40Ig蛋白可抑制淋巴细胞增殖,为干预同种异体复合组织移植排斥反应奠定了基础。

Objective To construct a eukaryotic expression vector encoding human OX40-IgG1 fusion gene, express OX40Ig fusion protein with high biological activity, and make preparation for the gene therapy of induction of composite tissue allograft transplants immune tolerance. Methods The cDNA of extracellular domain of human OX40 gene and IgG1 Fc gene were synthesized and inserted into pUC57 （＋） vector, and then subcloned into eukaryotic expression vector pDC315 （＋） to construct the plasmid pDC315-OX40Ig. The constructed recombinant plasmid was identified by PCR, restriction enzymes and DNA sequence analysis. After these identifications, the recombinant plasmid was transfected into NIH/3T3 cells with Lipofectamine 2000. Expression and secretion of QX401g was confirmed by SDS- PAGE and Western Blotting, and the inhibitory effect of OX40Ig on MLR in vitro was observed by MTT assay. Results A DNA fragment about 1320 bp was obtained. The OX40Ig gene sequence of pDC315-QX401g was consistent with GenBank. Restriction enzymes and PCR identification proved that the OX40Ig gene was correctly cloned into expression vector. SDS-PAGE and Western Blotting analysis showed that OX40Ig fusion protein was expressed in NIH/3T3 cells culture supernatant successfully. The relative molecular mass （Mr） of the expression protein was 48000, which was accorded with the predicted Mr value. It was confirmed that the OX40Ig protein can inhibit MLR in vitro. Conclusion The eukaryotic expression vector pDC315-OX40Ig was successfully constructed and OX40Ig fusion protein can inhibit lymphocytes proliferation. This study has laid the foundation for the intervention of composite tissue allograft rejective reaction.