应用重组腺病毒载体制备人CTLA4Ig融合蛋白

目的在实验室水平制备并鉴定人CTLA4Ig融合蛋白.方法将人CTLA4Ig cDNA片段构入重组腺病毒载体中,用该表达载体感染293细胞并在其中表达人CTLA4Ig,再应用SPA亲合层析柱自细胞培养上清中纯化出该融合蛋白,以WESTERN印迹验证后,观察了所制备CTLA4Ig对混合淋巴细胞反应的影响.结果经亲合层析柱洗下的蛋白为分子量为53.0kd的单一组分,经WESTERN印迹证实为CTLA4Ig,并发现所制备CTLA4Ig能呈剂量依赖性抑制混合淋巴细胞反应.结论本法成功制备了具有生物学活性的重组人CTLA4Ig融合蛋白.

Preparation of human CTLA4Ig fusion protein through recombinant adenovirus vector

Objective To prepare the human CTLA4Ig fusion protein for further experimental and clinical research.Methods The CTLA4Ig cDNA fragment was constructed into recombinant adenovirus vector through homologous recombination. Then the constructed vector was used to infect the cultured 293 cell, a package cell of recombinant adenovirus vector, and the target protein was expressed. The human CTLA4Ig fusion protein was purified from the cell cultured supernatant using SPA column and was identified by Western Blotting. Finally, the affection of the prepared CTLA4Ig on mixed lymphocyte reaction was observed.Results It was found that the molecular weight of the purified protein was about 53.0kd, which was identified as CTLA4Ig by Western Blotting. And the prepared CTLA4Ig could inhibit the MLR with a dosage dependent manner.Conclusion The human CTLA4Ig fusion protein was prepared successfully using the method mentioned.