抗-D抗体Fab段基因的克隆及其在大肠杆菌中的表达

Cloning and expression of a human monoclonal anti-D Fab fragment in E. coli with the use of bacteriophage vector

目的:于大肠杆菌表达抗-D抗体Fab段基因,为真核载体表达完整的人抗-D抗体奠定基础。方法:将已克隆测序的Fab段基因亚克隆到pComb3噬粒载体,用PCR和限制性内切酶酶切鉴定后于大肠杆菌进行表达,表达产物用SDS-PAGE和ELISA等方法分析。结果:SDS-PAGE电泳表明重组克隆表达了1条约48kD的蛋白条带,ELISA结果显示,所表达的抗体和Rh+的人红细胞呈阳性反应,和Rh-红细胞反应为阴性,阴性对照和Rh+及Rh-红细胞反应均为阴性。结论:于大肠杆菌表达了具有抗原结合特性的人抗-D抗体Fab段抗体分子。

AIM： To clone and express a human monoclonal anti - D Fab fragment in E. coli and make benefits for the expression of the whole immunoglobtdin molecules of anti - D. METODS： The gene of anti - D Fab fragment was cloned into the phagemid vector pComb3. After analyzing by PCR and restriction site analysis, the recombinant was expressed in E. coli and the expressed protein was analyzed by SDS - PAGE and ELISA. RESILTS： The result of SDS- PAGE confirmed that E. coli expressed a 48 kD protein. The ELISA result demonstrated that the cell culture supematant reacted with Rh ＋ group O human erythrocytes, but was not recognized by Rh- group O human erythrocytes. CONCLUSION： Expressed Fab fragment has the antigenic specificity for human erythrocytes.