重组人抗HBs-Fab抗体的纯化及结构分析

Purification and Analysis of Structure of Fab Fragment of Recombinant Human Anti-HBs

目的研究重组人抗HBs-Fab抗体的纯化工艺,并对其结构等特性进行分析。方法采用离子交换-分子筛层析法分离纯化由酵母工程菌(GS115/Fab)发酵的重组人抗HBsAg Fab抗体,并经ELISA检测其抗体活性,等电聚焦电泳法检测其等电点(pI),基质辅助激光解吸飞行时间质谱(MALDI-TOF-MS)检测其相对分子质量和肽质量图谱。结果纯化的重组Fab抗体的纯度可达90%以上,经Sephacryl-100进一步层析后,纯度达99%以上,总收率可达80%以上。Fab抗体具有较好的抗体活性,其pI值为7·6,为一碱性蛋白,相对分子质量为50494,比其理论值约多2579·35,经Endoglycosidase H内切酶消化后的相对分子质量为49609,证明Fab的一级结构中有糖基化现象,且分布于Fab抗体的H链和L链上。胰酶酶解肽段中有21个与理论肽段相符,另检测到1对二硫键正确。结论已建立了重组人抗HBs-Fab抗体的稳定的纯化工艺,且Fab抗体的一级结构正确。

Objective To explore the purification procedure of Fab fragment of recombinant human anti-HBs and analyze the structure of the fragment. Methods Purify the Fab fragment of recombinant human anti-HBs expressed in yeast ceils by ion exchangemolecular sieve chromatography. Determine tbe biological activity of the Fab fragment by ELISA, its pI by isoelectric focusing dectrophoresis and its relative molecular weight and peptide map by matrix assisted laser deaorption ionization time of flight mass spectrometry （MADLI-TOF-MS）. Results The purity of Fab fragment purified by ion exchange column chromatography reached more than 90%. However,after further purification by Sephacryl-100 chromatography,the purity reached more than 99%. The total recovery rate of Fab fragment reached more than 80%. The Fab fragment was a basic protein with a pI value of 7.6 and a relative molecular weight of 50 494,and showed good antigen binding activity. After digestion with cndoglyeosidase H, its relative molecular weight was 49 609. It proved the glycosylation in both H and L chains of the fragment. Peptide map proved that the Fab fragment was digested into 40 segments ,of which 21 showed the same amino acid sequences as those of theoretical segments. In addition,one disulfide bond joint correctly was observed. Conclusion A stable purification procedure of Fab fragment of recombinant human anti-HBs was developed,and the Fab fragment showed correct primary structure.