单克隆抗-D细胞株的建立及抗-D抗体Fab段的基因序列分析

目的:建立分泌抗-D抗体的人B淋巴细胞株,分析编码一株人单克隆抗-D抗体Fab段的基因序列。方法:以EB病毒(EBV)转化分泌抗-D抗体的人B淋巴细胞,建立分泌抗-D抗体的淋巴细胞株。设计扩增人lgM重链Fd段及κ轻链的引物,以聚合酶链反应进行扩增,对扩增产物进行分子克隆及测序。结果:分别用轻链引物和重链引物从一分泌lgM抗-D的单克隆细胞株扩增出一约650bp和700bp的特异带。序列分析表明,其核苷酸及其所推导的氨基酸(AA)序列符合人lgFab段的序列特征。结论:获得了抗-D抗体Fab段基因,为基因工程抗-D的研制及Rh新生儿溶血病的预防提供物质基础。

Establishment of human lymphocyte cell line secreting mon oclonal antibodies against Rhesus(D) antigen and sequence analysis of a human monoclo nal anti- D Fab fragment

AIM:To establish human lymphocyte cell line secreting monoclonal antibody against Rhesus(D) antigen and analyse the nucleotide and deduced amino acid sequences of a human monoclonal anti-D Fab fragment. METHODS:By using PCR method, the cDNA of human anti-(Rhesus D) antibody(lgM κ)Fab fragment was amplified from an Epstein- Barr-virus-transformed cell line. Cloning and subsequent sequence analysis of the Fab fragment was performed. The deduced amino acid sequence was compared and analysed with previously published sequences. RESULTS:A band of approximate 700 and 650 base pairs was amplified using lgM heavy chain primers and κ light chain primers, respectively. Sequence analysis indicated that the deduced amino acid sequences was in agreement with the characterization of the amino acid present in the human lg Fab fragment. CONCLUSION:The cloning and sequencing of a human anti-Rhesus (D) antibody Fab fragment cDNA will make benefits for production of recombinant anti-Rhesus (D) antibody and prevention of Rh haemolytic disease in the newborn.