1株人源抗狂犬病病毒单克隆抗体的基本特征

Basic characteristics of a human monoclonal antibody against rabies virus

目的评价从噬菌体库筛选获得的1株抗狂犬病病毒单克隆抗体的基本特征。方法从抗狂犬病病毒噬菌体库中筛选获得1个阳性克隆,扩增获得其抗体轻重链可变区,构建全分子抗体的表达质粒后在HEK293 EBNA1细胞中瞬时表达,亲和纯化后对该抗体进行体外抗狂犬病病毒中和活性、中和指数、逃逸株测序、差示扫描荧光法热稳定性分析,并与数据库中街毒株糖蛋白比对关键氨基酸位点。结果该抗体体外抗狂犬病病毒中和活性为691IU/mg;该抗体对狂犬病病毒CVS-11和CTN株的中和指数分别为8.52和7.05;CVS-11逃逸株糖蛋白测序分析表明,该抗体针对的关键氨基酸主要为N37 K、N194 K和K205 N。与1338株狂犬病病毒街毒株糖蛋白基因比对表明,37、194和205位上仅有1个街毒株在194位上与突变逃逸株氨基酸类型相同。该抗体Tm值为70.58±0.31℃。结论获得1株高中和指数的全分子人源抗狂犬病病毒中和抗体,该抗体具有较好的热稳定性。

Objective To evaluate properties of monoclonal anti-rabies virus antibodies obtained from phage library.Methods A positive clone was obtained from the phage library of anti-rabies virus,and the variable region of light and heavy chain of the antibody was amplified by PCR.The transient expression plasmid of the fully human antibody was constructed and then transiently expressed in HEK293 EBNA1 cells.The purified antibody was analyzed by in vitro anti-rabies virus neutralization activity,neutralization index,escape virus glycoprotein sequencing and thermal stability by differential scanning fluorimetry.Glycoprotein sequences of escape virus were compared with lyssavirus glycoprotein sequence in the database.Results In vitro neutralizing activity of the antibody was 691 IU/mg;the neutralization index of the antibody was 8.52 with CVS-11 and 7.05 with CTN.Sequence analysis of the escape mutant virus glycoprotein showed that the key amino acids of the antibody were N37 K,N194 K and K205 N.The amino acid sequence of mutated virus glycoprotein was compared with that of 1338 lyssavirus glycoprotein.The result showed that only one strain of lyssavirus had the same amino acid type at position 194 as that of mutated escape strain.Tm(melting temperature)of the antibody was 70.58±0.31℃.Conclusions A recombinant human neutralizing antibody with high neutralization index was obtained,which has high thermal stability.