人源抗狂犬病毒糖蛋白Ⅲ号表位链置换基因工程抗体研究

Generation of Human ScFv Antibodies for Antigenic Site Ⅲ of Rabies Virus Glycoprotein from Antibody-phage Libraries by Chain Shuffling

为获得针对狂犬病毒糖蛋白Ⅲ号表位的人源单抗,本研究采用噬菌体展示平台,对一株狂犬病毒糖蛋白Ⅲ号抗原表位的人源单抗CR4098采用链置换法进行改造。以CR4098单链抗体为骨架,从狂犬疫苗接种者外周血分离淋巴细胞,提核酸逆转录,PCR扩增抗体轻链可变区基因,替换CR4098的轻链基因,构建轻链置换文库。经纯化狂犬病毒aG株富集筛选,以上述筛选出的轻链阳性克隆为骨架,构建重链置换抗体库,富集筛选后通过ELISA和IFA鉴定阳性抗体克隆并进行序列测定。利用IgG表达载体VH/VK双质粒系统瞬时转染293T细胞实现IgG抗体的分泌型表达,通过亲和力测定和中和试验鉴定IgG抗体功能。结果显示,通过轻链置换,我们获得14株抗狂犬病毒scFv抗体,通过ELISA、IFA、亲和力测定及中和试验确定人源抗体RV3A5特异性结合狂犬病毒糖蛋白,对狂犬病毒CVS株和aG株均具有良好的中和活性,亲和力达到2.8×10-9 M。通过竞争ELISA对抗体结合表位进行鉴定,结果表明RV3A5特异性识别糖蛋白Ⅲ号抗原表位。通过链置换法成功获得1株全新的针对狂犬病毒糖蛋白Ⅲ号表位的高亲和力人源中和抗体,为狂犬病毒抗体制剂鸡尾酒治疗奠定了基础。

To obtain neutralizing high affinity human recombinant antibodies for antigenic site Ⅲ of rabies virus（RV）glycoprotein,we chose scFv phage display technology to optimize CR4098 with chain shuffling.Using pHAL14-CR4098 as vector,the combinatorial shuffling scFv antibody phage libraries were constructed to replace CR4098 light or heavy chain genes respectively by antibody genes derived from the blood of RV-vaccinated donors.After package by hyperphage,the chain shuffling scFv phage library was panned and selected by ELISA with purified rabies virus aG strain.The specific antibody was converted to full human IgG antibody with the VH/VK Express cassettes.Affinity and neutralizing test were performed to verify the function of the IgG molecules.Fourteen unique human ScFv antibodies specific for the glycoprotein of rabies virus were obtained by ELISA,IFA and DNA sequencing.Further tested showed that RV3A5 has a high affinity of 2.8×10^-9 M and high neutralizing activity to rabies virus both aG strains and CVS strains.Competitive ELISA showed that RV3A5 and CR4098for antigenic Site III competed with each other,indicating that they had overlapping or shared the epitope.Our results provide more candidates eligible for use in a mAb cocktail aimed at replacing RIG for rabies post-exposure prophylaxis.