抗H5N1亚型高致病性禽流感猴源噬菌体scFv的构建和表达

为探索治疗禽流感的方法,本实验从免疫H5N1亚型禽流感疫苗的猕猴淋巴细胞中提取总mRNA并扩增重链和轻链可变区(VH、Vλ和Vκ),用重叠延伸PCR方法构建VH-Linker-Vλ和VH-Linker-Vκ形式的单链抗体(scFv),采用噬菌体展示技术构建了噬菌体抗体库,从2.2×106的噬菌体库中淘选得到具有较高亲和力的H6株scFv,并通过ELISA、SDS-PAGE、western blot、间接免疫荧光(IFA)和竞争抑制ELISA对其进行鉴定,证实所获得的scFv为32ku,并且能与病毒发生特异性结合。IgBlast序列分析发现,所得到的scFv基因Vλ与人免疫球蛋白胚系基因同源性达91.5%,而VH同源性为75%。本研究所得到的猴源抗体可变区为人源化抗体的构建以及禽流感的治疗奠定了基础。

Expression of phage displayed single-chain antibody of rhesus macaque against H5N1 highly pathogenic avian influenza virus

In order to construct a subtractive single-chain antibody (scFv) against H5N1 highly pathogenic avian influenza virus, the variable cDNA fragment of H and L chain of the antibody was amplified from the total mRNA of immunized rhesus macaque lymphocytes. The single-chain variable fragments (VH-Linker-Vλ and VH-Linker-Vκ) were constructed by splicing overlap- extension (SOE) PCR and displayed on the phage surface. Panning of the phage library of 2.2 × 106 clones by ELISA allowed the isolation of H6, a high affinity scFv antibody. The scFv was further confirmed by SDS-PAGE and western blot, and its activity was analyzed by indirect immunofluorescence assay (IFA) and competitive inhibition ELISA, The scFV was about 32 ku which could specifically bind the virus. IgBlast results revealed that the Vλ gene sharing 91.5 % identity with those of human immunoglobulin germline genes, while the VH gene shared only 75 % identity. The scFv of rhesus macaque reported here provided a foundation for construction of the humanized antibody, and this approach of generating antibody fragments could be suitable for prophylaxis and therapeutics.