靶向甲型流感病毒血凝素蛋白的广谱抗体制备及鉴定

Preparation and identification of a broad-spectrum antibody targeting hemagglutinin protein of influenza A virus

目的制备靶向甲型流感病毒血凝素(hemagglutinin,HA)蛋白的广谱抗体FHA3,并对其进行鉴定。方法依据单链抗体(single-chain antibody fragment,scFv)序列信息,PCR扩增FHA3重链和轻链可变区序列,连入抗体哺乳细胞表达载体pFRT-IgG1κ,构建重组表达质粒pFRT-IgG1κ-FHA3。利用瞬时高效表达系统ExpiCHO以及亲和纯化技术获取抗体蛋白FHA3。ELISA检测FHA3与甲型流感病毒HA的结合。假病毒感染宿主细胞试验检测抗体FHA3的体外中和活性。结果蛋白质电泳结果显示制备获取了高纯度FHA3。FHA3与甲型流感病毒H1N1 HA、H2N2 HA、H3N2 HA、H5N1 HA、H7N9 HA以及H9N2 HA均识别结合,且呈浓度依赖效应。FHA3具有较好的体外中和活性,在低浓度下(5μg/ml)能有效阻断H5N1以及H7N9假病毒入侵靶细胞,在0.0125μg/ml浓度下能有效阻断H1N1假病毒入侵靶细胞。结论获得1株具有体外中和活性的靶向甲型流感病毒HA蛋白的广谱抗体。

Objective To prepare and identify a broad-spectrum antibody FHA3 targeting influenza A virus hemagglutinin(HA).Methods According to the single-chain antibody fragment(scFv)sequence,the heavy chain(VH)and light chain(VL)variable regions of FHA3 were amplified by PCR and a recombinant plasmid pFRT-IgG1κ-FHA3 was constructed by linking the expression vector pFRT-IgG1κ.FHA3 was expressed in the ExpiCHO system and purified by affinity purification.The binding activity of FHA3 to influenza A virus HA was detected by ELISA.The neutralizing activity of FHA3 was detected in vitro by infecting host cells with pseudovirus.Results SDS-PAGE showed that high-purity FHA3 was obtained.FHA3 could bind to H1N1 HA,H2N2 HA,H3N2 HA,H5N1 HA,H7N9 HA and H9N2 HA in a concentration-dependent manner.FHA3 had good neutralizing activity in vitro that was it could effectively block the invasion of H5N1 and H7N9 pseudoviruses into target cells at a low concentration of 5μg/ml and H1N1 pseudovirus at 0.0125μg/ml.Conclusions A broad spectrum antibody targeting HA protein of influenza A virus with neutralizing activity in vitro was obtained.