特异结合人禽流感病毒H5N1的scFv分子性能鉴定

目的从人源化噬菌体抗体库（human single fold scFv libraries I＋J）中筛选到能高亲和性、特异结合人禽流感病毒H5N1的单链抗体，为建立H5N1快速筛查试剂和人源化治疗单抗奠定基础。方法以H5N1病毒的血凝素（hemagglutitin，HA）蛋白和核蛋白（nucleoprotein，NP）为目的蛋白，对上述单抗噬菌体文库以亲和性为原理进行筛选，经过3轮筛选富集后，随机挑选了96个噬菌体克隆扩增培养，ELISA法挑选能特异性、高亲和性结合目的蛋白的噬菌体克隆，并换用HB2151宿主菌对阳性单链抗体克隆进行可溶性表达，ELISA法鉴定可溶性单链抗体的结合活性，PCR扩增阳性克隆的轻、重链基因片段，并对阳性单链抗体分子测序和序列分析。结果经过3轮筛选，分别从96个噬菌体克隆中挑选到了两株能特异结合NP蛋白、3株能特异结合HA蛋白的单链抗体，PCR扩增都得到了长为300、302和935bp的轻链、重链和轻链-连接片段-重链的基因片段，测序结果分析发现上述5条单链抗体片段在轻链的47、49、50、51、53、54、56、96、97、98和99位的氨基酸组成不同，而特异结合NP蛋白的单链在重链区域氨基酸组成完全相同，而特异结合HA蛋白的单链在重链的44、47、85、86、87、88和89位氨基酸组成不同。结论从噬菌体抗体库中筛选到的特异结合HA和NP蛋白的单链抗体片段，可为进一步研发H5N1快速筛选试剂和人源性治疗抗体奠定基础，也可为鉴定HA和NP蛋白中的抗原决定簇提供结构信息。

Screening and Detection of scFV Fragments Specific to HA and NP Proteins of H5N1 Virus

Objective To select single-chain variable fragment （scFv） antibody specific for human HSN1 virus from Human Single Fold seFv Libraries. Methods Human Single Fold scFv Libraries were panned against immobilized HA and NP of HSN1 in a microtiter plate. After three rounds of selection, 96 ScFV clones were determined to specifically bind to the target protein by ELISA. Positive clones were expressed in HB2151 for production of soluble antibody fragment~ The light and heavy chain genes of positive ScFv were amplified and sequenced. Results Two scFv clones specific for NP binding and three specific for HA binding were selected. The gene fragments of 300bp light chain, 302bp heavy chain and 935bp joint were amplified with PCR. The insertion gene fragments were correctly detected in positive scFV. The DNA sequencing analysis showed different a- mino acids at 47, 49, 50, 51, 53, 54, 56, 96, 97, 98 and 99 sites in light chains among 5 positive scFV. There were no difference in heavy chain of anti-NP ScFv, while 7 differences of ami- no acids at 44, 47, 85, 86, 87, 88 and 89 sites were detected in anti-HA SeFv. Conclusion The ScFvs specific to HA and NP may be used as reagent to detect and prevent infection of H5N1 virus and define antigenic determinant epitopes of HA and NP proteins.