H7亚型禽流感病毒纳米抗体制备与鉴定

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中图分类号：S852.659.5 文献标志码：A 文章编号：0366-6964（2026）01-0349-11

Abstract： This study aimed to express a nanobody targeting H7-subtype avian influenza virus using an E .coli system. Nanobody and antigen expression vectors were constructed via homologous recombination. Recombinant vectors were amplified using E .coliDH5a，followed bytransformation into E . coli BL21（DE3） and BEVS for high-yield protein production. Nanobodies and antigens were purified using streptavidin afnity chromatography and IMAC purification，respectively. Finally， immunofluorescence assay （IFA），enzyme-linked immunosorbent assay （ELISA）， hemagglutination inhibition（HI） and surface plasmon resonance （SPR） were employed to analyze nanobody titer， specificity， immunoreactivity，and antigen-nanobody interaction dynamics. Expression vectors were successfully constructed， followed by the production of a nanobody（D67），a fluorescent nanobody（D67-GFP）and a 55ku avian influenza antigen（H7-HA1）. In IFA，H7-HA1-positive cells incubated with the nanobody exhibited significantly stronger green fluorescence signals compared to the control group. ELISA revealed significant OD450 signals exclusively in the H7-HA1 antigen group，with P/N ratios decreasing as D67 concentrations diminished. HI assays demonstrated specific hemagglutination inhibition only for the H7 antigen group，achieving a titer of 8log2. SPR analysis quantified the interaction kinetics，yielding an association rate of 3.99×103L⋅（mol⋅s）-1 ，dissociation rate of 3.23×10-3s-1 ，and equilibrium dissociation constant of 8.1×10-7mol⋅L-1 ，confirming strong antigen-nanobody afinity. The H7-subtype-targeting nanobody D67 was successully prepared and validated through a multidimensional evaluation system，demonstrating its high-eficiency and specific binding to H7-HA1. This work provides a technical foundation for developing rapid detection methods for H7-subtype avian influenza.

Keywords：avian influenza;nanobody; biological activity；prokaryotic expression ∗ Corresponding author：FENG Saixiang，Email：fengsx@scau. edu.cn

禽流感是由禽流感病毒引发的烈性传染病，禽流感病毒囊膜表面存在两种重要蛋白质，其中一种为血凝素蛋白，简称HA，在空间上呈现为由HA蛋白构成的三聚体[1]。（剩余17027字）