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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. coli DH5α， followed by transformation into E. coli BL21（DE3） and BEVS for high-yield protein production. Nanobodies and antigens were purified using streptavidin affinity 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 15 ku nanobody （D67）， a 42 ku fluorescent nanobody （D67-GFP） and a 55 ku 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 3.99×103 L·（mol·s）⁻¹， dissociation rate of 3.23×10-3 s-1， and equilibrium dissociation constant of 8.1×10-7 mol·L⁻¹， confirming strong antigen-nanobody affinity.The H7-subtype-targeting nanobody D67 was successfully prepared and validated through a multidimensional evaluation system， demonstrating its high-efficiency and specific binding to H7-HA1. This work provides a technical foundation for developing rapid detection methods for H7-subtype avian influenza.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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