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Outline
This study aimed to achieve soluble prokaryotic expression of the N-terminal extracellular domain of the pseudorabies virus （PRV） receptor Nectin1 protein， and to screen high-affinity， high-specificity DNA aptamers targeting it， thereby providing a novel molecular tool for blocking PRV adsorption. The porcine Nectin1 gene was cloned into the prokaryotic expression vector pET28a containing a maltose-binding protein （MBP） solubility-enhancing tag to construct the recombinant plasmid pET28a-MBP-Nectin1. The correctly digested and sequenced recombinant plasmid was transformed into Escherichia coli BL21 （DE3） for induced expression. The recombinant protein was validated by Western blot and pull-down assays. A DNA aptamer specifically targeting rMBP-Nectin1 was selected using magnetic bead-coupled Systematic Evolution of Ligands by EXponential enrichment （SELEX）. The affinity and specificity of the selected aptamer Apt-Nectin1-6 were evaluated by magnetic bead-based ELASA. Nectin1 knockdown cell lines （sh-Nectin1-1 and sh-Nectin1-2） were used as controls to verify the binding ability of Apt-Nectin1-6 to cell surface Nectin1 by flow cytometry. Additionally， the inhibitory effect of the aptamer on on adsorption of pseudorabies virus to host cells was assessed by flow cytometry and viral genome copy number analysis. Western blot and pull-down assays confirmed the correct expression and biological activity of the recombinant MBP-Nectin1 protein. Through SELEX， the DNA aptamer Apt-Nectin1-6， exhibiting high affinity to rMBP-Nectin1 with an equilibrium dissociation constant （Kd） of （0.11±0.02） nmol and no cross-reactivity with rMBP-gD， rMBP-S1， rMBP-CD163， or rMBP alone， was successfully screened. Molecular docking analysis indicated that Apt-Nectin1-6 binds to Nectin1 via hydrogen bonds. Further flow cytometry， viral genome copy number quantification， and virus titration collectively demonstrated that Apt-Nectin1-6 inhibits PRV adsorption to host cells in a dose-dependent manner. （P<0.05）. In this study， a DNA aptamer Apt-Nectin1-6 with high specificity and affinity for the Nectin1 protein was successfully screened and verified to effectively block PRV adsorption， providing a foundation for developing novel anti-PRV strategies.
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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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