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Porcine reproductive and respiratory syndrome virus （PRRSV） is a major pathogen that poses a serious threat to the global swine industry. Its non-structural protein NSP4， as a 3C-like serine protease （3CLSP）， cleaves viral multiproteins at the junctions between other conserved domains， playing a critical role in viral transcription and replication. This study aims to elucidate the structural features and enzymatic activity of the NSP4 protein， providing a theoretical basis for the development of antiviral small-molecule drugs targeting NSP4. This study used the PRRSV NADC30-like strain as the research object. The PRRSV nsp4 gene was cloned into the pGEX-6p vector， induced for expression in Escherichia coli BL21（DE3）， and purified to obtain high-purity protein. Crystallization conditions were screened using the drop-plate method， and the 1.47 Å high-resolution crystal structure of NSP4 was determined using X-ray crystallography. Its enzymatic activity was measured in vitro using fluorescence resonance energy transfer （FRET）. The results showed that PRRSV NSP4 consists of three domains： the N-terminal β-barrel domain （I， residues 12-69）， the middle β-barrel domain （II， residues 89-153）， and the C-terminal α/β domain （III， residues 159-190）. Further active site prediction identified the substrate-binding pocket of NSP4 as composed of Ser-19， Met-20， and His-39， providing a target for structure-based inhibitor design. In vitro enzyme activity experiments showed that the catalytic efficiency of PRRSV NADC30-like strain NSP4 toward the substrate， Kcat/Km， was 411.38 L·（mol·s）-1， and the substrate affinity， Km， was 23.28 μmol·L-1. This study reports the high-resolution structure and enzyme kinetic characteristics of the PRRSV NADC30-like strain NSP4， laying an important foundation for structure-based antiviral drug design and screening.
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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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