| Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of PRRS, which caused reproductive failure in sows and respiratory problems in piglets. Glycosylated membrance protein (GP5) .nucleocapsid (N) protein coded by ORF5,ORF7 of PRRSV, are two major structural proteins of PRRSV. In this study, the N protein and GP5 were expressed as a fusion protein with glutathione-S-transferase (GST) or his-tag in E coli, and the recombinant protein of N and GP5 were successfully purified using different purification methods. Subsequently, the secondary structure of N and GP5 were characterized by cicular dichroism (CD) spectroscopy, and the relationships between the structure and function of N or GP5 were analyzed through their secondary structure.The soluble recombinant nucleocapsid protein (N) was obtained by constructing different expression vector. The N preotein was expressed as a fusion protein with glutathione-S-transferase (GST) in E coli BL21 strain by using prokaryotic expression vector pGEX-6P-l, the recombinant protein (GST-N) was obtained by a single step of affinity chromatography using Glutathinione Sepharose 4B, and the intact N (N') was successfully purified from the fusion protein by Prescission protease.The pET28-N expression vector was constructed using pET28a vector. The soluble fusion protein (P28-N) with his-tag located in N-terminal was expressed at 28% expression level in E coli BL21(DE3) host cells, and the purified P28-N protein was harvested by nickel chelate affinity chromatography method.The biochemical characteristics of GP5, dE (the signal peptide deleted), dEM (the transmembrance domain deleted) protein and many presumptive GP5 fusion proteins were analyzed by bioinformatics analysis methods, and the different recombinant expression vectors (pGEX-6P-dE, pGEX-6P-dEM, pET32-dEM) were constructed with expression vectors pGEX-6P-l and/or pET32a. The recombinant fusion proteins GST-dE, GST-dEM and P32-dEM (expressed by pGEX-6P-dE, pGEX-6P-dEM and pET32-dEM, respectively.) could amount to 16%, 32% and 50% of the total mass of bacterial proteins. The purified recombinant proteins GST-dE, GST-dEM and P32-dEM were gained through denaturation and refold of inclusion bodies, affinity chromatography on glutathione-agarose column, and gel filtration chromatography on Sephacryl S-100 HR on column.The secondary structure of P28-N, GST-dE, GST-dEM were performed by CD spectroscopy. The secondary structure of GP5 and N protein were plotted in terms of the results of CD spectroscopy and secondary structure predication by bioinformatics analysis.The helical content of P28-N was estimated by comparing its mean residue ellipticity, The CD analysis showed that the purified PET28-N shared a significant (26%) a-helical structure, p-sheet (23.7%), P-turn (19.8%), and random coil (30.3%), respectively. The third structure of NA57 (the mutant deleted N-terminal 57 ammo acids of the N protein) and its dimers of BJ-4 strains were protracted by homology modeling with N protein 3D structure template of PRRSV VR2332 strain from PDB. Finally, a modified N protein secondary structure of BJ-4 strain of PRRSV was deduced, and the amino acid amounts of a-helical, p-sheets,P-turn and random coil of N protein was approximately 40,12,14 and 57, respectively.The circular dichroism analysis showed that the purified GST-dEM contain a-helical (44%) (155aa) and P-sheet (14.4%) structure. The secondary structure of GP5 were plotted according to the results of GST protein 3D structure template from PDB data, different structural predication and CD analysis. The amino acid amounts of a-helical, p-sheets, p-turn and random coil of GP5 protein was approximately 58,56,14,and 72, respectively.The relationships between the structure and function of N or GP5 of PRRSV were elucidated through their secondary structure in aspects of structural biology. The secondary structure of N protein is helpful for understanding its functions. It was indicated that the structure of NLS (nuclear localization signal ),...
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