| Porcine reproductive and respiratory syndrome (PRRS) is one of the mostdevastating diseases of pig throughout the world,which is resulted from porcinereproductive and respiratory syndrome virus (PRRSV),and it leads to importanteconomic impact in production. So, it is very important to detect the disease as soon aspossible. Most of the methods are applied to detect PRRSV-specific antibodies in swinesera. ELISA is a common method, which used high purity proteins as the antigen.In this research, To gain high purity and high immunogenicity nonstructural protein7(nsp7). Nsp7is to be cleaved from the genome of PRRSV. The DNA fragmentencoding nsp7of PRRSV were cloned into the Hind Ⅲ and NdeⅠsites of pET-28avector to construct the expression plasmid pET-28a-nsp7. The recombinant vector wastransformed into E. coli BL21(DE3) strain, samples were collected after induction withIPTG. The specificity of the expressed protein was identified with SDS-PAGE, WesternBlotting and ELISA. The nsp7recombinant protein was further purified by thenickel-metal chelate chromatography, Q anion exchange chromatography, and nickelconcentrate. The indirect enzyme-linked immunosorbent assays was used to study theimmunogenicity of nsp7through compared with the LSI-ELISA kit. SDS-PAGEshowed that the size of the nsp7protein was34kDa, which was consistent with thetheoretical data. The nsp7recombinant protein was easily expressed and with goodsolubility. The purity of nsp7protein reached95%. Western-Blot analysis showed thensp7recombinant protein was able to react with porcine positive serum antibodiesagainst PRRSV. The nsp7-based ELISAs had higher correlation with the LSI-ELISA.we successfully established a system method for the expression and purification of nsp7and obtained nsp7protein with high purity and high immunogenicity, which lays afoundation for further study of nsp7on the serological diagnosis of PRRS. |
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