| Porcine reproductive and respiratory sydrome(PRRS) caused by PRRSV is a highly contagious and infectious disease, causing serious economic losses in many countries. Highly pathogenic porcine reproductive and respiratory sydrome virus(HP-PRRSV) is the variant of classical PRRSV, since the outbreak of HP-PRRSV, bring devastating loss to pig farm. Thus, establishing quick and senstive diagnostic methods are of great significance to prevent and control disease in time. GP5 is the important target protein in serological test, which as the main stuctural proteins of PRRSV. Pichia pastoris has been extensively used as a eukaryotic expression system of highly expressing heterologous protein. In this study, deleted transmembrane domain GP5(dGP5) was expressed abundantly by pichia pastoris and developing an indirect ELISA assay for detection GP5 antibody.First, we optimized the gene of HP-PRRSV HuN4 GP5 through codon bias, AT-rich region, G+C content adjustment and so on. The HuN4 wide-type and the optimized GP5 gene fragments encoding the same animo acid were inserted into the site between EcoRI and NotI site in pPIC9 K in our previous study. The recombinant plasmids were linearized and transformed into P. pastoris GS115 by twice electroporation. The PCR positive strains were screened by MD plates and different concentrations of YPD-G418 plates. Unfortunately, GP5 was not expressed in Pichia pastoris. So the optimized dGP5 gene fragment was further expressed through the same methods in Pichia pastoris and the high-expression positive strains were screened. To determine grope the best induced condition, different expressed conditions of dGP5 protein was optimized, such as experssion time, the induced concentration of methanol, PH value and temperature. The expressed protein was identified by SDS-PAGE and Western-blotting. The result showed that the optimized dGP5 gene fragments without transmenmbrane domain was expressed successfully in Pichia pastoris, and amino acid sequence was accord with the before optimization, modified base 75 bp, and the nucleotide homoology was 76.9 % between opitimized and original gene sequence, involving codon change of 62 amino acid, the G+C content transform from 49.68 % to 42.28 % after gene optimization. The optimal expressed conditions were the rotate speed 250 rpm/min, PH 6.5, the temperature 24 ℃, the induced concentration of methanol 1 % and growth time 120 h. Identified through SDS-PAGE and Western-blotting, the size of protein was about 19 KDa.Secondly, the dGP5 protein was purified by his column and the indirect ELISA method was established with the purified protein as coating antigen. Results showed the optimum coating concentration of antigen was 0.21 μg/well and incubated overnight at 4 ℃. The best serum dilution was 1:100 and reaction 37 ℃ 1 h. HRP-conjugated was diluted by 1:40000 and reaction 37 ℃ 1 h. The rGP5 was of perfect accuracy and had no cross-reactivity with PPV、RV、PRV、CSFV and TEGV. The variable coefficient of intre-assay and inter-assay were less than 10 %, which indicated the method had good repeatability. Compared with the LSI kit, the agreement rate of the two above methods could reach 87 %.In this study, dGP5 protein was highly expressed by pichia pastoris and an in direct ELISA was established. A simple and efficient method for detecting antibodies of HP-PRRSV was available in this study. |
PDF Full Text Request