Prokaryotic Expression Of CSFV NS3 Protein, And Application To Antibody Detection

NS3 gene fragment (about 2000 bp) was amplified by PCR from plasmid of pPOHCLV containing Hog Cholera Lapinized Virus (HCLV) cDNA, and cloned into the prokaryotic expression vector pET-32a(+) to obtain the recombinant prokaryotic expression vector pETNS3. The pETNS3 was transformed into E.coli Rosetta (DE3) and expressed optimally. The recombinant protein NS3 about 95 kDa was detected by SDS-PAGE and expressed mainly in the form of inclusion bodies. The result of Western Blotting showed recombinant protein NS3 was reactive with the antibody to CSFV. The nickel affinity chromatography was employed to purify the target protein, and purified recombinant protein NS3 (90%) was obtained.According to the《The ELISA Guidebook》, the purified recombinant protein NS3 (rp-NS3) was used as the coating antigen in this indirect enzyme-linked immunosorbent assay (ELISA) to detect specific antibodies against CSFV NS3 protein, and the optimal dilution of antigen and serum, and other conditions were determined by checkerboard titration. This NS3-ELISA, which has a good repeatability, is a promising tool for large scale monitoring of CSFV antibodies in pig herds, and the specificity and sensitivity were 97.3% and 98%, respectively.The NS3-ELISA developed in this study was employed for detection of CSFV antibodies, and comparison test of 325 serum samples with CSFV-Ab test kit of IDEXX showed the total coincidence rate was about 87%. Among them 42 inconsistent sera were further tested by Indirect Immunofluorescence Assay (IFA). Compared with the IFA, the NS3-ELISA has a higher coincidence rate than the CSFV-Ab kit of IDEXX does. A panel of sera collected at different time point post infection from pigs which experimentally infected with HCLV, and detected by both of NS3-ELISA and CSFV-Ab test kit of IDEXX to study the dynamic changes of antibodies to NS3 and glycoprotein E2. As the results showed that the antibody induced by NS3 is significantly later than antibodies against glycoproteins E2. We speculated that NS3-ELISA is not suitable for the early detection of CSFV antibody in infected pigs, but a more reliable indicator than antibodies against glycoproteins E2 to judge whether pig herds are infected with CSFV.