Cloning, Prokaryotic Expression of CSFV NS3 Gene, and Preliminary Establishment of an Indirect ELISA for Serum Antibody Detection

Abstract:

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 kD was detected by SDS-PAGE and expressed mainly in the form of inclusion bodies. The result of Western blotting showed recombinant protein NS3 has immunogenicity. The nickel affinity chromatography was employed to purify the target protein, and purified recombinant protein NS3 (90%) was obtained. An indirect ELISA was initially established to detect antibody against CSFV NS3 protein, and 221 sera samples of pig from different herds and ages were tested. The result was compared with CSFV-Ab test kit of IDEXX, and the result showed: the positive coincidence rate was 83.33%, the negative coincidence rate 89.38%, and the total coincidence rate 86.34%. 30 serum samples showed inconsistent, and were detected by Indirect Immunofluorescence Assay (IFA). The results showed: compared with IFA the accuracy rate of result detected by NS3 indirect ELISA and CSFV-Ab test kit of IDEXX was 56.67% and 43.33% respectively.