Porcine Reproductive And Respiratory Syndrome Virus (prrsv) N Gene Rt-pcr And Expression Of The Establishment Of The Method

Use the primer designed by ourselves, successfully build up a reverse transcription-PCR method of amplifying porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid protein gene. When obtained the full gene of PRRSV N protein, first checked whether the gene was the right gene that we need by analysis of UV Agarose Photograph and Analysis System and sequencing, After that, purify the gene by Wizard SV Gel and PCR Clean up System, then clone it into pGEM-T Vector. Transfer the full plasmid into JM109 bacteria, extract the plasmid which including the N protein gene band, cut it with restriction endonuclease, ligate the gene into plasmid pET-28a, and transfer the plasmid into JM109 bacteria. Find out the plasmid that consist of the gene band, transfer it into expressing bacteria BL-21. Select the bacteria that contain the positive plasmid, amplified at 37℃ and induced it to express with IPTG. 6 hours later, harvest and extract the total expressed protein, then identify it by SDS-PAGE and Western-blot. Purify the N protein by Ni2+ affinity chromatography and store at –20℃. In this research, the N protein expressed in E.coli could react with PRRS positive pig serum, it indicated that the protein could used as antigen of diagnostic assay for detecting antibodies.