Study On Prokaryotic Expression And Immunodetection Of Procine Interleukin-6 Gene

The PIL-6 cDNA fragment was inserted into pGEX-1?T plasmid to construct the expression plasmid pGPIL-6, the recombinant plasmid was digested by BamH I and Pst I to identify whether the PIL-6 cDNA fragment was inserted into the plasmid in correct orientation, the pGPIL-6 was transformed into E. coli DH5? competent cells. The mRNA and protein expression were assayed by RT-PCR and SDS-PAGE. The results were found that the specific 740bp DNA bases of IL-6 was detected by RT-PCR in the recombinant bacteria and a new protein band was found in SDS-PAGE with molecular mass of about 49 KDa which is consisted of a 23 KDa protein deduced from the IL-6 gene sequence and GST (26 KDa). These indicated that the porcine IL-6 gene was correctly transcribed and translated in the recombinant bacteria. The fusion protein was recovered from polyacrylamid gel and used to immunize the rabbit, and the liter of rabbit anti-porcine IL-6 serum from immunized rabbit was 1: 128,000.Biotinylated DNA was produced from the plasmid pGEX-IXT by PCR amplification with biotinylated sense primer and nonbiotinylated antisense primer. Avidin was used as the linker of biotinylated goat anti-rabbit IgG and biotinylated 700bp of pNA. Immuno-PCR for IL-6 was established by use of 1ng/l of the biotinylated DNA as template. This immuno-PCR has a detection limit of 1fg/ml of PIL-6 that is 105 times lower than that of indirect ELISA, and is the most sensitive method todetect IL-6 up to date.We used this immuno-PCR to detect IL-6 level in the sera of porcines which had been immunized with the trivalent vaccines and eukaryotic expression plasmids of pig IL-6 gene. The result showed that the inoculation of porcine IL-6 gene a;s adjuvant of the vaccines elevated the concentration of porcine IL-6 in the sera of immunized pigs and enhanced the immune responses of porcine. These suggest that cytokine genes, such as IL-6 and interferon gene could be used as effective immunoadjuvant to the immune responses of porcine to the conventional vaccines.