Cloning, Expression And Effect Of Vaccine Adjuvant Of Channel Catfish(Ictalurus Punctatus) Interleukin-1β

Cloning, expression and effect of vaccine adjuvant of Channel Catfish(Ictalurus punctatus) interleukin-1βCytokines are produced by immune cells, which play important roles in biological regulation, immune modulation and many other biological functions. Nowadays, they have been recognized as promising new immunoadjuvants. Recent years, researchers are keen to study cytokine recombinant proteins as vaccine adjuvants and many exciting results have been achieved. Such as, they found that many cytokines, including Interferon, TNF, GM-CSF et al, could enhance more or less the efficiency of inactivated vaccine, attenuated vaccine and subunit vaccine. In this research, we cloned and expressed channel catfish interleukin-1 β gene successfully, and studied the the protective effect as subunit vaccine adjuvants of channel catfish.1、Cloning and expression of channel catfish IL-1β geneSpecific primers for IL-1β1 and IL-1β2 gene were designed based on the catfish IL-1β1 and IL-1β2 gene sequences published in Genebank (Genebank accession numbers were:DQ160229, DQ160230) using the biological software and amplifying the regions by PCR. The purified PCR products were ligated with the pMD19-T vector. Recombinant plasmid was digested with BamH I / Xhol I to retrieve the fragments of interest, which were inserted into PET32a between BamH I / Xhol I sites and transformed into BL21 (DE3) for expression. For the optimization of effective expression, the bacteria were induced by the condition of different temperature, concentration of IPTG and the induced time. Finally, expressed the recombinant proteins with optimized conditions, and purified by nickel-nitrilotriacetic acid (Ni-NTA) columns. The purified recombinant proteins were stored at -80 ° C. Western-blot was used to detect the specificity of antibody. Results showed that:both proteins were expressed successfully and their molecular weights were about 48.1 kDa. The optimized temperature were 37℃, IPTG concentration were 0.5 mmol/L and the induction time were 4 h. Western-blot analysis showed that the anti-recombinant protein sera had good specificity.2、The immune effect of recombinant channel catfish IL-1β as subunit vaccine adjuvantsMix the recombinant proteins of IL-1β1 and IL-1β2 with the subunit vaccines pSCPI (1:1) respectively and then injected intraperitoneally into the channel catfish. After the first immunization, Sera were collected from the tail vein blood for 8 weeks at a one-week interval for ELISA, ACH50 activity, serum bactericidal activity and lysozyme activity. In addition, at the fourth week, challenged the fish with streptococcus iniae, mortality was monitored and the relative protection rates were calculated. Results showed that recombinant cytokines IL-1β1 as a subunit vaccine adjuvant can increase serum bactericidal activity of channel catfish, as well as ACH50 activity and lysozyme activity; and the further study of serum bactericidal activity showed that complements and antibodies were the major antibacterial substances. The antibody levels of both adjuvant vaccine and non-adjuvant vaccine group reached highest values at the fourth week. The antibody levels of β1+ β2 subunit vaccine group showed relatively higher than the others, while the effects between β1 and β2 subunit vaccine groups were equal. After challenged with s.iniae, the cumulative mortalities of the immune groups (PBS, pSCPI, pSCPI+β1, pSCPI+β2 and pSCPI+β1+β2) were:93.33%,46.67%,36.67%,33.33% and 23.33%, respectively. And the relative protection rates were:50.1%,61.71%,65.29% and 75.01%, respectively. The Real-time PCR showed that IL-1β and TNF-α relative transcript levels in the head kidney and spleen from adjuvant groups were higher than those without adjuvant and PBS groups.