| Interleukin-6(IL-6) is a kind of multifunctional cytokine which is produced by many different cell types. Recent studies have shown that chicken IL-6(chIL-6) can improve the immunity and increase the antibody level of vaccination. This study aims to produce chIL-6with high biological activity in Pichia pastoris, and find a method for recombinant chIL-6(rchIL-6) purification.First, the chIL-6gene was inserted into pET32a(+) and expressed in E coli, then the polyclonal antibody for detection of the expression of chIL-6in Pichia pastoris was prepared. The chIL-6gene was cloned into expression vector pPICZaA, then transformed into GS115by electroporation. One positive and high-copy clones screened by zeocin were induced by1%methanol and identified by western blotting,the result showed its expression level was162mg/L,accounting for64.5%of the total protein in the supernatant.After membrane ultrafiltration and dialysis, the chIL-6was purified with the DEAE anion-exchange chromatography, the concentration of purified protein was128mg/mL and recovery was75.9%.The biological activities of the recombinant chicken IL-6(rchIL-6) were determined using IL-6dependent cell lines7TD1and peripheral lymphocytes. MTT assay showed purified rchIL-6protein could stimulate the proliferation of7TD1hybridoma cells, these activities were highest when the concentration of rchIL-6was125pg/mL, above or below this concentration may adverse. In MTT assay, different concentrations of rchIL-6promoted peripheral lymphocytes proliferation in a concentration dependent manner.21day-old chickens were randomly assigned to three groups, including H9avian influenza (AI) vaccine with rchIL-6, H9AI vaccine and control. Flow cytometry showed the T lymphocyte ratio of CD4+/CD8+in the first group was higher than other groups, and the percentage of CD4+T lymphocyte was higher than CD8+T lymphocyte. Blood samples were collected at different time points after immunization; the HI antibody level of chickens in H9AI vaccine with rchIL-6was higher than other groups;the detoxification rate of chickens in H9AI vaccine with rchIL-6was significantly lower than other groups. These results suggested that chIL-6could be used as vaccine adjuvant. |
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