Cloning, Expression And Effects Of Vaccine Adjuvant Of Porcine IL-4 And IL-6 Gene

Vaccination is an important strategy to control animal infectious diseases in many countries,so vaccination is needed to not only strongly trigger immune responses, but be safe and lower side-effect. Cytokines produced by animals themselves in the use of immunoadjuvants are more advantageous than conventional adjuvants. IL-4 mainly inhibits cellular immune responses and promotes humoral immune responses especially induced by IgE. IL-4 is a specific revulsant of IgE. IL-6 is a versatile cytokine that plays an important role in the regulation of immune responses. It is engaged in regulating the biological activities, such as B cell differentiation, Ig production, peripheral T cells and thymic T cells differentiating into cytotoxic T lymphocytes. Furthermore, there is still lack of explorations of IL-4 and IL-6 gene at home or abroad in strengthening immune responses of Cysticercus cellulosae. In this study we co-stimulated lymphocytes from pig spleen and lymph code with PHA and LPS, then extracted total RNA from the cells and used it as a template to amplify IL-4 and IL-6 genes by RT-PCR. The two amplified fragments were subsequently ligated into the pGEM-T easy vector for sequencing. The results showed the cloned IL-4 gene and IL-6 gene respectively had 99%, 99.7% homology with the corresponding sequences obtained from the GenBank. The length of the IL-4 gene was 440bp (ORF was 402bp), encoding a peptide of 134 amino acids whose molecular weight is 15ku. The ORF length of IL-6 gene was 639bp, encoding a peptide of 212 amino acids with molecular weight 24ku. According to the ORF sequence of cloned IL-6 gene without signal peptide, we designed new primers to amplify truncated IL-6. After double digestion with EcoRⅠand NotⅠ, the amplified products and the pGEX-4T-1 vector were ligated together and transformed into E. coli cells, this construction was also sequenced to ensure fidelity. The mRNA and protein expression were assayed by RT-PCR and SDS-PAGE.The results were found that the specific 555bp DNA bases of IL-6 was detected by RT-PCR and a new protein band was found in SDS-PAGE with molecular mass of about 47ku which is consist of a 21ku IL-6 protein and GST(26ku). It indicated that the porcine IL-6 gene was correctly transcribed and translated in the recombinant bacteria.The highest bacterial yield and expression rate of inclusion body were obtained (33% of total bacterial proteins) at 42℃of initiative culture. After isolation of inclusion body, gel filtration chromatography with Sephadex G-100 was carried out for purification, the purity of IL-6 reached 90%. And then denatured recombinant IL-6 was renaturated. Renaturated IL-6 protein was injected into mice. By detection of biological activity of recombinant IL-6, it showed initially that IL-6 could increase immunity and enhance immune response of mice. Soluble protein of IL-6 was obtained by optimizing the culture temperature and expression inducing time. This protein was purified by GST filtration chromatography, the purity reached above 95%. Bioactivity of resultant IL-6 was monitored by mouse spleen lymphocytes proliferation assay. The result suggested that the expression products improved remarkably the proliferation of lymphocytes from spleen of mice stimulated by ConA. Furthermore, with the concentration of soluble protein increased, cell proliferation level is promoted and significantly different by t test, compared with control group. 　IL-6 gene was inserted into pcDNA3 eukaryotic expression vector and named pcDNA3-pIL-6. Then extracted pcDNA3-pIL-6 and pcDNA3-pIL-4 plasmids were used as adjuvants of Cysticercus cellulosae vaccine to immunize mice respectively. Antibodies were detected subsequently, which showed that pcDNA3-pIL-6 and pcDNA3-pIL-4 could significantly promote humoral immune response of pigs, much better than aluminum and 206 adjuvant, which could be employed as effective immune adjuvants. IL-4 production in mice spleen lymphocytes was detected by ELISA. The result demonstrated that recombinant IL-6 is lower than conventional adjuvants and pcDNA3 group and pcDNA3-pIL-4 produced the highest IL-4 level. So both pcDNA3-pIL-6 and pcDNA3-pIL-4 could be choosed as adjuvants of Cysticercus cellulosae vaccine, which depends on infectious degree of this kind of parasite.