| Adjuvant can enhance the immunogenicity of antigen and immune response, or change the type of immune response. In this study, we isolated Hz-Au peptide from the culture of Bacillus, it had low molecular weight and low toxicity. KM mice were used to evaluate the adjuvant effect of Hz-Au peptide on the immune responses to the ovalbumin(OVA) and glutathione s-transferase(GST) by ELISA method. We also studied on the immune dose, acute toxicity and mechanism underlying of Hz-Au peptide, the results were as follows:(1)Adjuvant effect of Hz-Au peptide on the immune responses to OVA and GST in mice. The OVA and GST protein with Hz-Au peptide, FA adjuvant, CTB adjuvant, were separately used to immunize KM mice. Compared with other groups, the group with Hz-Au peptide was detected the highest specific IgG antibody titer, which was significantly higher than the group with antigen alone(p<0.01). The specific antibody titers of anti-OVA IgG and anti-GST IgG were up to 2×105 with peritoneal injection route, which were higher than FA adjuvant group, the levels of specific antibody were a little lower with subcutaneous immunization. IgG subclasse anaylsis showed Hz-Au peptide only slightly increased titer of specific IgG2a Ab but pronounced abundance of IgGl compared with the immunogen only group, suggesting that the Hz-Au peptide preferentially arguments the Th2-dependent response. The specific IgG antibody provoked by Hz-Au peptide could last at a high level in mice for more than two months.(2)The immune dose study of Hz-Au peptide and immunogen. The adjuvant effect was positively correlated with Hz-Au peptide dose between 100μg and 5μg with peritoneal injection route. However, the proper dose of Hz-Au peptide for oral immune was from 200μg to 500μg, and the levels of specific IgG antibody were decreasd by antigen dose-dependent means.(3) The safety study of Hz-Au peptide. Mice acute toxicity study demonstrated that, it’s safe to orally administer Hz-Au peptide. All mice survived, the oral maximal tolerance dose of Hz-Au peptide was 30 mg per mouse. There were no visible changes found in organs by autopsy. Immunological analysis also showed there were no local reactions at the injection site.(4) Adjuvant effect of Hz-Au peptide on the immune responses to nucleic acid and peptide in mice. The DNA RH and peptide HCMV-IE1 with Hz-Au peptide, were separately used to immunize KM mice. In contrast to the immunogen only groups, higher levels of IgG were elicited in immunogen plus Hz-Au peptide groups(all p<0.01).(5)Digestion of immunogen by proteases. The protein OVA, GST, BSA were digested by trypsin and pepsin respectively. The result showed that Hz-Au peptide could protect protein from proteases digestion in vitro.(6) The antigen specific T lymphocyte proliferation assay. Hz-Au peptide could enhance the cellular immune responses in mice. When treated with Th and CTL epitope, splenocytes from immunized mice with OVA plus Hz-Au peptide showed markedly higher proliferative response than those from immunized with OVA only.(7) FACS analysis of TNF-a/IFN-y level in T lymphocyte. The data indicated that higher level IFN-y production appeared in CD4 and CD8 T cells when immunized with OVA plus Hz-Au peptide orally, which imply that Hz-Au peptide promoted the shift of immune balance to Thl type. When immunized mice by peritoneal injection, the IFN-γproduction level of Hz-Au peptide group was lower than OVA group, which imply that Hz-Au peptide promoted the shift of immune balance to Th2 type. |
