| Peripheral blood lymphocytes were collected from Holstein cow, and BoIL-2, BoIL-6 and BoGM-CSF were cloned from peripheral blood lymphocytes which stimulated with ConA by RT-PCR .The PCR products were subcloned into the pGEM T-easy vector and sequenced. Compared with the published sequence in GenBank, the BoIL-6 and BoGM-CSF gene had point-mutation with published gene but BoIL-2 had none. Analysis results showed a-coil of three sequences were all up 50%, and one or two glycosylation sites existed.VP1 of cattle FMDV was subcloned from PMD-18/VP1, and constructed into the expression plasmid pGEX-6p-1/VP1.Mature peptides of BoIL-2, BoIL-6 and BoGM-CSF were subcloned and constructed into the expression plasmid pGEX-6p-1/IL-2, pGEX-6p-1/IL-6, pGEX-6p-1/GM-CSF ,and coexpressed plasmids of one or two cytokine gene with VP1 were constructed into the expression plasmid pGEX-6p-1/BoIL-2/VP1, pGEX-6p-1/BoIL-6/VP1, pGEX-6p-1/BoGM-CSF/VP, pGEX-6p-1/BoIL-2/IL-6/VP1, pGEX-6p-1/BoIL-2/GM-CSF/VP1, respectively, and genes were connected with linker. In E.coli RS21,plasmids were highly expressed by IPTG induction at 30℃ .these proteins were in inclusion body and expression level were 37%, and the purity of refined proteins were 90%.Bioactivity assays showed that BoIL-2 protein in mice marrow cell and BoGM-CSF protein in mice thymus cell had bioactivity.Guinea pigs were separated into 17 groups and each group had five samples. All groups were inoculated with expressed proteins, respectively, for three times at two week intervals. Serum IgG were detected by ELISA every two weeks post-inoculation and neutralization antibody were tested by LP ELISA. Lymphocyte proliferation assays (LPA) were performed after the last inoculation and the expression level of cytokines IFN-γ, IL-2, IL-5, and IL-10 were analyzed by semi-quantitative RT-PCR. Three days before the experiment was finished, delayed-type hypersensitivity (DHT) was tested. The results showed that compared with control group, these proteins had good antigenicity which could stimulate humoral immune response as well as cell-mediated immune response in inoculated Guinea pig. Groups of BoIL-2/IL-6/VP1, /BoIL-2/GM-CSF/VP1 and BoIL-2+ IL-6/VP1, BoIL-2+ GM-CSF/VP1 were the best groups in all immunized groups ; The better effect groups were coexpressed proteins of one kind of cytokine with VP1 and mixed proteins of one kind of cytokine and VP1; groups of VP1,VP1 mixed with adjuvant and vaccine had the same effects. The results indicated that coexpressed protein and mixed protein had the same effect to Guinea pig. Neutralization assays indicated that groups of control, BoIL-2 and BoIL-6 had low neutralized antibody and other groups could markedly enhanced neutralized antibody to high level ; Compared with VP1 group, groups of BoGM-CSF , BoIL-2/VP1, BoIL-6/VP1, BoGM-CSF/VP, BoIL-2+VP1, BoIL-6+VP1, BoGM-CSF+VP, BoIL-2/IL-6/VP1, BoIL-2+BoIL-6/VP1 had a little higher neutralized antibody and groups of BoIL-2/GM-CSF/VP1, BoIL-2+BoGM-CSF/VP1 had a much higher level. Compared with Vaccine group, neutralized antibody level of BoIL-2/GM-CSF/VP1group and BoIL-2+BoGM-CSF/VP1 group had the same effects. Adjuvant Effects of cytokines with VP1 were tested by ELISA, LPA, LP ELISA, DHT and semi-quantitative RT-PCR. The results indicatedthat GM-CSF had the best effect as adjuvant of VP1 and IL-2 was better than IL-6; Adjuvant of multiple-cytokines had the better effect than one kind of cytokine.
|
PDF Full Text Request