| BoIFN-α genes were cloned from genomic DNAs of Chinese native bovine lines (yellow cattle, Holstein cow, Fuan water buffalo, Fuzhong water buffalo, Huanhu yak and wild yak) by PCR, and the PCR products were digested with Sph I and Hind III and subcloed into the expression vector pQE30 similarly digested. Sequence analysis showed that the 6 clones were composed of 498bp encoding a mature peptide of 166 amino acids with MW of 20kD. Compared with the published BoIFN-α genes in GenBank, only the IFN-α gene of Holstein cow had one point-mutation with BoIFN-αA subtype, and the left 5 clones were recommended to be added as new BoIFN-α subtypes, namely BoIFN-αD2, BoIFN-αI, BoIFN-αJ, BoIFN-αK and BoIFN-αL, respectively. SDS-PAGE and Western blot analysis showed that a specific targeted protein band was induced by IPTG against controls, and the expressed fusion protein is existed in inclusion body and was 25% of total proteins. Antiviral activities of rBoIFN-α was X105 U/mg and X106 U/mg in CEF/VSV and MDBK/VSV cell lines, respectively, and the rBoIFN-α proteins could protect against IBRV challenge in the MDBK cell line. These results showed that sequence changes don't affect their bioactivities and maybe related with breed evolution and geography situation, and the expressed rBoIFN-α proteins are good antiviral agents.BoIFN-γ cDNA was cloned from RNA of peripheral blood lymphocytes stimulated with ConA using RT-PCR and subcloned into the pGEM T-easy vector and sequenced. Mature peptide of BoIFN-y without signal peptide is subcloned and constructed into expression plasmids pET28a/BoIFN-y and pPICZa/BoIFN-y, respectively. In E.coli BL21, BoIFN-y was highly expressed by IPTG induction, and the higher expression level of 37% at 37癈 which was in inclusion body and the lower level of 18% at 30℃ but 25% of which was soluble; In Rpastoris GS115, BoIFN-y protein induced by methanol was directly secreted into cultural supernatant with only one specific targeted band of 23kD MW with expression of of 1.0g/L. Antiviral activity assays in CEF/VSV and MDBK/VSV cell lines, respectively, showed that rBoIFN-γ expressed in Rpastoris had 10 times higher antiviral activities than that expressed in E.coli in both cell lines, and for the same expressed proteins, antiviral activity in MDBK/VSV cell line is markedly higher than that in CEF/VSV cell line.VP1 of cattle FMDV was subcloned from PMD-18/VP1, and constructed into the expression plasmid pPICZa/VP1 and co-expression plasmid pPICZa/BoIFN-γ/VPl, respectively. In GS115, expression and co-expression were succeeded by methanol induction, with 28kD MW of VP1 protein and 52kD MW of co-expressed protein BoIFN-y/VPl analyzed by SDS-PAGE and Western blot analyses, and the expressed proteins were directly secreted into cultural supernatant with high level of l.Og/LBALB/c mice were inoculated with the rVPl, rBoIFN-y+rVP 1 and rBoIFN-y/VPl proteins expressed in P.pastoris, respectively, for three times at two week intervals. Serum IgG were detected by ELISA every two weeks post-inoculation, and lymphocyte proliferation assays (LPA) were performed after the last inoculation and the expression level of cytokines IFN-y, IL-2, IL-4 and 1L-10 wereanalyzed by semi-quantitative RT-PCR. Three days before the experiment was finished, delayed-type hypersensitivity (DTH) was tested. The result showed that rVPl expressed in P.pastoris had good antigenicity which could stimulate humoral immune response as well as cell-mediated immune response in inoculated mice, and rBoIFN-γ was a satisfactory immune adjuvant which could markedly enhance both Th1 and Th2 immunities, and serum IgG was markedly increased, T lymphocyte were proliferated to higher levels, more extensive DTH was observed, and expression levels of Thl and Th2 cytokines was much more promoted. Moreover, better immune enhancing effects was found in mice inoculated with co-expressed BoIFN-γ/VP1 than those co-inoculated with VP1 and BoIFN-γ.
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