The Gene Expression, Biological Activity Assay And Monoclonal Antibody's Preparation Of Bovine Interleukin-18 Mature Protein

The gene encoding interleukin-18 (IL-18) was firstly cloned by Okamura in 1995. IL-18 plays important roles in antitumor, anti-infection and immunoloregulation. And it has potential prospect in immunologic therapy and adjuvant. With the development of cow industry in China, the cow's disease especially mastitis is becoming severity. So a newly vaccine and its adjuvant are urgently demand to research. The gene encoding bovine IL-18(BoIL-18)was cloned and expressed in our laboratory in order to prevent and therapy the cow's diseases. In the present study, the efficiently expressed BoIL-18 protein with biological activity was obtained, and the anti-BoIL-18 monoclonal antibody was prepared by the expression product. It was included four parts as following:Experiment 1: The prokaryotic expression and biological activity assay of BoIL-18 fusion protein.Firstly, the double specific primers were designed by published gene sequence to PCR amplify the gene encoding BoIL-18 from recombinant plasmid pMDT-BoIL-18. Then the gene was subcloned into pGEX-6P-1 vector and transformed into Competent Escherichia coli BL21(DE3). The recombinant E.coli was induced by IPTG and the expression product was assayed by SDS-PAGE and Western blot methods and purified with GST resin by affinity chromatography. Finally the biological activities of purified product were detected. The results indicated that the recombinant protein was expressed successfully in E.coli by induced with 0.3 mmol/L IPTG for 8 h. the assay of SDS-PAGE and Western blot showed that engineering bacteria could express a 44 kD product. And densitometric scanning showed the expressed fusion protein was about 31.8% of total bacterial protein of BL21. The PBMC(peripheral blood mononuclear cells) proliferation indicated that the BoIL-18 fusion protein could enhance proliferation of PBMC when it was more than 0.10 mg/L. The ELISA method showed that the BoIL-18 fusion protein could induce IFN-γproduction in spleen lymphocyte when it was more than 0.20 mg/L, and the amount of BoIL-18 fusion protein and its inducing effect on IFN-γhad a direct proportion. And BoIL-18 fusion protein could inhibit the VSV activity by inducing IFN-γproduction in spleen lymphocyte. It can be concluded that BoIL-18 is expressed in E.coli BL21 and the expression product has functional activities.Experiment 2: The gene expression and biological activity assay of BoIL-18 mature protein in baculovirus expression vector.Firstly, the double specific primers were designed by published gene sequence to PCR amplify the gene encoding BoIL-18 from recombinant plasmid pMDT-BoIL-18. Then the gene was subcloned into pFastBac HTb vector and transformed into max efficiency DH10Bac Competent E. coli. The recombinant plasmid, that is recombinant Bacmid, was transfected into sf9 cells with the Cellfectin regent. Then the recombinant baculovirus was infected sf9 cells. The infected sf9 cells and its culture supernatant were collected and assayed by SDS-PAGE and Western blot with polyclonal antibody of BoIL-18 fusion protein expressed in pGEX-6P-1. The expression product was purified with Ni-NTA resin by affinity chromatography. Finally the biological activities of purified product, including of the proliferation of PBMC, inducing IFN-γproduction and inhibiting the VSV activity , were detected. The results indicated that the recombinant protein was expressed successfully in sf9. The intention banding was 44 kD by assay of SDS-PAGE and Western blot. And densitometric scanning showed the expressed fusion protein was about 21.3% of total protein of sf9. The PBMC(peripheral blood mononuclear cells) proliferation indicated that the BoIL-18 fusion protein could enhance proliferation of PBMC when it was more than 0.05 mg/L. The ELISA method showed that the BoIL-18 fusion protein could induce IFN-γproduction in spleen lymphocyte when it was more than 0.10 mg/L, and the amount of BoIL-18 fusion protein and its inducing effect on IFN-γhad a direct proportion. And BoIL-18 fusion protein could inhibit the VSV activity by inducing IFN-γproduction in spleen lymphocyte. So it can be concluded that BoIL-18 was expressed in sf9 and the expression product has functional activities.Experiment 3: The monoclonal antibody (McAb) of anti-BoIL-18 was prepared.In this study, BALB/c mice were immunized by BoIL-18 fusion protein expressed by E.coli.BL21 in experiment 1. Spleen cells of mouse immunized were fused with SP2/0 myeloma cells by the PEG regent. And hybridoma cell lines secreting monoclonal antibody, that is anti-Bo IL-18, were selected by indirect ELISA using recombinant BoIL-18 protein expressed by recombinant baculovirus in sf9 cells in experiment 2. Two positive cell lines, named 5G8 and 7B3 respectively, secreting anti-BoIL-18 McAb were obtained by limited- dilution method after five times cloning. The anti-BoIL-18 McAb were showed to be specificity by Western-blot assay, and the titer of 5G8 and 7B3 were 1×105,1×106 by indirect ELISA, respectively. The abilities of hybridoma cell lines secreting McAb remained the same after several generations or freezed two months later. And the subgroups of two hybridoma cell lines were IgG l.Experiment 4: The gene encoding BoIL-18 mature protein was expressed in baculovirus expression vector.Firstly, the double specific primers were designed by published gene sequence to PCR amplify the gene encoding BoIL-18 from recombinant plasmid pET-BoIL-18. Then the gene was subcloned into donor vector pVL1393, and transformed into TG1 Competent cells. The recombinant plasmid and lining virus Bm-BacPAK6 DNA were co-transfected into silkworm cells Bm5 with the Lipofectin regent. Then the recombinant baculovirus was selected by blue-white plaque. The correct recombinant baculovirus identified by PCR amplification was infected into 5d silkworm larva. The hemolymph of silkworm larva was assayed by SDS- PAGE and Western blot method. The results indicated that the BoIL-18 protein was expressed successfully in silkworm larva with the 18 kD intention banding by assay of SDS-PAGE and Western blot. And densitometric scanning showed the expressed protein was about 16.7% of total protein of silkworm larva.