CHO cell line expressing bovine beta interferon and application thereof
A technology of beta interferon and cell line, applied in the field of CHO cell line expressing bovine beta interferon and bovine beta interferon, can solve the problems of low product activity, complex production process and product composition, and achieve simple process and product composition. easy-to-control effects
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Embodiment 1
[0038] Example 1. Construction of a CHO expression system for efficiently and stably expressing recombinant bovine beta interferon
[0039] 1 Materials and methods
[0040] 1.1 Plasmids, cell lines and virus strains
[0041]The stable eukaryotic expression vector pCI-neo (pCN) was purchased from Promega, and neo was expressed under the control of the SV40 early promoter. The CHO-K1 cell line was purchased from ATCC (ATCC No. CCL-61), and the CHO-K1 cells were cultured in DMEM-Ham's F12 (DF12) medium (invitrogen) containing 10% FBS.
[0042] The pMD18-T vector was purchased from TaKaRa Company; prokaryotic expression plasmid pET-32a(+) (Novagen Company), passaged bovine kidney cell line (MDBK) (ATCC No.CCL-22) and BHK-21 cells (ATCC No.CCL-22) 10) All are preserved by our laboratory; the primary chicken embryo fibroblasts (Chickenembryo fibroblasts, CEFs) adopt the routine method of 10-day-old SPF chicken embryos [17] Preparation; expression-enhanced green fluorescent protei...
Embodiment 2
[0078] Example 2. BoIFN-β CHO The study of the biological activity of
[0079] Put 102316AU·mL -1 The BoIFN-β obtained by the CHO expression system of Example 1 CHO (Titrate its antiviral activity on MDBK cells with VSV*GFP) for 10-fold serial dilution (ie 0.1, 1.02, 10.2, 1.02×10 2 , 1.02×10 3 and 1.02×10 4 AU·mL -1 , Figure 4 Swimming lanes A to F), respectively stimulated MDBK cells for 24 hours, and harvested MDBK cells. Then the chicken Mx protein mouse polyclonal antibody was used as the primary antibody, and the goat anti-mouse IgG was used as the secondary antibody to detect the expression of bovine Mx protein (molecular weight about 78kDa) by immunoblotting, and the results were as follows Figure 4 Shown: BoIFN-β CHO The lower limit of detectable Mx protein can be induced is 10.2AU·mL -1 ( Figure 4 Lane C), and between 10 and 10 3 AU·mL -1 ( Figure 4 In the range of lanes C to F), the amount of induced Mx protein and BoIFN-β CHO Activity units are po...
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