Generation Of Several CHO Cell Lines Stably Expressing Bovine, Porcine And Chicken Beta Interferon And Evaluation Of Their Bioactivity

Type I interferons are important in the therapy and related fundamental study of human and animals virus and tumor diseases. Production technology of recombinant interferon using genetic engineering has great advantages. But, the production systems widely used at present have trouble in purification and renaturation, and there are great differences in the structure and bioactivity of the products with the nature interferon. At same time, the bioactivity assays of interferon available are time consuming, tedious, and inaccurate to operate and bad in specificity, therefore the improvement should be made right now.Aiming directly at to the problems above, this study is willing to construct a series CHO cell line expressing chicken, porcine and bovine IFN-β, and develop convenient, sensitive and precise bioactivity assay of interferon, then evaluate the bioactivity of all these interferon. All these will be useful for the fundamental study and application of chicken, porcine, and bovine type I interferon.1. Establishing an Antiviral Assay for the Bioactivity of Interferon Based on Recombinant VSV*GFP virus In this study, recombinant VSV*GFP expressing green fluorescence protein (GFP) and MDBK cells were used in the antiviral assay (AVA) of interferon. This method was carried on 96-well cell culture plate, quantifying the half virus replication reduction by quantifying GFP was used to replace quantifying the half CPE reduction by cell dyeing. In order to quantifying GFP, 50μL cell lysis buffer was added directly into the well containing supernatant (100μL) of MDBK cells infected with VSV*GFP, then the cells was lysed to release GFP following the inactivation of virus, finally the relative quantification of GFP was assayed by assaying the relative fluorescence of it using Microplate Fluorescence Reader (Bio-Tek). Compared with cell dyeing, the operation was greatly simplified. The concentration of GFP had good linear relationship with RFU whether it was in medium with or without phenol red. But phenol red had some effect on the quantification of GFP, so that quantifying GFP in medium without phenol red was more sensitive and accurate. If using standard interferon sample, the time of virus replication has little effect on the result of antiviral activity calculation. This method has good reproducibility (the coefficient of variation of inter-assay and intra-assay if using standard interferon sample), and assay results were almost same to that of the assay by cell dyeing. In addition, the cells forming confluent monolayer when inoculating virus had advantage to improve sensitivity and accurate, and GFP was stable in wet circumstance at 4℃at least 1 week without effect on assaying results.2. Mx promotor-luciferase reporter gene bioassay for chicken type I interferon Chicken Mx promoter (Mxp) was cloned, and the reporter gene of luciferase (luc) was placed under the Mxp to construct the pMxp-luc for the bioactivity assay of type I IFN. Chicken embryo fibroblast cell line was transiently transfected with pMxp-luc. Twenty four hours later, the cells were treated with chicken IFN-αand IFN-βwhich were generated by transient transfection and triturated by classical antiviral assay. After treatment for 6 hours, the expression of luciferase was detected and the expression level of luciferase had good linear correlation with the antiviral activity of IFN well. This Mxp-luc based bio-assay system provided a faster, safer and more precise method for the high-throughly quantitive bio-assay of type I IFN compared to traditional antiviral assay method.3. A bioassay for bovine and porcine type I interferon using a indicator cell line We cloned chicken Mx promoter (Mxp), and linked luciferase (Luc) reporter gene downstream of it. MDBK cells stably transfected with pMxp-luc were isolated and useful in assaying the bioactivity of bovine and porcine type I Interferon. MDBK-Mxp-luc assay for bovine and porcine IFN-α/βhad a useful dose response range between about 0.1 and 100 AU·mL^-1. The reproducibility was good and smaller than 10%. The luciferase expression level was highest at 5—6 h after treatment with IFN, then decreased to about 5 times at 12 h, this was completely different from other reporter gene assays using human, murine and fish Mx promoter (increasing even after 48 h), and the reason was worth exploring. The whole procedure of MDBK-Mxp-luc assay could be finished in 14 h which is the quickest having been reported. The assay was safe (no live virus used) and suit for high-flux operation. MDBK-Mxp-luc cells could also be used to study the molecular mechanism of interaction between virus infection and bovine type I IFN system.4. Recombinant baculovirus expressing bovine beta-interferon and evaluation of its bioactivity A recombinant baculovirus containing the ORF of bovine interferon-β (BoIFN-β) gene, rBac-BoIFN-β, was generated to express recombinant BoIFN-β(rBoIFN-β) in sf9 insect cells. The expression of rBoIFN-βin rBac-BoIFN-βinfecting sf9 cells and its supernatants was confirmed by indirect immunofluorescence assay and Western blot. The antiviral activity of rBoIFN-βin the supernatant can reach 10⁶⁰ AU·mL^-1 evaluated by the assay with VSV*GFP that expressed green fluorescence protein and rBoIFN-βcould stimulate the expression of luciferase reporter gene controlled by chicken Mx promoter. All the results showed that rBac-BoIFN-βconstructed here could express high level recombinant BoIFN-βin secreted form that had the bioactivity of natural type I IFN.5. Generation of a CHO cell line stably expressing recombinant bovine beta interferon and evaluation of its bioactivity The ORF of bovine interferon-β(BoIFN-β) gene was subcloned to stable eukaryotic expression vector pCI-neo (BoIFN-βwas expressed under the control of CMV promoter), and was stably transfected into CHO-K1 cells. Under the pressure of G418, a CHO cell clone (CHO-BoIFN-β) stably expressing recombinant BoIFN-βwas selected. In 25cm2 flasks with 5 mL cell culture medium, the expression of recombinant BoIFN-βby CHO-BoIFN-βcells accumulated up to 8.4×10⁵ AU·mL^-1, and could reach to 5.0×10⁵AU·mL^-1 or 0.5 AU/one cell, per day, and was stable for at least 20 passages without the pressure of G418. The recombinant BoIFN-βcould induce the expression of bovine Mx protein in MDBK cells and stimulated the transcription and expression of luciferase reporter gene under control of chicken Mx promoter.6. Generation of a CHO cell line stably expressing recombinant porcine beta interferon and evaluation of its bioactivity The ORF of porcine interferon-β(PoIFN-β) gene was subcloned to stable eukaryotic expression vector pCI-neo (PoIFN-βwas expressed under the control of CMV promoter), and was stably transfected into CHO-K1 cells. Under the pressure of G418, a CHO cell clone (CHO-PoIFN-β) stably expressing recombinant PoIFN-βwas selected. In 25cm2 flasks with 5 mL cell culture medium, the expression of recombinant PoIFN-βby CHO-PoIFN-βcells accumulated up to 7.7×10⁵AU·mL^-1, and could reach to 4.6×10⁵AU·mL^-1 or 0.46 AU/one cell, per day, and was stable for at least 20 passages without the pressure of G418. The recombinant PoIFN-βcould induce the expression of porcine Mx protein in PK15 cells, and could stimulated the transcription and expression of luciferase reporter gene under control of chicken Mx promoter. In addition, it could also fully protect PK15 cells against the infection of 1000 TCID₅₀ transmissible gastro-enteritis virus (TGEV) and rabies virus (PRV). All the results showed CHO-PoIFN-βcells could stably express high level recombinant PoIFN-βwhich has the bioactivity same to porcine nature type I interferon.7. Generation of a CHO cell line stably expressing recombinant chicken interferon beta and evaluation of its bioactivity The ORF of chicken interferon-β(ChIFN-P) gene was subcloned to stable eukaryotic expression vector pCI-neo (ChIFN-βwas expressed under the control of CMV promoter), and was stably transfected into CHO-K1 cells. Under the pressure of G418, a CHO-K1 cell clone (CHO-ChIFN-β) stably expressing recombinant ChIFN-βwas selected. In 25cm² flasks with 5 mL cell culture medium, the expression of recombinant ChIFN-βby CHO-ChIFN-βcells accumulated up to 6.9×10⁴ AU·mL^-1, and could reach to 4.3×10⁴ AU·mL^-1 or 0.043 AU/one cell, per day, and was stable for at least 20 passages without the pressure of G418. The recombinant ChIFN-βcould induced the expression of chicken Mx protein in chicken embryo fibroblast cells (CEFs), and could stimulated the transcription and expression of luciferase reporter gene under control of chicken Mx promoter. In addition, it could also fully protect CEFs against the infection of 1000 TCID50 avian influenza virus (H5N1 subtype).In order to quantify the bioactivity of interferon, reporter gene assay system based on a MDBK cell line and antiviral assay based on VSV*GFP were established to assay the bioactivity of interferon accurately, sensitively, rapidly and specifically. In addition, the interferon expressed by CHO cells here were almost same to nature interferon compared with other expression systems, which was critical for its structure stability and its bioactivity. While the expressing level was similar to baculovirus expression system, and the production, purification artwork was very simple to be used in clinical treatment of animals even with simple or without purification, so that the cost is to be very low. This study provide good technical platform and study foundation for deeply exploring the mechanism of infection and anti-infection between etiological agents, and the production and application of type I interferon.