Recombinant cell line for efficiently and stably expressing feline interferon-omega2 and application of recombinant cell line
A recombinant cell line and stable expression technology, applied in the field of genetic engineering, can solve unpredictable problems and achieve high-efficiency and stable expression
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Embodiment 1
[0023] Example 1RFEIFN-Ω expression plasmid construction and identification
[0024] Get the FeiFN-ω2 sequence from GenBank (login number: DQ420221), which is introduced into endozyme sites AVR II and PAC I at its 5 'end and 3' end, and after AVR II enzyme dug point, GCCACC Sequence, end add 6 × HIS sequence; artificial synthesis of FeiFN-ω2 gene, connect it to the modified UCOE expression vector (Pucoe-M), transformed TOP 10 sensitive state cells to obtain Pucoe-m / RFEiFN-ω2 expression plasmid ( figure 1); Double-digested with Avr II expression plasmid and the Pac I restriction enzyme, plasmid digestion products identified on 1% agarose gels ( figure 2 ) Display, pUCOE-M / rFeIFN-ω2 expression plasmids were successfully constructed.
Embodiment 2
[0025] Example 2 Plasmid transfection and cell selection
[0026] Axygen Plasmid Max Kit using extraction kit pUCOE-M / rFeIFN-ω2 expression plasmid, linearized by the restriction enzyme PvuI, the reference Amaxa Cell Line Nucleofector TM Kit V kit described method, the linearized plasmid was stably transfected into CHO DG44 cells. After transfection cells were serum-free culture 48h, addition of selection medium containing 10 nM MTX in the culture was continued, and then gradually increasing the MTX concentration from 500 nM to 100nM, then cell clones were screened by limiting dilution.
Embodiment 3
[0027] Preparation Example 3 and chromosome probes embodiment
[0028] Cell clones obtained in the primary screening colchicine was added (final concentration 0.3μg / mL), 37 ℃ incubation was continued for 3h, cells were harvested by 0.075mol / L KCl hypotonic treatment, and chromosomes prepared according to conventional methods and G-banding. In linearized pUCOE-M / rFeIFN-ω2 plasmid as a template, using DNA probes prepared by random primer specific legal, used in conjunction with cell clones inserted rFeIFN-ω2 specific target gene, the probes were purified and labeled with reference " Random PrimedDNA Labeling Kit "(1104760001, Roche), and" PCR Purification Kit "instructions operate.
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