Novel therapeutic enzyme fusion protein and use thereof
A fusion protein and non-fusion technology, applied in the direction of fusion polypeptide, immunoglobulin, peptide/protein components, etc., can solve the problems of inability to maintain the activity of fusion protein, increase the duration of fusion protein, instability, etc.
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Embodiment 1
[0187] Embodiment 1: Preparation of fusion protein expression vector
[0188] For the production of enzyme fusion proteins, expression vectors (IDS cDNA, catalog number EX-C0003-M02, Gencopoeia; ARSB cDNA, cat. - sulfatase (IDS, SEQ ID NO: 1) and arylsulfatase B (ARSB, SEQ ID NO: 3) were inserted into -, the synthetic linker (SEQ ID NO: 5) and the IgG4 Fc region (SEQ ID NO: 3) respectively ID NO: 7) The expression vector of the fusion protein was prepared by overlapping PCR. Since the overlapping PCR technique includes sequences overlapping the primers when amplifying each of the enzyme and linker-Fc, the resulting PCR products will include overlapping sequences. To amplify the fusion protein, the following PCRs were performed: 1) primary PCR (25 cycles consisting of 95°C for 1 minute; 57°C for 30 seconds; 68°C for 3 minutes) and 2) secondary PCR (25 cycles , which consisted of 95°C for 1 minute; 57°C for 30 seconds; and 68°C for 4 minutes).
[0189] Specifically, for IDS...
Embodiment 2
[0204] Example 2: Transformation of CHO cell lines using expression vectors for fusion proteins
[0205] The recombinant expression vector pX0GC-enzyme-Fc prepared in Example 1 was introduced into the DG44 / CHO cell line (CHO / dhfr-) (Urlaubet et al., Somat. Cell. Mol. Genet., 12, 555 to 566, 1986)—— In which the DHFR gene is disrupted, and thus its nucleic acid biosynthesis process is imperfect - a transformant is obtained, and an enzyme fusion protein (enzyme-Fc) is expressed in the transformant.
[0206] Specifically, the DG44 / CHO cell line was grown to confluence such that the cells covered about 80% to about 90% of the bottom of the vessel, and the cells were washed 3 times with Opti-MEM (Gibco, Cat. No. 51985034).
[0207]Meanwhile, a mixture of Opti-MEM (3 mL) and pX0GC-enzyme-Fc (expression vector, 5 μg) and a mixture of Opti-MEM (3 mL) and lipofectamine 2000 (Gibco, catalog number 11668-019, 20 μL) was left at room temperature 30 minutes. Then, the two mixtures were...
Embodiment 3
[0209] Embodiment 3: Confirm the expression of IDS-Fc, ARSB-Fc fusion protein by ELISA
[0210] Part of the transformed cells prepared in Example 2 was mixed with 1×10 7 A concentration of two cells was transferred to each 175-T cell culture flask and cultured until the cells nearly covered the bottom of the culture vessel, and then 15 mL of serum-free Ex- cell medium (purchased from Sigma according to custom order, catalog number 14360C) was added to each flask, and it was placed in an incubator (33°C, 5% CO 2 ) for 48 hours. Each cell culture was transferred to a 50 mL tube, centrifuged and the supernatant collected again and the expression levels of the fusion proteins (IDS-Fc and ARSB-Fc) measured.
[0211] First, the expression level of IDS-Fc was performed by applying an indirect ELISA method. Human α-IDS antibody (R&D Systems, Cat. No. AF2449) diluted in PBS at a concentration of 1 μg / mL was added to a 96-well ELISA plate (Nunk, Cat. No. 44-2404-21) in an amount of...
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