Fusion protein, encoding gene and uses thereof
A technology of fusion proteins and genes, applied in the direction of peptide/protein components, hybrid peptides, drug combinations, etc., to achieve the effect of prolonging half-life, improving therapeutic effect, and reducing the production of antibodies
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Embodiment 1
[0040] Example 1, Preparation of Albumin and Urate Oxidase Fusion Protein
[0041] 1. Acquisition of human serum albumin coding gene
[0042] Using the human fetal liver cDNA library (purchased from Clontech, product number 637230) as a template, primers were designed to amplify the human serum albumin gene (HSA) without the signal peptide coding sequence. The specific primers are as follows:
[0043] HSA5:5′-TCG CTCGAG AAAAGAGATGCACACAAGAGTGAGG-3' (XhoI),
[0044] HSA3:5′-TT GGATCC ACCTCCACCGGAACCTCCACCTCCTAAGCCTAAGGCAGCTTGACTTG-3' (BamHI).
[0045] PCR amplification system: human fetal liver cDNA 1.0 μL, HSA5 primer and HSA3 primer 0.5 μL each, dNTP 2 μL, KOD DNA Polymerase 0.1 μL, 10×KOD buffer 2 μL, MgSO 4 2 μL, ddH 2 O 35 μL, total volume 50 μL. The PCR reaction conditions were: 94°C for 30s, 53°C for 30s, 72°C for 2min, a total of 30 cycles.
[0046] The PCR amplification product was detected by agarose gel electrophoresis, and the results showed that a band of...
Embodiment 2
[0071] Embodiment 2, the determination of the immunogenicity of fusion protein in rat body
[0072] Male SD rats were divided into two groups, HSAUOA fusion protein group and rUO protein group (for the preparation process of rUO protein, see: Wu Weili, Fang Hongqing, etc. Expression, purification and active form identification of uric acid oxidase. Journal of the Academy of Military Medical Sciences, 2005, 29(2): 124-126.), five rats in each group were intravenously injected with the HSAUOA fusion protein prepared in Example 1 above and the rUO protein prepared according to the above literature method. The dosage of HSAUOA fusion protein group was 0.9 mg / kg body weight, and the dosage of rUO group was 0.3 mg / kg body weight. Administered once a week for four weeks. Blood was collected before the first administration and 14, 21, 28, and 35 days after the first administration, and the serum was separated, put in a 4°C refrigerator for testing, and observed continuously for 2 mon...
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