Sterilized/permeability-strengthened protein-human acid fibroblast growth factor fusion protein, preparation method and application thereof
A fibroblast and growth factor technology, applied in the field of genetic engineering antibacterial protein preparation, can solve the problems of difficult protease degradation, long half-life, short half-life, etc., and achieve the effects of inhibiting bacterial infection, improving physiological state, and accelerating wound healing
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Embodiment 1
[0039] 1. Bactericidal / permeability increasing protein BPI 23 and the cloning of human acidic fibroblast growth factor haFGF
[0040] Complete total RNA was extracted from human peripheral blood leukocytes and fetal brain tissue, reverse-transcribed to synthesize cDNA, and used as BPI 23 and haFGF two gene amplification templates. According to the cDNA sequences of BPI (Genebank accession number: NM_001725) and haFGF (Genebank accession number: S67291) published by Genebank, 4 specific primers P1-P4 were designed using Primer Premier 5.0 and DNA club biological software. The primer sequences are shown in SEQ ID NO : as shown in 2 to 5; using cDNA as template, P1 and P2 as primers to amplify BPI including signal peptide 23 cDNA sequence, the upstream P1 contains the BamH I restriction site, and the downstream P2 contains the Xho I restriction site; P3 and P4 are primers for amplifying the haFGF cDNA sequence, the upstream P3 contains the EcoR I restriction site, and the down...
Embodiment 2
[0051] Example 2 Optimization of Induced Expression Conditions
[0052] According to the above method, inoculate and culture the strain with higher expression level, induce expression in PMSF induction medium with a final concentration of 0.5mM, take samples every 24h from the beginning of induction until the 7th day, and compare the expression at different times by SDS-PAGE amount; then with the best induction time, at different temperatures (24°C, 26°C, 28°C, 30°C), different pH values (4.0, 5.0, 6.0, 7.0), different methanol concentrations (0.5%, 1.0%, 1.5%, 2.0%) to induce expression, and SDS-PAGE to compare the expression levels in various situations to determine the best induction expression conditions.
[0053] The results showed that: 28℃, pH5.0, final concentration of methanol 1.0wt%, final concentration of PMSF 0.5mM, induction 4d were the best expression conditions, and the expression level could reach about 25μg / ml.
Embodiment 3
[0054] Example 3 Expanded expression, purification and concentration of fusion protein
[0055] The optimal expression induction conditions (28°C, pH 5.0, final methanol concentration of 1.0%, PMSF final concentration of 0.5mmol / L, induction expression time of 96h) were used to amplify the expression, and the supernatant was collected and filtered through a 0.22μm microporous filter. After membrane filtration, follow the Bio-Rad protein purification system and HisTrap from Amersham Bioscenes TM Purify according to the HP Kit instructions, elute the target protein with PBS solutions containing different concentrations of imidazole, collect the eluate and SDS-PAGE, dialyze the eluted protein containing the target protein with a dialysis bag with a molecular weight cut-off of 3KD, desalt and freeze-dry Concentrate and store at -70°C for later use.
[0056] SDS-PAGE analysis of the purified eluate collected showed that the eluate of 100mmol / L imidazole contained a large amount o...
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