We transformed the fip-fve gene into Pichia pastoris GS115 for inducible and constitutive expression to obtain feasible bioactvie recombinant Fip-fve. The fip-fve gene was cloned from Flammulina velutipes fruting body by PCR and ligated to pPIC9 to construct inducible expression vector pPIC9-FIP-fve, and promotor pgap was used to replace the paox1 to construct constitutive expression vector pPIC9-PGAP-FIP-fve. These two vectors were used to transform P. pastoris by PEG method. The fip-fve was expressed after histamine-absence screening and yeast colony PCR. The inducible expression level reached 158.2 mg/L at the fourth day and the constitutive expression level was 46.3 mg/L and 29.5 mg/L using glucose and glycerol, respectively. The SDS-PAGE and Western blotting both proved the correctness of rFip-fve, and the hemagglutination test indicats the rFip-fve's bioactivity.
Electrophoresis, Polyacrylamide Gel ; Flammulina ; chemistry ; Fungal Proteins ; biosynthesis ; Genetic Vectors ; Pichia ; metabolism ; Polymerase Chain Reaction ; Promoter Regions, Genetic ; Recombinant Proteins ; biosynthesis

Objective:To investigate the correlation between concomitant diabetes mellitus and clinical features in patients with COVID-19.Methods:Fifty patients with COVID-19 admitted to the Sixth People′s Hospital of Shenyang were divided into 2 groups with and without diabetes mellitus. Indexes such as hospitalization days, duration of positive virus-PCR result, secondary infection, duration of fever, rate of liver dysfunction, and rate of cardiac injury were compared between 2 groups.Results:The average hospitalization durations of diabetes group and control group were(24.0±6.8) and(18.4±5.9) days, respectively, and the duration of positive virus-PCR testing results were(16.0±4.5) and(12.3±4.4) days respectively, suggesting longer hospital stay and longer time for virus clearance in the diabetes group than those in the control group(both P<0.05). The rates of secondary infection, severe subtype, liver dysfunction and cardiac injury in the diabetes group increased, though without significant difference( P>0.05). Conclusion:Prolonged duration of positive virus-PCR result and average days of hospitalization in patients with COVID-19 may be associated with diabetes mellitus.