Fig 1.
Scale-up expression of rTP4 in P. pastoris in a 500-liter fermenter.
(A) The dissolved oxygen (%), pH, RPM and temperature (°C) of broth during incubation of a P. pastoris X-33 transformant in BSM. (B) Western blot analysis profiles of recombinant rTP4 expression. M: Molecular weight marker; sTP4: Synthetic TP4 with concentrations of 10, 5, 2.5, 1.25 and 0.635 ng/μL; rTP4: Recombinant TP4.
Fig 2.
The effects of rTP4 on gram-negative and gram-positive strains by coincubation assay.
After incubation, the culture medium was measured at a wavelength of 600 nm. A low OD600 value indicates that bacterial growth was inhibited. Vector control: Empty pPICZαA vector. TP4: Protein expressed by the pPICZαA-TP4 vector.
Fig 3.
Flow cytometry phagocytosis assay.
Flow cytometry analysis using cell size forward-scattered light (FSC) and side-scattered light (SSC) parameters was performed. (A) RAW264.7 cells alone, (B) RAW264.7 cells coincubated with E. coli, and (C) RAW264.7 cells treated with rTP4 and coincubated with E. coli. (D) The mean fluorescence intensity (MFI) of RAW264.7 cells was analyzed, and the asterisks indicate significant differences.
Table 1.
The antimicrobial activity and effect of temperature of rTP4.
The rTP4 was expressed by P. pastoris X-33 and supernatant were collected after induction and applied to disk diffusion assay. After incubation, the inhibition zone diameters were measured on the culture plate. Representative radial diffusion assays are shown. (A) The antimicrobial activity of rTP4. (B) The effect of temperature on rTP4 antimicrobial activity.
Fig 4.
Effect on feed parameter analysis.
(A) Weight for day 2 and (B) day 36 analysis between groups. (C) weight gain, (D) FE (feed efficiency), and (E) PER (protein efficiency ratio) of G. g. domesticus fed diets containing rTP4 (0.75, 1.5, 3.0, 6.0 or 12%) spray-dried powder for 5 weeks. Each bar represents the mean value from three determinations, with the standard error (SE). Data (mean ± SE) with star marker represent differ significantly (P<0.05) among treatments.
Fig 5.
Histological analysis of tissue sections.
Representative photomicrographs of tissue sections stained with H&E. A: Spiraline-A group (No. 50); B: 3% rTP4 group (No. 99); C: Basal diet group (No. 90).
Fig 6.
Effect of orally administered 3% rTP4, Spiraline-A and basal diet on the intestinal microflora in G. g. domesticus.
Control: Basal diet group; AMP: 3% rTP4 group; antibio: Spiraline-A group.
Table 2.
Effect of weight gain ratio, feed efficiency (FE), protein efficiency ratio (PER) and survival rate of G. g. domesticus fed with diets containing rTP4 (0.75, 1.5, 3.0, 6.0 and 12%) spray-dried powder for 5 weeks1.