Table 1.
Numbers of transgenic chickens in generations of G3 and G4.
Fig 1.
Expression of rHLY in transgenic chickens.
(A) RT-PCR analysis of the expression of rhLY in G3 and G4 hens. Total RNA was isolated from the heart, liver, spleen, lung, kidney, intestine and oviduct of the G3 and G4 hens. NC (negative control) was the RNA of the oviduct from a non-transgenic hen. The 460bp fragments represented the RT-PCR products of hLY, and the 81bp fragments represented the RT-PCR products of GAPDH. M, DNA ladder. (B) Western blot of egg whites from a G3, a G4 and a non-transgenic hen (NC). Samples were separated by SDS-PAGE and hybridized with anti-hLY antibody and anti-cLY antibody, separately. hLY, commercial hLY (positive control for anti-hLY); cLY, commercial cLY (positive control of anti-cLY).
Fig 2.
Immunofluorescence detection of hLY expression in oviduct sections.
Sections of the magnum portions of the oviducts from a G3, a G4, and a wild type White Leghorn hen were immunolabeled with the anti-hLY antibody (red). The nuclei were stained with DAPI (blue). Scale bar: 25 μm.
Fig 3.
Purification of rhLY by cation-exchange chromatography and gel filtration chromatography.
(A) Purification of rhLY by cation-exchange chromatography through the Hiscreen SP FF prepacked column. Lyophilized powder (20 g) from transgenic egg whites was dissolved in 20 mM sodium phosphate buffer (pH 8.5) and applied to the column. F, the flow through; P, the elution peak. (B) Examination of the flow-through and elution peak from the Hiscreen SP FF prepacked column by 15% SDS-PAGE and Coomassie blue. (C) Western blot of the flow-through and elution peak from the Hiscreen SP FF prepacked column. (D) Purification of rhLY from the first chromatographic step by the Mono S 5/50 GL prepacked column. The P fraction from the first step was loaded on the column after being desalted by 20 mM sodium phosphate buffer (pH 7.0). P1, P2, P3, P4 and Mix represented the elution peaks. (E) Examination of the elution peaks from the second chromatographic step by 15% SDS-PAGE and Coomassie blue staining. (F) Western blot of the elution peaks from the second step. (G) Gel-filtration chromatography of the partly purified rhLY from the second step through the SuperdexTM 75 10/300GL prepacked column. The P3 fraction from the second chromatographic step was applied to the column after being concentrated with an ultrafiltration centrifugal tube. P1 and P2 represented different elution peaks. (H) Examination of the rhLY from the third chromatographic step by 15% SDS-PAGE and Coomassie blue staining. (I) Western blot identification of the elution peaks from Gel-filtration chromatography. hLY, commercial hLY (positive control for anti-hLY); cLY, commercial cLY (positive control for anti-cLY).
Table 2.
Compare the properties of the purified rhLY and commercial hLY.
Fig 4.
Antibacterial activity of rhLY, hLY, and cLY against M. lysodeikticus.
The picture showed the inhibition zones of the agar disc diffusion of commercial hLY (A), commercial cLY (B), purified rhLY (C), and sterile water (D, negative control).
Table 3.
Antibacterial activity of rhLY, hLY and cLY against M. lysodeikticus determined with the turbidimetric method.
Fig 5.
Optimal conditions of purified rhLY and hLY for their antibacterial activity.
(A) The optimal temperatures of the rhLY and hLY against M. lysodeikticus were measured in phosphate buffered saline (pH 7.18) at 25°C, 40°C, 60°C, and 80°C respectively. (B) The optimal pH values of the rhLY and hLY for the antibacterial activity was measured in phosphate buffer with different pH values (from 2–12), separately. hLY, commercial hLY; rhLY, purified rhLY. The experiment for each group was repeated at least three times, and the results were presented as mean ± S.D.
Fig 6.
Thermostability of purified rhLY and hLY at 100°C, 80°C and 60°C.
The lysozyme samples were first incubated for different periods of time at 100°C (A), 80°C (B), and 60°C (C), and their antibacterial activity against M. lysodeikticus was measured in phosphate buffered saline (pH 7.18) at room temperature. Lysozyme activity without heat treatment was defined as 100% activity. hLY, commercial hLY; rhLY, purified rhLY. The experiment for each group was repeated at least three times, and the results were presented as mean ± S.D.
Fig 7.
Stability of purified rhLY and hLY under different pH conditions.
The lysozyme was incubated in phosphate buffer of different pH values (from 2–12) for 20 minutes. Then the antibacterial activity against M. lysodeikticus was measured in phosphate buffered saline (pH 7.18) at room temperature. The lysozyme activity incubated in phosphate buffer at pH 7 was defined as 100% activity. hLY, commercial hLY; rhLY, purified rhLY. The experiment for each group was repeated at least three times, and the results were presented as mean ± S.D.