Figure 1.
The structure of insertion sites of the H9N2-AIV-NA and NDV-F gene under the control of the bi-directional promoters on MZC13NA/F genome (GX0101/Δmeq/Kan−/gpt−/NA/F).
Table 1.
List of primers used for construction of different recombinant plasmids.
Figure 2.
Demonstration of H9N2-NA or NDV-F expressing cells in IFA with monospecific sera in CEF transfected with different plasmids or their combinations (×200).
(a) IFA with mouse anti-NA polyclonal serum to NA in CEF co-transfected with pPpp38-NA and pBud-pp38-pp24; (b) IFA with chicken polyclonal anti-F serum to F in CEF co-transfected with pP1.8kb-F and pBud-pp38-pp24; (c) IFA with mouse anti-NA polyclonal serum and chicken polyclonal anti-F serum to NA and F in CEF co-transfected with pPpp38-NA/1.8kb-F and pBud-pp38-pp24; (d) Detection of NA and F in CEF transfected with pPpp38-NA/1.8kb-F only.
Figure 3.
Demonstration of H9N2-NA or NDV-F expressing cells in IFA with specific antibodies in GX0101-CEF transfected with different plasmids (×200).
(a) IFA with mouse anti-NA polyclonal serum to NA in GX0101-CEF transfected with pPpp38-NA; (b) IFA with chicken polyclonal anti-F serum to F in GX0101-CEF transfected with pP1.8kb-F; (c) IFA with mouse anti-NA polyclonal serum to NA in GX0101-CEF transfected with pPpp38-NA/1.8kb-F; (d) IFA with chicken polyclonal anti-F serum to F in GX0101-CEF transfected with pPpp38-NA/1.8kb-F; (e,f) IFA with mouse anti-NA polyclonal antibody against NA in GX0101-CEF; (g,h) IFA with chicken polyclonal anti-F serum against F protein in GX0101-CEF.
Table 2.
Comparison of the expression of NDV-F or AIV-NA in the presence or absence of pp38/pp24 dimers via IFA.
Figure 4.
Southern blot of PCR products amplified from GX0101 and its recombinant genomes to demonstrate foreign genes.
(a) Amplification of MZC13NA/F and GX0101 US2 gene by PCR. The US2 gene was amplified from DNAs prepared from the MZC13NA/F-infected CEF cells, or GX0101-infected CEF cells. The reaction products were submitted to agarose gel electrophoresis and stained with ethidium bromide; (b) The same DNAs and the NA ORF amplified from DNAs prepared from MZC13NA/F-infected CEF cells were processed for Southern blot analysis with the NA probe; (c) The same DNAs and the F ORF amplified from DNAs prepared from MZC13NA/F-infected CEF cells were processed for Southern blot analysis with the F probe.
Figure 5.
Growth curves (one-step growth kinetics) of GX0101 and its recombinant MZC13NA/F.
After inoculation of CEFs with 100-infection until maximal titers were reached. The data of the growth curve of MZC13NA/F were cited from reference [40]. The results represent the mean±standard deviations of three independent replicates.
Figure 6.
Plaque sizes of Meq-deleted MDV GX0101 and its recombinant MZC13NA/F in infected CEF cells at second passage (×200).
The mouse anti-pp38 monoclonal antibody H19 bound to the MZC13NA/F (a) or its parent GX0101 (b) infected CEF cells by IFA, or to the CEF cells (c). MZC13NA/F plaque size was similar to GX0101 at some level, 96 hours post-infection showing bulging and aggregation of rounded cells.
Figure 7.
Standard curve of NA, F and pp38 assay.
Copy number for the plasmid pMD18-NA, -F or -pp38 were respectively determined spectrophotometrically and diluted serially from 109 copies/µL to 101 copies/µL for use as standard controls. Standard curve (y = 39.369−3.122x, y = 31.533−3.332x and y = 37.614−3.299x) for NA, F and pp38 genes quantification (R2 = 0.9918, 0.9906 and 0.9978; Efficiency = 100±1.5%) was analyzed with the GraphPad Software 5.0®. The concentration refers to the template copy number per reaction.
Table 3.
The absolute quantification (AQ) values of cDNAs of different genes (copies/ul).
Figure 8.
Demonstration of H9N2-NA or NDV-F expressing cells in IFA with monospecific sera in the MZC13NA/F-infected CEF (×200).
(a) Distribution of NA was detected by staining with mouse anti-NA polyclonal antibody and Fluorescein Isothiocyanate-labelled anti-mouse secondary antibody (green fluorescence); (b) Distribution of F expression was detected using NDV virus-specific chicken antiserum and phycoerythrin-conjugated goat anti-chicken IgG (red fluorescence); (c) The merge including the visualization of red and green images showed that the co-expression proteins were visible in the cytoplasm or on the cell surface; (d) Photo was taken under regular light from the same plaque in the same visual field.
Figure 9.
Western blot analyses of recombinant NA and F proteins co-expressed in MZC13NA/F infected CEF cells.
CEF lysates prepared from the MZC13NA/F-infected CEF cells were subjected to SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The blotted membrane was blocked and reacted with mouse anti-pp38 monoclonal antibodies H19, mouse anti-NA polyclonal serum and chicken antiserum raised against NDV followed by HRPO-conjugated goat anti-mouse or chicken IgG antibodies and ECL Western blotting detection reagents. The prestained molecular size marker was included in the same gel and three experiments were accomplished independently.
Table 4.
Comparison of relative expression levels of H9N2-NA and NDV-F to MDV-pp38 in MDV- GX0101 and its recombinant with H9N2-NA and NDV-F genes.