Experimental infection of chickens with MDV
Twenty 1-day-old SPF chickens were obtained from Shandong Academy of Agricultural Sciences Poultry Institute SPF Chicken Research Center (Jinan, China). They were randomly divided into two equal groups as an infection group and a control group. Two groups of chickens were raised separately in isolators with filtered air under positive pressure. At the first day of the experiment, 2,000 plaque forming units (PFU) of vvMDV (GX0101), which normally cause tumors at the tenth week, was inoculated intra-abdominally (i.a.) into each chicken in the infection group. The control group was inoculated with PBS. At the tenth week, serum was collected from seven chickens each group. The experiment ended after ten weeks. All chickens were carbon dioxide euthanized and their liver and spleen were collected.
Natural infection of chickens with MDV
Seven 160-days-old laying hens were gotten from a local flock. They were confirmed as MDV natural infection and absence of other common viral diseases by PCR detection. Their serum was collected for detecting P53 antigen and antibody. Their tissue samples, including liver, spleen, pancreas and bursa of Fabricius, were collected and fixed with 4% formaldehyde for 48h for histopathological and immunohistochemical studies.
Histopathology and Immunohistochemical
The fixed liver, spleen, pancreas and bursa of Fabricius were dehydrated, waxed, and cut into 3μm slices, followed by Haematoxylin and eosin (H-E) staining for histopathology observation. In immunohistochemical (IHC) processing, tissues were cut into sections of 3μm thickness and mounted on microslides treated with 0.1% poly-L-lysine. The sections were deparaffinized in xylene and rehydrated in a graded series of ethanol solutions into PBS, then were pre-treated in citrate buffer (0.01mol/L; pH 6.0; 100°C) for antigen retrieval by microwaving and cooled at room temperature for 20 min . Endogenous alkaline peroxidase was quenched with 3% hydrogen peroxide solution in methanol for 30 min . Nonspecific antibody binding sites were prevented by incubating the sections with 5% fetal bovine serum for 60 min at room temperature. Following this, the anti-P53 polyclonal antibody (BOSTER, China) was diluted into 1:200 as primary antibody and immersed overnight at 4°C in a black humid chamber. The secondary antibodies were HRP (horse radish peroxidase)-conjugated goat anti-mouse IgG (CWBIO, China). Immunoreactivity was then visualized with diaminobenzidine (DAB) staining. The sections were counterstained with hematoxylin and mounted. Negative controls were incubated with PBS instead of the primary antibody in the immunohistochemical analysis.
Total RNA isolation and reverse transcription
Total RNA of liver and spleen tissue samples (50mg/tissue) were isolated by using TRIzol reagent (Takara, Japan) following the manufacturer’s instructions. Extracted total RNA (1ug) was reverse transcribed to cDNA with PrimeScript™ RT Reagent Kit (Roche, Switzerland). According to the published complementary DNA (cDNA) sequence of the chicken p53 (GeneBank accession number: nm205264), specific primers were designed to amplify the DNA-binding domain (DBD) of p53, which were shown in Table 1. Using PCR (Polymerase Chain Reaction) techniques, p53 cDNA genes sequences were amplified. The PCR reaction was performed by using thermal cycler (Takara, Japan) under the following conditions: 95℃ for 5min; followed by 35 cycles of 95℃ for 30s, 56℃ for 30s, 72℃ for 45s, and then a final 72℃ for 10min extension cycle. DNA templates that were acquired from the MDV positive chickens were amplified using primer pair p53-1/-2. The SPF chicken were regarded as negative control. DNA fragments were successfully amplified with sizes of 687bp. The target fragment was gel extracted and connected to pEASY-T1 vector. Then the recombinant plasmid was transformed in bacteria and made sequencing analysis. Finally, the sequencing results were compared with the wild type p53 cDNA sequences reported previously.
Enzyme-linked immunosorbent assay (ELISA) for P53 and P53 antibody
The antigen and antibody levels of chicken P53 were monitored by ELISA (Mlbio, China). All processes were implemented in accordance with the instructions, and the absorbance (OD) of each sample was finally measured at a wavelength of 450 nm.
Statistical analyses were conducted with GraphPad. The Non-paramatic T test was used for statistical analysis of differences about the level of P53 and P53 antibody. Statistical significance was designated as *p < 0.05 or ** p < 0.01.