Rabbit hemorrhagic disease virus VP60 recombinant antigen with two-site chimeric pasteurella PlpE epitopes as well as preparation and application of rabbit hemorrhagic disease virus VP60 recombinant antigen
A rabbit hemorrhagic virus, Pasteurella technology, applied in the field of biomedicine
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Embodiment 1
[0028] Example 1: Preparation of VP60 recombinant protein chimeric with PlpE epitope
[0029] (1) Plasmids, strains and culture vectors Plasmids pFastBac1 and pFastBac1-VP60 were constructed and preserved by our laboratory; E. coli Competent DH5α was purchased from Nanjing Nuoweizan Biotechnology Co., Ltd.; E. coli Competent DH10 Bac was purchased from Shanghai Weidi Biotechnology Co., Ltd.; Pasteurella multocida C51-17 strain was preserved and provided by our laboratory; sf9 insect cells were preserved and cultured by our laboratory.
[0030] Protein amino acid sequence analysis was carried out using the PlpE gene sequence in the whole genome sequence of Pasteurella multocida C51-17 strain published by Genbank as the reference sequence. B-cell epitope distribution of PlpE was determined using Bepipred-2.0 (http: / / tools.immunepitope.org / bcell / ).
[0031] The B cell epitope prediction results of the above PlpE are as follows: figure 1Shown: PlpE primary structure conta...
Embodiment 2
[0034] Example 2 Hemagglutination test of two-site chimeric VP60-PlpE recombinant protein
[0035] Add VP60-PlpE and wild-type baculovirus cultures to two wells of a 50-well U-shaped plate, 50 μL per well. Then 50 μL of 1% human type O red blood cell suspension was added to each well, and after 30 min at 4 °C, the hemagglutination was observed. The results showed that VP60-PlpE recombinant protein had obvious hemagglutination phenomenon ( Figure 4 ), that is, it is considered that the VP60-PlpE recombinant protein can still form RHDV-like particles and effectively display the neutralizing epitope of RHDV.
Embodiment 3
[0036] Example 3 Two-site chimeric VP60-PlpE recombinant protein reacts with PlpE hyperimmune serum
[0037] The VP60-PlpE recombinant protein and VP60 recombinant protein (control) were subjected to SDS-PAGE gel electrophoresis and then transferred to NC membrane. After blocking with 5% skim milk, add 1 / 500 diluted mouse anti-PlpE serum preserved in our laboratory, incubate at 37°C for 1 h; rinse with PBST 5 times, add 1 / 10000 diluted HRP-coupled goat anti-mouse IgG, incubated at 37°C for 1 h; rinsed with PBST 5 times, and developed with ECL. Western blot results showed that there was a band of about 68 kDa in the VP60-PlpE lane ( Figure 5 ), but the VP60 lane as a control does not have a band of the expected size, which indicates that the VP60-PlpE recombinant protein can specifically react with mouse anti-PlpE serum, that is, the partial immunogenic fragment of PlpE is smoothly integrated into VP60 .
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