Porcine reproductive and respiratory syndrome virus Nsp2 protein nano-antibody and application thereof
A technology for respiratory syndrome and nano-antibodies, applied in the direction of anti-viral immunoglobulin, applications, antibodies, etc.
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Embodiment 1
[0036]Example 1 Construction of expression diagnostic antigen recombinant plasmid pET-NSP2 and expression identification of recombinant antigen protein
[0037] 1 method
[0038] 1.1 The cultivation of PRRSV HuN4 strain
[0039] Digest Mark-145 cells with 0.25% trypsin, add MEM growth medium containing 6% calf serum, pack into Kirschner flasks for static culture, and replace with MEM maintenance medium containing 3% calf serum after 24 hours of attachment. After the cells form a monolayer, pour off the culture medium. After diluting the lyophilized PRRSV HuN4 virus with the maintenance solution, take 10ml and add it to the cell monolayer for adsorption at 37°C for 30 minutes, add 100ml of the maintenance solution, and continue culturing at 37°C.
[0040] 1.2 Viral RNA extraction
[0041] Freeze and thaw the virus culture repeatedly three times, take 300 μl of the virus stock solution, add 600 μl of TriZol (purchased from Shanghai Sangong Company), shake vigorously several t...
Embodiment 2
[0064] Example 2 Screening and Identification of Porcine Reproductive and Respiratory Syndrome Virus Nsp2 Protein Nanobody
[0065] 1 Materials and methods
[0066] 1.1 Plasmids, strains and experimental animals
[0067] BL21(DE3) competent cells were purchased from Thermo Company; pCANTAB 5E phage, E.coli TG1 and helper phage M13KO7 were donated by Professor Zhou Enmin’s laboratory at Northwest A&F University; 6-8 week-old BALB / c mice were purchased from Beijing Experimental Animals center; adult big brown Bactrian camels were purchased from Inner Mongolia Agricultural University.
[0068] 1.2 Main reagents
[0069] PRRSV antibody-positive pig serum was preserved by our laboratory; mouse anti-His monoclonal antibody (MAb) was purchased from Life Technology Company; goat anti-mouse IgG / horseradish enzyme-labeled and goat anti-rabbit IgG / horseradish enzyme-labeled were purchased from Beijing Zhongshan Jinqiao Company; Ni-NTA Agarose was purchased from GE Company; rabbit anti...
Embodiment 3
[0109] Example 3 Intracellular nanobody activity detection and antiviral function research
[0110] 1 material
[0111] 1.1 Plasmids, cells and viruses
[0112] The pCAGGS vector and the pEGFP-N2 vector are kept in this laboratory; the pET-Nsp2 plasmid is constructed from Example 1; the pCANTAB 5E-Nbs plasmid is constructed from Example 2; PAM immortalized cells and Marc-145 cells are preserved in this laboratory); The highly pathogenic PRRSV HuN4 (GenBank ID: EF635006) is preserved in our laboratory.
[0113] 1.2 Main reagent consumables
[0114]The mouse anti-GFP monoclonal antibody was purchased from Beyond Company, the mouse anti-His monoclonal antibody was purchased from TIANGEN Company, and the mouse anti-HA monoclonal antibody was purchased from Sigma Company; Beads were purchased from Chromotek; goat anti-mouse IgG (DyLight800) antibody was purchased from Thermo Fisher; PCR enzyme Primer Star was purchased from Takara Company; restriction endonucleases EcoRI and X...
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