Porcine reproductive and respiratory syndrome virus nsp2 protein nanobody and its application

A technology of respiratory syndrome and nano-antibody, applied in anti-viral immunoglobulin, antibody, application, etc.

Active Publication Date: 2021-10-08
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In contrast, nanobodies are rarely reported in animal viral diseases

Method used

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  • Porcine reproductive and respiratory syndrome virus nsp2 protein nanobody and its application
  • Porcine reproductive and respiratory syndrome virus nsp2 protein nanobody and its application
  • Porcine reproductive and respiratory syndrome virus nsp2 protein nanobody and its application

Examples

Experimental program
Comparison scheme
Effect test

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 XH...

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Abstract

The invention discloses a porcine reproductive and respiratory syndrome virus Nsp2 protein nanobody antibody and application thereof. The nanobody is named Nb12, and its amino acid sequence is shown in SEQ ID NO.3. The present invention separates camel peripheral blood lymphocytes after immunizing camels with truncated recombinant protein Nsp2, uses RT-PCR to amplify the immunized Bactrian camel VHH gene, connects it into pCANTAB 5E phage vector, and constructs Bactrian camel heavy chain antibody A total of 44 strains of Nsp2-specific nanobodies were screened from the variable region library using phage display technology after three rounds of panning. Tests have shown that these nanobodies can specifically react with Nsp2 protein, and Nb12 has a strong binding force. The results of the antiviral test showed that the nanobody Nb12 showed inhibitory effect on virus replication on both PAM cells and Marc-145 cells when the inoculated dose was 0.01 MOI. Therefore, the proposal of the present invention provides a new technical means for the subsequent research on PRRSV Nsp2 and the prevention, control and diagnosis of PRRSV.

Description

technical field [0001] The invention relates to a nanobody and its application, in particular to a porcine reproductive and respiratory syndrome virus Nsp2 protein nanobody and its application, and the invention belongs to the field of biotechnology. Background technique [0002] Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome, PRRS) is a viral infectious disease caused by PRRS virus (Porcine reproductive and respiratory syndrome virus, PRRSV). Respiratory diseases of pigs cause irreparable economic losses to the pig industry. Due to the high variability and antibody-dependent enhancement of PRRSV, existing vaccines cannot provide complete immune protection to pig herds, so it is necessary to try to study new antiviral strategies. PRRSV nonstructural protein 2 (Nonstructural protein 2, Nsp2) is the largest nonstructural protein of PRRSV, its cis structure and trans structure both have protease activity, which is crucial to the vi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/10C12N15/13A61K39/42A61P31/14
CPCA61K2039/505A61P31/14C07K16/10C07K2317/22C07K2317/33C07K2317/35C07K2317/569
Inventor 蔡雪辉刘永刚孙明霞宋欢
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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