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A nanobody targeting porcine pseudorabies virus gd protein, preparation method and application

A porcine pseudorabies virus and nano-antibody technology, applied in the biological field, can solve the problems of no commercial products and no nano-antibodies, and achieve the effects of simplifying the production process, reducing production costs, and broad application prospects

Active Publication Date: 2022-06-07
重庆市动物疫病预防控制中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are no relevant research reports on the application of nanobodies in PRV diagnostic methods, and there are no commercial products.

Method used

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  • A nanobody targeting porcine pseudorabies virus gd protein, preparation method and application
  • A nanobody targeting porcine pseudorabies virus gd protein, preparation method and application
  • A nanobody targeting porcine pseudorabies virus gd protein, preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Soluble expression and purification of porcine pseudorabies virus gD protein

[0036]The well-grown PK15 cells were inoculated with PRV HN1201 virus (HN1201 strain (Pseudorabiesvirus, strainHN1201), the deposit number is CCTCCNO.V201311; deposited in the China Center for Type Culture Collection; the deposit address is Wuhan University, Wuhan, Hubei Province, and the deposit date is 2013 May 20), using conventional methods in the art to isolate porcine pseudorabies virus genomic DNA as a template, using primers for PCR amplification, using TAKARA's high-fidelity enzyme HS DNA Polymerase with GC Buffer, amplification conditions: 94 ℃ 3min; 94 ℃30s, 68℃90s, 30cycles; 72℃5min. The PCR product was named PRV-gD

[0037] gD upstream amplification primer: GGATCCATGCACGTCGCA (SEQ ID NO.1)

[0038] gD downstream amplification primer: GCGGCCGCCTAGCGACGCGG (SEQ ID NO.2)

[0039] The PCR product PRV-gD with the correct sequencing was cloned into PET-28a (purchased from ...

Embodiment 2

[0040] Example 2 Screening and identification of specific nanobodies against gD protein of PRV

[0041] After the purified soluble PRV-gD protein was fully emulsified with combined Freund's adjuvant, the Bactrian camels were immunized subcutaneously through the neck, and immunized for a total of 6 times at intervals of 2 weeks between each immunization. After the sixth immunization, blood was collected to separate the serum, and gD protein was used as the antigen to determine the antibody titer to monitor the immunization effect. Negative control is camel serum before immunization, get the serum that has been centrifuged after immunization is finished, and ELISA method detects its antibody titer ( figure 2 ), the results showed that the antibody titer in serum reached 1:256000. Lymphocyte RNA was extracted using a kit (purchased from Invitrogen), and the extracted RNA was used as a template using Oligo (dT) 20 primer reverse transcriptase ( III) Synthesize cDNA, and furthe...

Embodiment 3

[0042] Example 3 Preparation of PRV-DND-HRP fusion protein

[0043] The PRV-DND sequence was ligated into pEGFP-N1-HRP vector (Sheng, Y., et al., Nanobody-horseradish peroxidasefusion protein as an ultrasensitive probe) using primers FDND (SEQ ID NO. 4CTGCAGGAAGTACAGCTGGTGGAGTC) and RDND (SEQ ID NO. 5GCGGCCGCTTTCAGAACCAGCTTGGTCCC) to detect antibodies against Newcastle disease virus in the immunoassay. J Nanobiotechnology, 2019.17(1):p.35.), constructed into a pEGFP-DND-HRP expression plasmid, which also carries a secretion signal peptide, HA tag, polyclonal Restriction sites, PRV-DND, horseradish peroxidase and His tag, were sequenced correctly and stored for later use.

[0044] Take HEK-293T cells in good condition and plate them in a 6-well plate at a density of 2-3×10 5 cells / ml, 2ml / well, cultured in a 37°C incubator; when the cells reached 80% confluence, the lipid method was used to transfer pEGFP-DND-HRP into HEK-293T cells (Roche X-tremeGENE HP DNA Transfection Reag...

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Abstract

The invention discloses a nanobody PRV‑DND specifically targeting PRV, and discloses the amino acid sequence of the nanobody. At the same time, a preparation method of the nanobody PRV-DND or fusion protein PRV-DND-HRP is provided. In particular, the use of fusion proteins formed by coupled labeling enzymes as competitive probes provides a competitive ELISA for the detection of PRV antibodies in samples. Based on the advantages of small molecular weight and easy genetic engineering of nanobodies, nanobodies can be combined with enzymes or fluorescent Fusion expression of proteins, etc., which can be directly used in immunological detection, without the need for antibody labeling and the use of secondary antibodies, thus simplifying the production process and greatly reducing production costs. Therefore, screening and preparing nanobodies of PRV antigen proteins, and using them as materials for immunological detection, have very broad application prospects.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a nanobody targeting porcine pseudorabies virus (Porcine Pseudoraibies Virus, PRV) gD protein, a preparation method and an application. Background technique [0002] Porcine Pseudoraibies (PR) is an acute infectious disease of pigs, also known as Aujeszky disease, caused by Porcine Pseudoraibies Virus (PRV). As the natural host of PRV, pigs are more sensitive to virus infection and are the only animal species that can survive acute infection and have latent infectivity. After infection, newborn piglets are characterized by high mortality and neurological disorders, pregnant sows have abortions and reproductive disorders, older pigs are mainly respiratory diseases, and piglets characterized by neurological symptoms have a mortality rate greater than 20%, and pigs are infected with wild-type virus. The probability is as high as 50%, causing huge losses to my country's pig industry. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/08C12N15/13C07K19/00C12N9/08G01N33/535G01N33/569G01N33/68G01N33/58
CPCC07K16/085C12N9/0065G01N33/535G01N33/56994G01N33/581G01N33/6854C12Y111/01007C07K2317/569C07K2317/92C07K2319/61G01N2469/20G01N2333/032
Inventor 董春霞曾政费磊骆璐胡健孙燕蒋佳利贺青松王萍钟莉
Owner 重庆市动物疫病预防控制中心
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