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Nano antibody of PRRSV N protein, and preparation method and application of nano-antibody

A nano-antibody and protein technology, applied in the biological field, can solve the problems of large-scale, time-consuming, and cumbersome operations that are difficult to clinical at the grassroots level, and achieve good market transformation prospects, simplify production processes, and reduce production costs.

Pending Publication Date: 2021-03-09
NORTHWEST A & F UNIV
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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 PRRSV diagnostic methods, and there are no commercial products.
[0004] So far, the serological detection method for porcine PRRS is currently mainly indirect ELISA, which requires high-purity antigen and enzyme-labeled secondary antibody, resulting in high production costs, cumbersome operation, and long time-consuming for commercial kits. hampers clinical application
For example, IDEXX’s PRRSV antibody detection kit is widely used in the market at present, and its detection effect is good, but due to its high price, it is difficult to promote its use on a large scale in clinical grassroots

Method used

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  • Nano antibody of PRRSV N protein, and preparation method and application of nano-antibody
  • Nano antibody of PRRSV N protein, and preparation method and application of nano-antibody
  • Nano antibody of PRRSV N protein, and preparation method and application of nano-antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Screening and identification of specific nanobodies against PRRSV N protein

[0029] (1) Bactrian camel immunity

[0030] Mix 2mg of PRRSV N recombinant protein with Freund's adjuvant in equal volume and emulsify evenly, immunize a Bactrian camel, once every two weeks, a total of 5 immunizations, Freund's complete adjuvant is used for the first time, and all other 4 times Freund's incomplete adjuvant.

[0031] (2) Construction of phage library

[0032] Four days after the fifth immunization, blood was collected, peripheral blood lymphocytes of camels were separated and total RNA was extracted, and the operation was performed according to the instructions of the QIAGEN RNA extraction kit; The NotI restriction site was ligated into the pMECS phage display vector; the ligation product was electrotransferred into TG1 competent cells, after activation, it was spread on LB-AMP agar plate, cultured overnight at 37°C, and the bacterial lawn was collected to make gly...

Embodiment 2

[0038] Example 2 Preparation of Nanobody Nb1 and Horseradish Peroxidase Fusion Protein

[0039] pEGFP-N1-HRP vector (Sheng, Y., et al., Nanobody-horseradish peroxidasefusion protein as an ultrasensitive probe to detect antibodies against Newcastle disease virus in the immunoassay. J Nanobiotechnology, 2019.17(1): p.35.), DNA Sequences include secretion signal peptide, HA tag, multiple cloning restriction site, horseradish peroxidase and His tag.

[0040] Ligate the camel-derived VHH gene to the pMECS vector, use the obtained pMECS-VHH as a template, and use Nb-F:AA CTGCAG ATGGAGACCGACACC, Nb-R: ATAAGAAT GCGGCCGC TTAGTGGTGATGGTG primer amplifies the VHH sequence, which introduces the enzyme cutting site Pst I ( CTGCAG ) and Not I ( GCGGCCGC ). The VHH gene was obtained by double digestion with Pst I and Not I, and connected to the pEGFP-N1-HRP vector. The ligation product was transformed into Escherichia coli and cultivated, and a single colony was picked, and after se...

Embodiment 3

[0048] Example 3 Identification of specific nanobody Nb1 against PRRSV N protein

[0049] Identification of specific nanobodies against PRRSV N protein by enzyme-linked immunosorbent assay (ELISA)

[0050] (1) Coating plate: Coat the ELISA plate (400ng / well) with prokaryotic expressed PRRSV N protein and chicken Newcastle disease virus (NDV) NP protein (irrelevant protein control).

[0051] (2) Blocking: wash the plate 3 times with PBS'T, add 200 μL of 2.5% skimmed milk powder to each well and incubate at room temperature for 1 h.

[0052] (3) Antibody incubation: wash the plate 3 times with PBS'T, add PRRSV-N-Nb1-HRP fusion protein to the coated ELISA plate, and incubate at room temperature for 30 minutes.

[0053] (4) Color development: wash the plate 3 times with PBS’T, add commercialized TMB color development solution (Tiangen Biochemical Technology Co., Ltd.), and after 10 minutes of color development in the dark, add 3M concentrated H 2 SO 4 .

[0054] (5) Reading: r...

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Abstract

The invention discloses a nano-antibody of PRRSV N protein, and a preparation method and application of the nano-antibody, and belongs to the technical field of biology. The amino acid sequence of thenano-antibody is shown as SEQ ID No.1 in a sequence table. The invention also discloses an expression preparation method of the nano-antibody and horseradish peroxidase (HRP) fusion protein, and theapplication of the nano-antibody and the HRP fusion protein in detection of PRRSV antibodies in pig serum is simultaneously evaluated. The anti-PRRSV N protein nano-antibody and the HRP fusion proteinare applied to establishment of competitive ELISA for detecting PRRSV antibodies in pig serum, and the established method is simple to operate, short in sample detection time, and free of an enzyme-labeled secondary antibody. A key material is provided for subsequent development of a product for applying the nano antibody to PRRSV antibody detection.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a nanobody of PRRSV N protein and its preparation method and application. Background technique [0002] Porcine Reproductive and Respiratory Syndrome (PRRS), commonly known as porcine blue ear disease, is a reproductive disorder and respiratory disease of pigs of all ages caused by porcine reproductive and respiratory syndrome virus (PRRS virus, PRRSV) The severe infectious disease characterized by it has brought huge economic losses to the global pig industry. PRRSV is an enveloped, single-stranded positive-sense RNA virus that contains more than 10 open reading frames (ORFs) ORF1a, ORF1b, ORF2a, ORF2b, ORF3, ORF4, ORF5, ORF5a, ORF6, and ORF7. The content of ORF7 (nucleocapsid protein, N protein) in PRRSV virions is higher than other structural protein components, accounting for 20%-40% of viral proteins; the amino acid sequence homology of N protein between different strains reach...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13C07K19/00C12N15/85G01N33/535G01N33/569G01N33/68
CPCC07K16/10C12N9/0065C12N15/85G01N33/56983G01N33/535G01N33/6854C12Y111/01007G01N2333/08G01N2469/20C07K2317/569C07K2319/61
Inventor 孙亚妮段红赵钦周恩民
Owner NORTHWEST A & F UNIV
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