Pig Delta coronavirus N-protein monoclonal antibody and epitope and application thereof

A monoclonal antibody, coronavirus technology, applied in the field of bioengineering, can solve the problems of differential diagnosis, no differential diagnosis of PDCoV-specific antibodies, etc.

Active Publication Date: 2019-05-24
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The newly discovered PDCoV belongs to a new type of coronavirus, that is, the deltacoronavirus subgroup (DeltaCoronavirus). It has been confirmed that the virus infection has been prevalent in pigs in my country, and the clinical symptoms caused by its infection are very similar to those of PEDV and TGEV infection. It is difficult for people to make a differential diagnosis with the naked eye in clinic. Effective drugs or vaccines, and no specific antibodies for differential diagnosis of PDCoV infection

Method used

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  • Pig Delta coronavirus N-protein monoclonal antibody and epitope and application thereof
  • Pig Delta coronavirus N-protein monoclonal antibody and epitope and application thereof
  • Pig Delta coronavirus N-protein monoclonal antibody and epitope and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Preparation of porcine Delta coronavirus nucleoprotein (PDCoV N protein) antibody

[0029] 1. Materials and Methods

[0030] 1.1 Materials

[0031] 1.1.1 Virus strains, cells and experimental animals

[0032] PDCoV strain JS, ST cells, and SP2 / 0 cells were all preserved by the Shanghai Veterinary Research Institute of the Chinese Academy of Agricultural Sciences; 3-4-week-old female Balb / c mice were purchased from Shanghai Slack Company.

[0033] 1.1.2 Main reagents and antibodies

[0034] DMEM was purchased from Gbico, fetal bovine serum for cell culture was purchased from BI Company; complete Freund's adjuvant, incomplete Freund's adjuvant, HT, HAT, PEG2000 were purchased from Sigama-Aldrich; reverse transcription kit, protein pre-stained Marker , BCA protein quantification kit was purchased from Thermo Fisher; pCold I, pCold TF, E.coli BL21 competent cells, LA Taq Premix, In-Fiusion HD Cloning Kit, T4 Ligase were purchased from Takara Company; Ni-NTA was...

Embodiment 2

[0057] Example 2 Identification of Monoclonal Antibody Epitopes

[0058] 1. Method

[0059] 1.1 Segmented amplification and cloning of PDCoV N protein

[0060]According to the above test results, the N1 gene after the PDCoV N protein segmentation was continued to be further truncated and segmented, and the N1 segment was divided into three overlapping small segments, each segment was named N1-1, N1 -2, N1-3. Corresponding homologous recombination primers were designed for each fragment (see Table 1 for the length and primer sequence of each fragment). XhoI and EcoR I restriction sites were introduced, respectively. Then use the primer pairs for each truncated fragment, and use the N gene of PDCoV as a template to perform PCR amplification on each segmented gene. The PCR reaction system is 50 μL, including: 25 μL LA TaqPremix, 22 μL ddH2O, upstream and downstream 1 μL of each primer and 1 μL of cDNA template. The reaction program of PCR amplification is: pre-denaturation a...

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Abstract

The invention discloses a pig Delta coronavirus N-protein monoclonal antibody which is secreted from hybridoma cell strain with the collection number CCTCC NO:C2017257, further discloses a pig Delta coronavirus detection kit containing the monoclonal antibody, and pig Delta coronavirus N-protein epitope of which the amino acid sequence is as shown as the SEQ ID NO.15. The pig Delta coronavirus N-protein monoclonal antibody can recognize the N-protein of PDCoV specifically, and can be used for detecting and diagnosing the pig Delta coronavirus rapidly flexibly.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a porcine Delta coronavirus N protein monoclonal antibody and its antigenic epitope and application. Background technique [0002] Porcine Delta Coronavirus (PDCoV) is a new type of coronavirus that can cause diarrhea in piglets. PDCoV was first reported in Hong Kong, China in 2012. Researchers detected the presence of PDCoV-positive for the first time in excrement samples from piglets. samples, and completed the sequencing of the full-length genes of the PDCoV strains in two of the samples (HKU15-44 strain and HKU15-155 strain), confirming that the virus belongs to a new type of coronavirus, but the pathogenicity of the virus Sex is unknown. In 2014, the virus was detected in fecal samples of piglets with diarrhea in the United States, and it was confirmed that the virus could cause watery diarrhea in piglets and adult pigs, and lead to the death of suckling piglets. Th...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N5/20G01N33/577G01N33/569C07K14/165C12R1/91
Inventor 童光志周艳君李慧春张文超陈鹏飞李先斌虞凌雪姜一峰高飞李丽薇
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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