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Construction method and application of anti-PEDV (Porcine Epidemic Diarrhea Virus) N protein VHH (variable region of heavy chain) bacteriophage display library

A phage display and construction method technology, applied in the direction of antiviral immunoglobulin, library, chemical library, etc., to achieve the effect of rich diversity, easy expression, and high affinity

Inactive Publication Date: 2019-04-26
NORTHWEST A & F UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But there is no relevant report about the nanobody of PEDV at present; Therefore, it is a problem to be solved urgently for those skilled in the art to provide a kind of construction method of the single domain heavy chain antibody VHH phage display library of anti-porcine influenza virus N protein

Method used

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  • Construction method and application of anti-PEDV (Porcine Epidemic Diarrhea Virus) N protein VHH (variable region of heavy chain) bacteriophage display library
  • Construction method and application of anti-PEDV (Porcine Epidemic Diarrhea Virus) N protein VHH (variable region of heavy chain) bacteriophage display library
  • Construction method and application of anti-PEDV (Porcine Epidemic Diarrhea Virus) N protein VHH (variable region of heavy chain) bacteriophage display library

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Embodiment 1

[0067] 1) Determination of antigen vaccination dose, immunization program and serum antibody titer

[0068] Multi-point vaccination is performed under the skin of the neck of the Bactrian camel. Each immunization is 5mg of PEDV N protein. The immunization program is the second immunization on the 14th day after the first immunization, the third immunization on the 21st day, and the fourth immunization on the 28th day. Venous blood was collected before the first immunization and after each immunization, and the antibody titer in the serum was detected by indirect ELISA method to reach 1:128000. After the antibody reached the experimental requirements, blood was collected from the vein.

[0069] The antibody titer of PEDV N protein in camel serum was detected by ELISA. The antibody titer of the serum after the last immunization was as follows figure 1 As shown, the abscissa represents serum dilution, and the ordinate represents OD 450nm The absorbance value shows that the antibody t...

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Abstract

The invention discloses a construction method and an application of an anti-PEDV (Porcine Epidemic Diarrhea Virus) N protein VHH (variable region of heavy chain) bacteriophage display library. The construction method comprises the following steps: carrying out extraction and reverse transcription of immune two-humped camel peripheral blood lymphocyte and lymphocyte total RNA (Ribonucleic Acid); designing two pairs of PCR (Polymerase Chain Reaction) amplification primers to obtain a target fragment; connecting the obtained target fragment with a phagemid carrier pCANTAB-5E; converting a connection product into a TG1 competent cell, and obtaining the bacteriophage display library; successfully constructing the VHH bacteriophage display library of the anti-PEDV N protein; calculating the capacity and the diversity of the library; and carrying out quality evaluation on the constructed library. The library established with the construction method has the characteristics of large library capacity, rich diversity and the like, and lays a foundation for establishing a clinic diagnosis method of the PEDV and researching a virus infection mechanism.

Description

Technical field [0001] The present invention relates to the field of biotechnology, and more specifically to a method for constructing a single domain heavy chain antibody VHH phage display library against the N protein of porcine fluid diarrhea virus. Background technique [0002] Porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV) is the causative factor of porcine epidemic diarrhea (PED), which can cause an acute, destructive, and highly contagious intestinal disease, which is characterized by Severe watery diarrhea, vomiting, dehydration, little or no food, acid-base imbalance and high mortality of suckling piglets. PEDV is a member of the Coronavirus genus (Coronavirus) of the Coronavirus family. It is an enveloped single-stranded positive-stranded RNA virus. The genome of PEDV is about 28kb and consists of four typical structural proteins, including nucleocapsid Protein (N), membrane protein (M), glycosylated spike protein (S) and envelope protein (E), ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C40B40/02C40B50/06
CPCC07K16/10C07K2317/569C40B40/02C40B50/06
Inventor 肖书奇李爽马志倩田阳升王天宇
Owner NORTHWEST A & F UNIV
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