Recombinant canine sourced antibody scFv-Fc against H3N2 canine influenza virus

A technology of scfv-fc and VH-F is applied in the field of genetic engineering to achieve the effects of short preparation cycle, simple operation and mature production technology

Active Publication Date: 2020-09-25
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no report on the preparation of recombinant caninized antibodies against canine influenza virus for treatment

Method used

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  • Recombinant canine sourced antibody scFv-Fc against H3N2 canine influenza virus
  • Recombinant canine sourced antibody scFv-Fc against H3N2 canine influenza virus
  • Recombinant canine sourced antibody scFv-Fc against H3N2 canine influenza virus

Examples

Experimental program
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Embodiment 1

[0035] Example 1: Construction of pET-32a-scFv-Fc expression vector

[0036] 1 Virus strains, plasmids, cell lines and reagents

[0037] H3N2 subtype canine influenza virus strain A / Canine / Jiangsu / 06 / 2010 (H3N2) (06 for short), isolated and preserved by the laboratory, its GenBank sequence number is from JN247615 to JN247623. The strain and its sequence reference website: http: / / www.ncbi.nlm.nih.gov / nuccore. Rosetta (DE3) was purchased from Shanghai Weidi Biotechnology Co., Ltd. Escherichia coli DH5α competent cells and pClone007 Blunt Simple Vector Kit were purchased from Beijing Qingke Xinye Biotechnology Co., Ltd. (hereinafter referred to as Qingke). The pET-32a(+) prokaryotic expression vector is kept by the laboratory. Canine kidney cells (Madin-Darby canine kidney, MDCK) were purchased from the US ATCC and preserved by the laboratory.

[0038] Animal peripheral blood lymphocyte separation kit and TMB single-component chromogenic solution were purchased from Beijing S...

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Abstract

The invention discloses a recombinant canine sourced antibody scFv-Fc against the H3N2 canine influenza virus (H3N2 CIV). The recombinant canine sourced antibody scFv-Fc is prepared by the following steps: extracting the total RNA of an infection cell and performing reverse transcription to obtain cDNA; amplifying VH, VL and Fc genes; performing PCR fusion, and extracting recombinant plasmids T-scFv and/or T-CE-Fc; performing amplifying to obtain scFv and/or Fc on the basis of the T-scFv and/or T-CE-Fc, linking the scFv and/or Fc target fragments into a cloning vector, and selecting a positivestrain; performing amplification by using the scFv as a template and CE-scFv-F and/or CE-scFv-R as a primer, and performing homogenous recombinant linkage on the product and the vector, transforminga Rosetta competent cell, and selecting a positive strain named ET-32a-scFv-Fc after resistance plate screening. The recombinant canine source antibody scFv-Fc can be used for preparing similar biological preparations. The recombinant canine sourced antibody against H3N2 sub-type CIV lays a material foundation for preventing and controlling canine infection caused by the H3N2 CIV and dealing withpotential menace of the H3N2 CIV on human health and public hygiene.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a recombinant caninized antibody scFv-Fc for H3N2 canine influenza virus. Background technique [0002] Canine influenza virus (CIV) is an RNA virus that can cause respiratory symptoms such as cough, runny nose, and fever in dogs. It is a highly contagious respiratory infectious disease virus that can cause epidemics in dogs. In 2004, the horse-origin H3N8 subtype influenza virus was first reported in the United States to spread horizontally in dogs, which changed the view that dogs are resistant to influenza viruses. Subsequently, canine respiratory disease caused by H3N2 subtype influenza virus appeared in South Korea in 2007. In 2015, the H3N2 subtype of CIV broke out in Chicago, the United States, and then spread to other continents in the United States and even Canada. Currently, the main canine influenza virus subtype in China is H3N2. [0003] CI...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/70C12N15/11A61K39/395A61P31/16
CPCC07K16/1018C12N15/70A61P31/16C07K2317/622A61K2039/505
Inventor 刘永杰赵丹韩德敏董雨豪赵艳兵
Owner NANJING AGRICULTURAL UNIVERSITY
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