Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

A kind of anti-h5 subtype avian influenza nanobody protein and its coding gene and application

A nanobody, avian influenza virus technology, applied in the direction of antiviral immunoglobulin, application, antibody, etc., can solve the problems of high production cost and low yield, and achieve the effects of low cost, high sensitivity and simple operation

Active Publication Date: 2022-03-29
SOUTH CHINA AGRI UNIV
View PDF22 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to overcome the shortcomings and deficiencies such as the presence of inclusion bodies in the prokaryotic expression system of avian influenza antibodies, low yield, renaturation, and high production costs that limit the application of avian influenza antibodies in production, the first object of the present invention is to provide a Anti-H5 Subtype Avian Influenza Virus Nanobody Protein

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A kind of anti-h5 subtype avian influenza nanobody protein and its coding gene and application
  • A kind of anti-h5 subtype avian influenza nanobody protein and its coding gene and application
  • A kind of anti-h5 subtype avian influenza nanobody protein and its coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 Construction of phage display library

[0058] (1) Immunize Xinjiang Bactrian camel (Lanzhou Animal Husbandry and Veterinary Research Institute, Gansu) with the hemagglutination inhibition test antigen (purchased from Harbin Veken Biotechnology Development Co., Ltd.) of avian influenza virus H5 subtype Re-8 strain, and immunize once every three weeks , a total of four times of immunization, the peripheral lymphatic blood of the immunized donor was extracted, and the peripheral blood lymphocytes (PBMC) were separated for RNA extraction and reverse transcription to obtain cDNA;

[0059] (2) The first round of nested PCR reaction: the first round of nested PCR reaction was carried out using the cDNA obtained in step (1) as a template, wherein the reaction system was: 2 μL of cDNA template, 10 μL of 2×Taq Plus Master Mix, primers CALL001 and 1 μL each of CALL002, with ddH 2 O was added to 20 μL; the amplification reaction conditions were: 95°C for 5 minutes; 95°C...

Embodiment 2

[0071] Example 2 Screening of phage nano-library

[0072] (1) The hemagglutination inhibition test antigen (purchased from Harbin Veken Biotechnology Development Co., Ltd.) of the H5 subtype Re-8 strain was coated with sodium carbonate-sodium bicarbonate buffer solution (0.05mol / L, pH=9.6). 100 μL per well on a microtiter plate, in which the antigen of the hemagglutination inhibition test of the H5 subtype Re-8 strain accounts for 10% by volume, put it at 37°C for 2 hours; discard the liquid in the well, and wash once with 300 μL PBS Then add 300 μL BSA with a mass percentage of 3% and place at room temperature to block for 1 h; discard the blocking solution, add 100 μL of the phage display library obtained in Example 1, and incubate at room temperature for 1 h; discard the liquid in the well and unbound phage, and then use Wash 5 times with PBS containing 0.05% Tween 20 by volume, 5min / time; add 100 μL of eluent containing 0.2M glycine (pH=2.7) and mix well, collect the eluat...

Embodiment 3

[0075] Example 3 ELISA screening positive monoclonal

[0076] (1) The phages screened out in the third round of Example 2 were first mixed with 5 mL of TG1 bacteria (OD 600 =0.5), after incubating at 37°C for 30 minutes, take 100 μL from the infecting bacterial solution for 10 N-th power gradient dilution, re-absorb 100 μL of the bacterial solution and streak on 2×YT / Amp / Glu solid medium respectively, at 37°C Cultivate overnight, then inoculate a single colony in 50mL of 2×YT / Amp / Glu liquid medium, culture overnight at 220rpm 37°C, pipette 1mL of bacterial liquid into 100mL of 2×YT / Amp / Glu liquid medium the next day, 220rpm 37°C Shake to OD 600 = about 0.6, add helper phage M13KO7 with multiplicity of infection MOI = 20:1, let stand at 37°C for 20 minutes, shake at 200 rpm for 30 minutes, carry out helper phage infection, centrifuge at 3000g to discard the supernatant including glucose in the medium, and use fresh 2 Resuspend in ×YT / Amp / Kana medium, shake culture overnight a...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention belongs to the technical field of biopharmaceuticals, and in particular relates to an anti-H5 subtype avian influenza nanobody protein and its coding gene and application. The amino acid sequence of the anti-H5 subtype avian influenza virus nanobody protein is shown in SEQ ID NO.1. The nanobody protein is specifically directed against H5 subtype avian influenza virus and can solve the problems of low antibody affinity, low purity during production and low sensitivity during detection in the development process of existing H5 subtype avian influenza diagnostic reagents. In the present invention, the anti-H5 subtype avian influenza virus nanobody protein is fused and expressed in Escherichia coli with a maltose-binding protein tag, which can realize rapid detection of antigens, and is applied to the development of a rapid detection kit for H5 subtype avian influenza, and The fusion protein can be used in the development of products for preventing and treating A(H5) subtype avian influenza.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, and in particular relates to an anti-H5 subtype avian influenza nanobody protein and its coding gene and application. Background technique [0002] Avian influenza virus (AIV) belongs to the genus Orthomyxoviridae of the Orthomyxoviridae family. It is a class of single-stranded negative-sense segmented RNA viruses with an envelope. Different types are divided into A, B and C types, which are also called A, B and C types in China. According to the antigenic difference of hemagglutinin (HA) and neuraminidase (NA), influenza A virus can be divided into different subtypes. To date, there are 17 antigenically distinct influenza A hemagglutinin subtypes, which are further classified as Group I or Group II hemagglutinins (Group I: H1, H2, H5, H6, H8, H9, H11, H12, H13, H16 and H17 viruses; Group II: H3, H4, H7, H10, H14, H15 viruses). [0003] Hemagglutinin (HA) exists as a glycoprotein tr...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/10C12N15/13C07K19/00C12N15/62C12N15/70C12N1/21G01N33/569G01N33/68A61K39/42A61K39/44A61P31/16C12R1/19
CPCC07K16/1018C07K16/005C12N15/70G01N33/56983G01N33/6854A61K39/42A61K39/44A61P31/16C07K2317/569C07K2317/94C07K2319/24G01N2333/11G01N2469/10A61K2039/505
Inventor 廖明许斯祺张奇潘志超冯赛祥陈爱华江金飞代绘琳谢倩梅罗开健
Owner SOUTH CHINA AGRI UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com