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Anti-H5 subtype avian influenza nano antibody protein as well as coding gene and application of nano antibody protein

A technology of nano-antibody and bird flu virus, applied in the direction of anti-viral immunoglobulin, antibody, application, etc., can solve the problems of high production cost and low output, and achieve the effect of low cost, high sensitivity and high purity

Active Publication Date: 2021-07-16
SOUTH CHINA AGRI UNIV
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  • 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

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  • Anti-H5 subtype avian influenza nano antibody protein as well as coding gene and application of nano antibody protein
  • Anti-H5 subtype avian influenza nano antibody protein as well as coding gene and application of nano antibody protein
  • Anti-H5 subtype avian influenza nano antibody protein as well as coding gene and application of nano antibody protein

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...

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Abstract

The invention belongs to the technical field of biological medicines, and specifically relates to an anti-H5 subtype avian influenza nano antibody protein as well as a coding gene and application of the nano antibody protein. The amino acid sequence of the anti-H5 subtype avian influenza virus nano antibody protein is as shown in SEQ ID NO. 1. The nano antibody protein specificity aims at the H5 subtype avian influenza A virus and can solve the problems of low antibody affinity, low purity in the production process and low sensitivity in the detection process in the research and development process of an existing H5 subtype avian influenza diagnostic reagent. According to the present invention, the anti-H5 subtype avian influenza virus nano antibody protein and a maltose binding protein tag are further subjected to fusion expression in escherichia coli, rapid antigen detection can be achieved, the fusion protein can be applied to development of a rapid H5 subtype avian influenza A detection kit, and the fusion protein can be used for development of products for preventing and treating H5 subtype avian influenza A.

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

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Application Information

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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
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