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Single-domain antibodies against hepatitis A viruses and derived proteins of single-domain antibodies

A hepatitis A virus and single-domain antibody technology, applied in the field of biotechnology or immunology, can solve problems such as poor repeatability, large differences in antibody affinity, and poor stability between production batches

Active Publication Date: 2020-05-12
REGENECORE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the test must be based on RT-PCR, and the detection cost is high; the SPA synergistic agglutination test utilizes the A protein (SPA) in the cell wall of Staphylococcus aureus to specifically bind to the Fc segment of HAV-IgG. Reverse indirect agglutination occurs when HAV-Ag is contained in
The sensitivity and specificity of this test are low, and the results are judged to be largely influenced by human factors, so it can only be used as a preliminary detection method when the equipment is limited; the general ELISA method uses the solid-phase carrier to adsorb immunologically active proteins. Add the serum of HAV antibody to be tested to the microplate of μ chain. If the serum to be tested is in the acute stage of hepatitis A infection, the IgM antibody in the serum will be captured by its antibody human μ chain antibody, then add the enzyme-labeled antibody, and the substrate will develop color. After that, it is judged that the serum to be tested is a hepatitis A infected patient
However, this method can only detect the IgM antibody in the serum of patients who have been infected with hepatitis A virus, and cannot directly quantify or semi-quantify the hepatitis A virus in the patient's body; The content of hepatitis A virus in serum or other samples, but there are few quantitative or semi-quantitative ELISA kits for direct detection of hepatitis A virus on the market, and the capture antibody of the existing direct detection kit is rabbit polyclonal antibody , due to the large difference between the preparation batches of rabbit polyclonal antibody, the affinity of the antibody is very different, which leads to the poor stability and poor repeatability of the production batch of the kit detection

Method used

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  • Single-domain antibodies against hepatitis A viruses and derived proteins of single-domain antibodies
  • Single-domain antibodies against hepatitis A viruses and derived proteins of single-domain antibodies
  • Single-domain antibodies against hepatitis A viruses and derived proteins of single-domain antibodies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Example 1: Construction of a single-domain antibody library against hepatitis A virus:

[0074] (1) Use 1ml of hepatitis A virus inactivated vaccine to immunize an Inner Mongolia Alxa Bactrian camel, once a week, a total of 7 consecutive immunizations, during which B cells are stimulated to express specific nanobodies; (2) After the immunization Finally, extract 100ml of camel peripheral blood lymphocytes and extract total RNA; (3) synthesize cDNA and use nested PCR to amplify VHH; (4) use restriction endonucleases Pst I and Not I to digest 20 μg pMECS phage display vector and 10 μg VHH and ligated the two fragments; (5) Transformed the ligated product into electroporation-competent cells TG1, constructed a phage display library for the capsid protein of hepatitis A virus and determined the storage capacity, the size of the library storage capacity was about 2×10 9 ; At the same time, the correct insertion rate of the target fragment in the built library was detected by...

Embodiment 2

[0075] Embodiment 2: the expression of hepatitis A virus capsid protein:

[0076] (1) The sequence information of the hepatitis A virus capsid protein was found from the protein database of NCBI (AccesionNumber: P08617.1); (2) After Uniprot analysis, VP2-VP3-VP1 was selected as the target expression fragment; (3) Add kozak enhancer sequence (5'-GGATCGAACCCTT-3') and IgG kappa signal peptide (METDTLLLWVLLLWVPGSTGD) at the 5' end of VP2-VP3-VP1, and add 6*His tag fusion expression at the 3' end; (4) Examples of use The scheme described in 8 carries out eukaryotic expression; (5) purify with reference to the scheme described in Example 9 using nickel column affinity chromatography; (6) carry out SDS-PAGE analysis with reference to the method of Example 9, and determine that the protein purity is at 95 % or more, and the concentration is more than 0.5mg / mL, keep it at low temperature for later use.

Embodiment 3

[0077] Example 3: Screening for single domain antibodies against hepatitis A virus:

[0078] (1) Take 200 μL of recombinant TG1 cells to culture in 2×TY medium, add 40 μL of helper phage VCSM13 to infect TG1 cells during the period, and culture overnight to amplify phages, use PEG / NaCl to precipitate phages the next day, and centrifuge to collect amplified phages ; (2) NaHCO diluted in 100mM pH 8.3 3 500ug of neutravidin protein in the medium was coupled to the microtiter plate, placed overnight at 4°C, and a negative control well was set up at the same time; (3) the next day, 100 μL of biotin-labeled hepatitis A virus capsid protein (VP2- VP3-VP1-Biotin), incubate at room temperature for 2 hours, add 100 μL PBS to the negative control well; (4) After 2 hours, add 100 μL of 3% skim milk, and block at room temperature for 2 hours; (5) After the end of blocking, add 100 μl amplification Post-phage library (approximately 2×10 11 phage particles) at room temperature for 1 h; (6)...

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Abstract

The present invention relates to the technical field of biotechnology or immunology and relates to single-domain antibodies against hepatitis A virus and derived proteins of the single-domain antibodies. An amino acid sequence of CDR1 of the single-domain antibodies is shown in any one of SEQ ID NO:83-SEQ ID NO:117; an amino acid sequence of CDR2 is shown in any one of SEQ ID NO:118-SEQ ID NO:143;and an amino acid sequence of CDR3 is shown in any one of SEQ ID NO:144-SEQ ID NO:174. Beneficial effects are that: the traditional method for indirect detection of hepatitis A virus antigen (HAV-Ag)by detecting hepatitis A virus antibodies (HAV-IgM and HAV-IgG) has many detection steps and many influence factors. The present invention provides a detection method based on ELISA that directly detects the hepatitis A virus antigen (HAV-Ag) without RT-PCR.

Description

technical field [0001] The invention relates to the technical field of biotechnology or immunology, and relates to a single-domain antibody against hepatitis A virus and its derivative protein. Background technique [0002] Antibody (Ab) or Immunoglobulin (Ig) is a type of glycoprotein in blood and tissue fluid, which is produced by plasma cells that proliferate and differentiate after receiving antigen stimulation from B cells. It mainly exists in body fluids such as serum and can It specifically binds to the corresponding antigen and is an important effector molecule that mediates humoral immunity. In addition to mediating specific humoral immune responses as important effector molecules, antibodies play an important role in the prevention and treatment of diseases, especially infectious diseases. Behring won the Nobel Prize in Medicine and Physiology for creating serum therapy. Since then, antibodies artificially prepared by polyclonal, monoclonal and genetically enginee...

Claims

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Application Information

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IPC IPC(8): C07K16/10G01N33/569
CPCC07K16/1009G01N33/56983C07K2317/565G01N2333/10C07K2317/569C07K2319/30Y02A50/30
Inventor 苏志鹏姚尧王乐飞
Owner REGENECORE BIOTECH CO LTD
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