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Nanometer antibody for specifically aiming at H3N2 influenza A virus and application thereof in diagnosis

A H3N2, nanobody technology, applied in the field of biomedicine or biopharmaceuticals, to achieve good specificity

Active Publication Date: 2014-07-23
北京科卫临床诊断试剂有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Ordinary antibodies can be quickly degraded by the digestive system, preventing them from entering the brain or the periphery of some tumors. Many diseases cannot be treated with monoclonal antibody drugs

Method used

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  • Nanometer antibody for specifically aiming at H3N2 influenza A virus and application thereof in diagnosis
  • Nanometer antibody for specifically aiming at H3N2 influenza A virus and application thereof in diagnosis
  • Nanometer antibody for specifically aiming at H3N2 influenza A virus and application thereof in diagnosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Construction of H3N2 Nanobody Library:

[0043] (1) Mix 0.1 mg of inactivated natural influenza A H3N2 antigen with an equal volume of Freund's adjuvant to immunize a Xinjiang Bactrian camel once a week for a total of 7 times to stimulate B cells to express antigen-specific nano Antibody; (2) After 7 times of immunization, extract 100 mL camel peripheral blood lymphocytes and extract total RNA; (3) Synthesize cDNA and amplify VHH by nested PCR; (4) Use restriction enzymes PstI and NotI Digest 20 ug pComb3 phage display vector (supplied by Biovector) and 10 ug VHH and ligate the two fragments; (5) Transform the ligated product into electroporation-competent cells TG1, construct the H3N2 nanobody library and measure the library capacity, the library size is 1.0 ×10 9 . At the same time, we randomly selected 24 clones for colony PCR detection, and the results showed that the insertion rate of the built library was above 90%. figure 1 Colony PCR results are...

Embodiment 2

[0044] Example 2: Nanobody screening process against H3N2:

[0045] (1) Dissolve in 100 mM NaHCO 3 , 20 ug of inactivated H3N2 antigen in pH 8.2 was coupled to the NUNC microtiter plate, and placed overnight at 4 ℃; (2) the next day, 100 uL of 0.1% casein was added, and blocked at room temperature for 2 h; (3) after 2 h , add 100 uL phage (5×10 11 tfu immunized camel nanobody phage display gene library), and reacted at room temperature for 1 h; (4) Washed 5 times with 0.05% PBS+Tween-20 to wash away non-specific phage; (5) Washed with 100 mM TEA (triethylamine) Dissociate the phage that specifically binds to H3N2, and infect Escherichia coli TG1 cells in logarithmic phase growth, culture at 37°C for 1 h, produce and purify the phage for the next round of screening, and repeat the same screening process for 3-4 Rounds, gradually enriched.

Embodiment 3

[0046] Example 3: Using phage enzyme-linked immunosorbent method (ELISA) to screen specific single positive clones:

[0047] (1) From the cell culture dish containing phage after the above 3-4 rounds of selection, pick 96 single colonies and inoculate them in TB medium containing 100 micrograms per milliliter of ampicillin (1 liter of TB medium contains 2.3 grams of phosphoric acid Potassium dihydrogen, 12.52 grams of dipotassium hydrogen phosphate, 12 grams of peptone, 24 grams of yeast extract, 4 milliliters of glycerol), after growing to the logarithmic phase, add IPTG with a final concentration of 1 millimolar, and culture overnight at 28 °C. (2) Use the infiltration method to obtain the crudely extracted antibody, and transfer the antibody to an antigen-coated ELISA plate, and place it at room temperature for 1 hour. (3) Wash off the unbound antibody with PBST, add a mouse anti-HA tag antibody (anti-mouse anti-HA antibody, purchased from Beijing Kangwei Century Biotech...

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Abstract

The invention discloses a nanometer antibody for specifically aiming at an H3N2 influenza A virus, a gene sequence for encoding the nanometer antibody and an application of the nanometer antibody in diagnosis, solving the problem that the production and detection of a high-purity high-affinity high-sensitivity antibody can not be obtained in the research process of the traditional H3N2 influenza A diagnostic reagent. The nanometer antibody for specifically aiming at the H3N2 influenza A virus, which is disclosed by the invention, can be used for fast detecting the antigen by being efficiently expressed in escherichia coli and being improved and is applied to the research of an H3N2 influenza A fast diagnosis kit.

Description

technical field [0001] The invention belongs to the technical field of biomedicine or biopharmaceuticals, and relates to the research and development of a nanobody specific for influenza A (H3N2) virus, a transformation technology of the nanobody and a rapid diagnostic kit. Background technique [0002] Influenza A virus can be divided into 16 subtypes (H1-H16) according to the difference of the hemagglutinin (HA) antigen on its surface, and 9 subtypes (N1) according to the difference of the neuraminidase (NA) antigen. -N9). Influenza viruses produce a new influenza virus (antigenic shift) through the cumulative change of surface protein antigenicity (antigenic drift) or gene rearrangement of different influenza viruses (antigenic shift) to evade the immune recognition of the infected host, thereby spreading and multiplying in the infected population . Influenza A (H3N2) is a respiratory disease caused by the influenza A (H3N2) virus. The H3N2 influenza outbreak in Hong ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/11C12N15/70C12N1/21G01N33/569
Inventor 万亚坤朱敏欧卫军王保君吴启运沙海王蕾
Owner 北京科卫临床诊断试剂有限公司
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