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CD44V6 nano antibody and application thereof as leukemia research reagent

A nanobody and antibody technology, applied in the field of biomedicine, can solve the problems of long development cycle, low stability, harsh storage conditions, etc., achieve good bioavailability, small molecular weight and stability, and achieve the effect of mass preparation

Active Publication Date: 2021-05-28
姜国胜
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In view of the above research background, the purpose of the present invention is to provide a CD44V6 nanobody, which is based on the special structure of the VHH single domain antibody of the alpaca heavy chain antibody, which has the advantages of both traditional antibodies and small molecule drugs, and almost perfectly overcomes the traditional antibodies. Due to its long development cycle, low stability, and harsh storage conditions, it has gradually become an emerging force in the new generation of therapeutic biomedicine and clinical diagnostic reagents.

Method used

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  • CD44V6 nano antibody and application thereof as leukemia research reagent
  • CD44V6 nano antibody and application thereof as leukemia research reagent
  • CD44V6 nano antibody and application thereof as leukemia research reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 CD44 antigen preparation:

[0052] (1) Protein information: Uniprot Accession: https: / / www.uniprot.org / uniprot / P16070; Species name: Homo sapiens (Human); protein length: Q21-Q320; protein purity: 90%; expression system: Escherichia coli expression system.

[0053] (2) Experiment content:

[0054] 1) Acquisition of CD44-pet28a recombinant vector: The acquisition and construction of the target gene was synthesized by a third-party company. After comparison of the sequencing results, the synthetic sequence was completely correct and conformed to the experimental design.

[0055] 2) Expression and purification of CD44 recombinant protein: Inoculate 50uL of the expression strain into 5mL of LB medium (containing a final concentration of 50ug / mL kanamycin), culture overnight at 37°C and 220rpm. Inoculate 5mL of bacterial liquid in the test tube into 400mL of culture medium (inoculate 2 bottles in total, 400mL per bottle), add kanamycin (mother solution concentra...

Embodiment 2

[0058] Example 2 Antibody Preparation

[0059] 1. Use complete Freund's adjuvant or incomplete Freund's adjuvant to emulsify 1-2mg, a total of 1.6mL of antigenic protein for alpaca immunization.

[0060] 2. Selection of alpacas: Choose alpacas that are healthy, strong, in good spirits, and of moderate size. The selected alpacas have bright wool and no symptoms of injury or discomfort. Choose good animals, and pre-raise them for about 1 week to eliminate some unqualified animals, so that the later experiments can be carried out smoothly. Finally, a 1-2-year-old healthy ewe alpaca was selected, number 7764.

[0061] 3. Immunization: Select the alpaca and ensure that the animal is suitable, and start the immunization experiment after recording the ear number. Before each immunization, 5 mL of blood was drawn for the detection of immune titer. During immunization, the left and right sides of the alpaca neck lymph nodes are injected each time, and 0.8 mL of 1.6 mL (about 1-2 mg)...

Embodiment 3

[0063] Example 3 CD44 Nanobody Screening

[0064] 1. Lymphocyte separation: Collect 50 mL of lymphocytes in total, add diluent and separation liquid for lymphocyte separation, and use lysate to lyse the remaining red blood cells, then add Trizol (5 mL) to lyse the lymphocytes.

[0065] 2. Extraction of RNA: Extract RNA according to the RNA extraction process, and dissolve it with 50uL RNase free-water. Take 2uL to measure the concentration, which is 790ng / uL. From the perspective of A260 / 280, the purity of RNA extraction is achieved. There is a small amount of chloroform remaining, but it does not affect transcription. , RNA transcription can be performed.

[0066] 3. Transcribe cDNA: Transcribe the RNA according to the steps of the transcription kit, transcribe 10 tubes, and the cDNA volume is 400uL.

[0067] 4. The first round of nested PCR verification: take different amounts of cDNA for PCR verification to obtain the final PCR template volume. Taking this cDNA as an exam...

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Abstract

The invention particularly relates to a CD44V6 nano antibody and application thereof as a leukemia research reagent. The invention provides an alpaca-derived CD44V6 monoclonal antibody. The alpaca-derived CD44V6 monoclonal antibody comprises target antigen alpaca immunization, a nano-antibody rapid screening technology and nano-antibody large-scale expression and purification. Through verification, the CD44V6 prepared by the scheme has a good effect of inducing differentiation and maturation of leukemia cells, and can be applied as a leukemia research reagent.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a CD44V6 nanobody, the antigenic protein of the nanobody and the application of the protein as a leukemia research reagent. Background technique [0002] The information disclosed in this background section is only intended to increase the understanding of the general background of the present invention, and is not necessarily taken as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to those skilled in the art. [0003] Recombinant antibody technology has developed rapidly in the past 20 years. By combining antibodies with specific antigens, on the one hand, they are used to establish enzyme-linked immunosorbent assay kits for detecting antigens, and on the other hand, they can directly bind to target antigens or carry therapeutic drugs to achieve target cells. The specific enrichment on the cell, or through...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13C12N15/81C12N15/12A61K39/395A61K47/55A61K49/00A61K51/10A61P35/02C12R1/84
CPCC07K16/2884C12N15/815C07K14/70585A61K47/55A61K49/0058A61K51/1027A61P35/02C07K2317/569C07K2317/22C07K2317/73C07K2317/92C07K2317/94Y02A50/30
Inventor 姜国胜王希娣张丹凤高伟陈梅英
Owner 姜国胜
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