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Anti-jellyfish toxin nanobody koto54, preparation method and use

A technology of KOTO54 and nano-antibody, which is applied in the preparation of preventive or therapeutic anti-jellyfish toxin preparations. In the field of preparation of nano-antibody KOTO54, it can solve the problems of treatment and prevention of jellyfish stings, achieve excellent anti-jellyfish toxin, and realize industrial production , excellent prophylactic or therapeutic effects

Active Publication Date: 2022-07-01
THE NAVAL MEDICAL UNIV OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] There are currently no treatments and preventive medicines for jellyfish stings
However, there are no relevant reports on nanobodies against jellyfish stings

Method used

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  • Anti-jellyfish toxin nanobody koto54, preparation method and use
  • Anti-jellyfish toxin nanobody koto54, preparation method and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1. Construction of Nanobody Library

[0037] (1) 0.5 mg of jellyfish toxin CfTX1 [Brinkman D, Burnell J. Partial purification of cytolytic venomproteins from the boxjellyfish, Chironex fleckeri [J]. Toxicon, 2008, 51(5): 853-863.] was equal volume with Freund's adjuvant Mix and immunize a Xinjiang Bactrian camel once a week for a total of 6 consecutive immunizations. During the immunization process, B cells are stimulated to express specific nanobodies;

[0038] (2) After 6 immunizations, extract 200 mL of camel peripheral blood lymphocytes and extract total RNA;

[0039] (3) Synthesize cDNA and amplify VHH by nested PCR

[0040] (4) 20 μg pMECS phage display vector and 10 μg VHH were digested with restriction endonucleases Pst1 and NotI, and the two fragments were ligated;

[0041] (5) Transform the ligation product into the electrotransformation competent cell TG1, construct a phage display library and measure the storage capacity, the size of the storage ca...

Embodiment 2

[0042] Example 2. Nanobody screening process

[0043] (1) Take 200 μL of recombinant TG1 cells to culture in 2TY medium, add 50 μL of helper phage VCSM13 to infect TG1 cells during the period, and culture overnight to amplify the phage, use PEG / NaCl to precipitate the phage the next day, and collect the amplified phage by centrifugation;

[0044] (2) will be dissolved in 150mmol / L pH 8.2NaHCO 3 150 μg of jellyfish toxin was coupled to the ELISA plate, placed at 4°C overnight, and a negative control was established at the same time;

[0045] (3) The next day, 100 μL of 5% BSA was added, and it was blocked at room temperature for 2 hours;

[0046] (4) After 2h, add 100 μL of amplified phage (1×10 11 tfu-immunized camel nanobody phage display gene library) for 1 hour at room temperature;

[0047] (5) Wash five times with PBS+0.05% Tween 20 to wash off bound phage;

[0048] (6) Dissociate the specifically bound phage with trypsin with a final concentration of 25 mg / mL, and inf...

Embodiment 3

[0049] Example 3. Screening of specific positive clones by phage-enzyme-linked immunosorbent assay (ELISA)

[0050] (1) Select 200 single colonies from the cell culture plates after the above 3 rounds of screening and inoculate them in 96 deep-well plates containing 100 μg / mL ampicillin TB medium, and set up a blank control, and cultivate to log phase at 37°C After that, IPTG with a final concentration of 1 mmol / L was added and incubated at 28°C overnight;

[0051] (2) Utilize osmotic burst method to obtain crudely extracted antibody, and transfer the antibody to an ELISA plate coated with antigen, and place it at room temperature for 1 h;

[0052] (3) Wash off the unbound antibody with PBST, add 100 μL of Mouse anti-HA tagantibody (mouse anti-HA antibody, purchased from Covance) diluted 1:2000, and place at room temperature for 1 h;

[0053] (4) Wash off the unbound antibody with PBST, add 100 μL of Anti-mousealkaline phosphatase conjugate (goat anti-mouse alkaline phosphata...

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Abstract

The invention relates to the technical field of biomedicine, and provides a nanobody KOTO54 against jellyfish toxin, a preparation method and application. The nanobody is a VHH antibody and has the amino acid sequence shown in SEQ ID NO. 1. The present invention is analyzed by affinity The nanobody of the present invention has good affinity. It has been proved by small animal experiments that the mice in the antibody protection group pre-injected with the nanobody of the present invention have no symptoms of neurotoxicity after the injection of jellyfish toxin. It shows that the nanobody of the present invention has excellent anti-jellyfish toxin effect, has excellent preventive or therapeutic effect on jellyfish stings, and has broad clinical application prospects.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a nanobody KOTO54, its preparation method and use, especially the use in the preparation of an anti-jellyfish toxin preparation for prevention or treatment. Background technique [0002] Jellyfish is an important plankton in the aquatic environment. It is one of the oldest and mysterious creatures in the world. It has existed on the earth about 650 million years ago and is distributed in almost all sea areas. In terms of taxonomy, it includes cnidaria jellyfish, cruciate jellyfish, and cubic jellyfish; in terms of number, the number of jellyfish has exploded in recent decades, which not only leads to the destruction of marine ecosystems, but also causes catastrophic damage to marine fisheries. damage, and the increasing number of jellyfish stings, with thousands of people injured every year, has become a very difficult problem. [0003] Since the 1940s, Pubmed ha...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/18C07K16/00C12N15/13C12N15/70A61K39/395A61P39/02
CPCC07K16/18C07K16/005C12N15/70A61P39/02C07K2317/569A61K2039/505
Inventor 胡适雷长海傅文燕
Owner THE NAVAL MEDICAL UNIV OF PLA
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