Anti-jelly fish toxin nano antibody KY031 as well as preparation method and application thereof

A nano-antibody and anti-jellyfish technology, which is applied in the preparation of jellyfish anti-venom preparations, treatment or prevention of jellyfish stings, its preparation, nano-antibody KY031, can solve the problems of treatment and prevention of jellyfish stings, and achieve excellent prevention or treatment. Therapeutic effect, broad clinical application prospects, simple construction and expression process

Active Publication Date: 2021-08-06
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 is no relevant report on nanobodies against jellyfish stings

Method used

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  • Anti-jelly fish toxin nano antibody KY031 as well as preparation method and application thereof
  • Anti-jelly fish toxin nano antibody KY031 as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1. Construction of Nanobody Library

[0037] (1) Mix 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.] with Freund's adjuvant in equal volume 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 the 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) Digest 20 μg of pMECS phage display vector and 10 μg of VHH with restriction enzymes Pstl and NotI and connect the two fragments;

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

Embodiment 2

[0042] Example 2. Nanobody screening process

[0043] (1) 200 μL of recombinant TG1 cells were cultured in 2TY medium, during which 50 μL of helper phage VCSM13 was added to infect TG1 cells, and cultivated overnight to amplify the phages, and the next day, PEG / NaCl was used to precipitate the phages, and the amplified phages were collected by centrifugation;

[0044] (2) Dissolve in 150mmol / L pH 8.2NaHCO 3 150 μg of jellyfish toxin in the medium was coupled to the microtiter plate, placed overnight at 4°C, and a negative control was set up at the same time;

[0045] (3) Add 100 μL of 5% BSA the next day, and block for 2 hours at room temperature;

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

[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 at a final concentra...

Embodiment 3

[0049] Example 3. Screening specific positive clones with phage enzyme-linked immunoassay (ELISA)

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

[0051] (2) Utilize the osmotic bursting 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;

[0052] (3) Unbound antibodies were washed away with PBST, and 100 μL of Mouse anti-HA tagantibody (mouse anti-HA antibody, purchased from Covance) diluted 1:2000 was added, and left at room temperature for 1 hour;

[0053] (4) Unbound antibodies were washed away with PBST, and 100 μL of Anti-mousealkaline phosphatase conjuga...

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Abstract

The invention relates to the technical field of biological medicine, and provides an anti-jelly fish toxin nano antibody KY031, a preparation method and application. The nano antibody is a VHH antibody and has an amino acid sequence shown in SEQ ID NO.1, affinity analysis shows that the nano antibody has good affinity, and small animal experiments prove that after jelly fish toxin is injected into mice in an antibody protection group injected with the nano antibody in advance, no poisoning symptom occurs in the mice, and no toxin lethal condition exists after continuous observation for one month. The nano antibody has an excellent anti-jelly fish toxin effect, has an excellent prevention or treatment effect on jelly fish stings, and has broad clinical application prospects.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to a nanobody KY031, its preparation method and use, especially the use in preparing jellyfish anti-poison preparations and treating or preventing jellyfish stings. 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 existed on the earth about 650 million years ago and is distributed in almost all sea areas. In terms of classification, it includes cnidaria, cruciate jellyfish, and cubic jellyfish; in terms of quantity, the number of jellyfish has exploded in recent decades, not only causing damage to marine ecosystems, but also catastrophic to marine fisheries In addition, the number of jellyfish sting incidents is increasing, and tens of thousands of people are injured every year, which has become a very difficult problem. [0003] Since the 1940s, Pubmed has p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N15/13C12N15/70A61K39/395A61P39/02
CPCC07K16/18C12N15/70A61P39/02C07K2317/569C07K2317/92C07K2317/22C07K2317/76A61K2039/505
Inventor 胡适雷长海
Owner THE NAVAL MEDICAL UNIV OF PLA
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