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Anti-sea snake neurotoxin nanobody, preparation method and use

A nano-antibody and neurotoxin technology, applied in the field of biomedicine, can solve the problems of weakened anti-toxin neutralization activity, loss of antigenicity and epitope, difficult to natural sea snake toxin, etc., to achieve good affinity, excellent anti-sea snake toxin effect, excellent The effect of prophylactic or therapeutic action

Active Publication Date: 2021-07-20
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although early injection of antivenom preparations such as special antivenom or antibody drugs is the most effective emergency treatment, the method of directly using snake venom toxins to immunize animals to obtain antivenom or to screen poly / monoclonal antibodies has the following disadvantages: First It is difficult to catch sea snakes, it is difficult to obtain a large amount of natural sea snake toxins for animal immunity, and the separation of neurotoxins from venom glands is far from meeting the needs of preparing anti-venom preparations; the second is that snake venoms are lethal to animals, so attenuation treatment is required. However, attenuated toxins often lose their antigenicity and epitopes, resulting in the weakening of the neutralization activity of antitoxins prepared by using attenuated toxins; third, animal-derived antiserum and other products are likely to cause strong allergic reactions in patients, and even death
However, there are no relevant reports on nanobodies against sea snake neurotoxins

Method used

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  • Anti-sea snake neurotoxin nanobody, preparation method and use
  • Anti-sea snake neurotoxin nanobody, preparation method and use
  • Anti-sea snake neurotoxin nanobody, preparation method and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1. Construction of Nanobody Library against Sea Snake Neurotoxin SN36

[0041] (1) 0.5 mg sea snake neurotoxin SN36 (Hu Shi et al., Screening, preparation and biological activity research of fully human monoclonal antibody against short-chain neurotoxin from flat-chin sea snake. PLA Medical Journal 42.7 (2017): 612-616.) The antigen was mixed with Freund's adjuvant in equal volumes, and a Xinjiang Bactrian camel was immunized once a week for a total of 6 consecutive immunizations. During the immunization process, B cells were stimulated to express specific nanobodies;

[0042] (2) After the 6 immunizations, extract 200 mL of camel peripheral blood lymphocytes and extract total RNA;

[0043] (3) Synthesize cDNA and utilize nested PCR to amplify VHH. The primer sequences used in this step are shown in Table 1:

[0044] Table 1 PCR primer sequences

[0045]

[0046] (4) Digest 20 μg of pMECS phage display vector and 10 μg of VHH with restriction enzymes Pstl a...

Embodiment 2

[0048] Example 2. Nanobody screening process against sea snake neurotoxin SN36

[0049] (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 this period, and cultivate overnight to amplify the phages, use PEG / NaCl to precipitate the phages the next day, and centrifuge to collect the amplified phages;

[0050] (2) Dissolve in 150mmol / L pH 8.2 NaHCO 3 The sea snake neurotoxin SN36150ug 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;

[0051] (3) Add 100 μL of 5% BSA the next day, and block for 2 hours at room temperature;

[0052] (4) After 2 hours, add 100 μL of amplified phage (1×10 11 tfu immunized camel nanobody phage display gene library) at room temperature for 1 hour;

[0053] (5) Wash 5 times with PBS+0.05% Tween 20 to wash off the bound phage;

[0054] (6) Use trypsin at a final concentration of 25 mg / mL to dissoci...

Embodiment 3

[0055] Example 3. Screening specific positive clones with phage enzyme-linked immunoassay (ELISA)

[0056] (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 culture them to the logarithmic phase at 37°C After that, add IPTG with a final concentration of 1mmol / L, and culture overnight at 28°C;

[0057] (2) Use 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;

[0058] (3) Unbound antibody was washed away with PBST, 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;

[0059] (4) Unbound antibodies were washed away with PBST, 100 μL of Anti-mousealkaline phosphatase conjugate (goat anti-mouse al...

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Abstract

The present invention relates to the technical field of biomedicine, and provides an anti-sea snake neurotoxin nanobody, a preparation method and an application thereof. The anti-sea snake neurotoxin nanobody is a VHH antibody, has the amino acid sequence shown in SEQ ID NO.1, and encodes The nucleotide sequence of the nanobody is shown in SEQ ID NO.2. Through affinity analysis, 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 injected with the nanobody of the present invention in advance can be After the sea snake neurotoxin SN36, no neurotoxic symptoms appeared in any mice, and the toxin-free death was observed continuously for one month, indicating that the nanobody of the present invention has an excellent anti-sea snake toxin effect, and has excellent preventive or therapeutic effects on sea snake bites , with broad prospects for clinical application.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to a nanobody anti-sea snake neurotoxin, its preparation method and application, especially the application in the preparation of sea snake anti-venom preparation and the treatment or prevention of sea snake bites. Background technique [0002] As a maritime power, building a powerful navy is a key issue in my country's military development. However, the poisonous and harmful marine organisms that exist in the coastal waters will pose a potential threat to naval soldiers. Therefore, research on the prevention and treatment of marine biological injuries is of great significance to the medical support and medical work of cross-sea landing operations. Therefore, it is necessary to start anti-marine Scientific application research of bioinjury. [0003] The flat-chinned sea snake is a typical poisonous marine creature belonging to the Cobra family. It is commonly known a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/18C12N15/13A61K39/395A61P39/02
CPCA61K2039/505A61P39/02C07K16/18C07K2317/569C07K2317/76C07K2317/92
Inventor 胡适刘烁吾雷长海李天
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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