Anti-sea snake neurotoxin SN160 nano antibody, preparation method and application

A nano-antibody and neurotoxin technology, applied in the preparation of sea snake anti-venom preparations, treatment or prevention of sea snake bites, can solve the problems of strong patient, unstable quality, and difficulty in catching sea snakes, and achieve simple construction and expression process and excellent prevention Or therapeutic effect, the effect of broad clinical application prospects

Active Publication Date: 2019-02-15
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 the most effective first-aid treatment is to inject anti-venom preparations such as special anti-venom serum or antibody drugs as soon as possible, at present, anti-venom preparations such as special anti-venom serum and other anti-venom preparations mainly come from immunized animals, and the method of directly using snake venom toxins to immunize animals to obtain anti-venom The method of venomous serum or screening poly / monoclonal antibodies has many disadvantages: first, it is difficult to capture sea snakes, it is difficult to obtain a large amount of natural sea snake toxins for animal immunity, and the separation of neurotoxins from venomous glands is far from meeting the needs of preparing anti-venom preparations
Second, because snake venom toxins are lethal to animals, attenuation treatment is required, and attenuated toxins often lose their antigenicity and epitopes, resulting in weakened neutralization activity of antitoxins prepared with attenuated toxins
Third, animal-derived antivenom and other products are likely to cause strong allergic reactions in patients, and even death
Therefore, it is difficult to prepare the antivenom serum of sea snake venom and its quality is unstable. Only a few countries in the world can always have sea snake antivenom preparations
However, there is no relevant report on nanobodies against sea snake neurotoxin SN160

Method used

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  • Anti-sea snake neurotoxin SN160 nano antibody, preparation method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Example 1. Construction of Nanobody Library against Sea Snake Neurotoxin SN160

[0041] (1) 0.5 mg sea snake neurotoxin SN160 (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 SN160

[0049] (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, the phages were precipitated with PEG / NaCl, and the amplified phages were collected by centrifugation;

[0050] (2) Dissolve in 150mmol / L pH 8.2NaHCO 3 The sea snake neurotoxin SN160 in 150 μg was coupled to a 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 five times with PBS+0.05% Tween 20 to wash off bound phage;

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

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 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;

[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 antibodies were washed away with PBST, and 100 μL of Mouse anti-HAtagantibody (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-mousealkalinephosphate conjugate (goat ant...

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Abstract

The invention relates to the technical field of biological medicine and provides an anti-sea snake neurotoxin SN160 nano antibody, a preparation method and an application. The anti-sea snake neurotoxin SN160 nano antibody is a VHH antibody and has an amino acid sequence shown as SEQ ID NO. (sequence identifier number) 1; a nucleotide sequence encoding the nano antibody is shown as SEQ ID NO. 2; affinity analysis shows the nano antibody has good affinity; small animal experiments prove that none of mice in an antibody protection group injected with the nano antibody in advance appears a neurotoxic symptom after injection of sea snake neurotoxin SN160; after continuous observation for a month, no death caused by the toxin happens; and therefore, the nano antibody has an excellent anti-sea snake toxin effect, an excellent prevention or treatment effect on sea snake bites and wide clinical application prospects.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to a nanobody against sea snake neurotoxin SN160, its preparation method and application, especially the application in the preparation of sea snake antivenom preparation and the treatment or prevention of sea snake bites. Background technique [0002] Since entering the new era, my country's marine undertakings have continued to advance, and aquaculture and fishing, marine resource utilization and research have developed rapidly; and as a major maritime country, my country needs to continuously strengthen its naval construction if it wants to build a world-class navy. However, the poisonous and harmful marine organisms that exist in coastal waters pose potential threats to fishermen, marine resource developers, researchers, and naval fighters in production operations. Therefore, research on the prevention and treatment of marine biological injuries is of great signif...

Claims

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Application Information

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IPC IPC(8): C07K16/18C12N15/13C12N15/63A61K39/395A61P39/02
CPCA61K2039/505A61P39/02C07K16/18C07K2317/569
Inventor 胡适刘烁吾傅文燕雷长海李天
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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