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Specific antibody of new coronavirus S protein NTD region as well as preparation method and application of specific antibody

A specific, protein-based technology, applied in the direction of viruses/bacteriophages, microbial-based methods, antiviral agents, etc., can solve the problems of large-scale mass production of high finished products, unfavorable large-scale mass production, and limited antibody specificity. Low production cost, conducive to large-scale popularization and application, and high antibody stability

Active Publication Date: 2022-06-07
BIOISLAND LAB +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The above-mentioned technology based on the immune system of mice and other animals to generate and separate antibodies to the S protein on the surface of the new coronavirus has the following disadvantages: (1) The specificity of the antibody is limited: the S protein has a large structure and complex spatial folding. Traditional mouse monoclonal antibodies It is difficult to identify the complex spatial structure of the S surface, and it is difficult for researchers to obtain antibodies against each domain; (2) The stability of the antibody is poor: the above-mentioned mouse monoclonal antibody needs to be stored, transported, tested and tested at low temperature (4 degrees Celsius). Verification and other work can maintain the concentration, specificity and other characteristics of the antibody, which is not conducive to the development of large-scale application research; (3) large-scale mass production of finished products: the above-mentioned mouse monoclonal antibody is a full-length immunoglobulin, which needs to be produced in mammals Cells and other expensive expression systems carry out recombinant expression followed by separation and purification. The operation is complicated and expensive, which is not conducive to large-scale mass production

Method used

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  • Specific antibody of new coronavirus S protein NTD region as well as preparation method and application of specific antibody
  • Specific antibody of new coronavirus S protein NTD region as well as preparation method and application of specific antibody
  • Specific antibody of new coronavirus S protein NTD region as well as preparation method and application of specific antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0131] Example 1: Preparation of antigens

[0132] The conventional method for preparing antigens in the prior art is to recombinantly express and purify the NTD region of the S protein as an antigen, and then immunize animals such as mice, rabbits, and monkeys. However, the above-mentioned prior art has the following disadvantages: the immune systems in animals such as mice, rabbits, and monkeys have limited ability to generate specific spatial structures for recognizing antigens; The NTD region of recombinantly expressed and purified S protein is not a membrane protein, and its existing state is not as good as the natural state, so the obtained antibody may not be able to correctly recognize the antigen.

[0133] For the NTD domain of the new coronavirus S protein, the antigen preparation method of the present invention is specifically:

[0134] (1)串联、构建编码S蛋白NTD区域(即第1-304位氨基酸,具体为: MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIH VSGTNGTKRFDNPVLPFNDGVYFA...

Embodiment 2

[0139] Example 2: Alpaca immunization injection

[0140] In this example, alpacas were immunized with the antigen prepared in Example 1. Specific steps are as follows:

[0141] (1) The antigens prepared in Example 1 were equally divided into 4 parts;

[0142] (2) The alpacas were immunized 4 times in total, and the antigens were subcutaneously injected into the animals. The first immunization was recorded as the first day, and the subsequent immunizations were on the 10th, 19th, and 28th days respectively; On the 28th day, before the fourth immunization injection, about 200 mL of alpaca venous peripheral blood was collected, and on the 42nd day, that is, 14 days after the fourth immunization, about 300 mL of alpaca venous peripheral blood was collected.

[0143] Compared with the traditional immunization technical solutions for animal antibodies such as mice and rabbits, the method provided in this example collects a large amount of alpaca venous peripheral blood, which is c...

Embodiment 3

[0144] Example 3: Construction of Antibody Libraries

[0145] Using the two batches of alpaca venous peripheral blood collected in Example 2 as raw materials, a high diversity Nanobody library was constructed. The two batches of alpaca venous peripheral blood were treated in the same way, specifically:

[0146] (1) Using density gradient centrifugation to separate lymphocytes from the peripheral blood of alpaca veins;

[0147] (2) Extracting the total mRNA of lymphocytes and reverse transcribing into cDNA;

[0148] (3) Using appropriate DNA primers, the above-mentioned cDNA is used as a template, and the VHH fragments of alpaca immunoglobulin IgG2 and IgG3 are obtained by polymerase chain reaction (PCR) amplification, that is, the DNA fragments of Nanobodies;

[0149] (4) connecting the DNA of VHH to the phage surface display screening vector to form a VHH-pIII fusion protein expression vector plasmid library; wherein, pIII is a protein present on the phage surface flagella;...

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Abstract

The invention relates to an antigen binding protein, an antibody or an antibody active fragment obtained by immunizing a camelidae animal by using an S protein NTD region of SARS-Cov-2. According to the invention, the antibody which specifically recognizes and is combined with the NTD region of the S protein on the surface of the new coronavirus is screened, identified and prepared by virtue of an immune system of a camelidae animal, the obtained antibody is high in affinity and specifically recognizes and is combined with the NTD region, and the region is a part with relatively great difference between the S protein of the new coronavirus and other coronavirus sequences, so that the new coronavirus S protein can be specifically recognized and combined with the NTD region. The method can be used as a marker for distinguishing a new coronavirus from other coronaviruses. The antibody is developed aiming at the NTD region of the S protein, so that the new coronavirus and other coronaviruses are expected to be specifically distinguished, and the diagnosis of patients and the implementation of symptomatic treatment measures are facilitated.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a specific antibody for the NTD region of the S protein of the new coronavirus, and a preparation method and application thereof. Background technique [0002] Antibodies are proteins mainly secreted by plasma cells and used by the immune system to identify and neutralize foreign substances such as bacteria and viruses, which are called antigens. The binding of antibodies and antigens is entirely dependent on non-covalent interactions. This specific binding mechanism allows antibodies to capture foreign microorganisms as well as infected cells, further inducing other immune mechanisms to attack them, or directly neutralize their targets . Antibodies and antibody-related products have been widely used in research fields such as life science and medicine. Many experimental techniques derived from antigen-antibody specific binding have laid an important foundation for scientific resear...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13C12N15/70C12N5/10C40B50/06C40B40/10G01N33/569C12N1/21C12N7/01A61K39/42A61P31/14A61K51/10A61K49/00C12R1/19C12R1/92
CPCC07K16/10C07K16/005C40B50/06C40B40/10C07K14/7051C12N5/0636C12N7/00G01N33/56983C12N15/70A61P31/14A61K51/1006A61K49/0058C07K2317/22C07K2317/569C07K2317/92C07K2317/565C07K2317/14C07K2317/94C07K2317/24C07K2319/33C12N2510/00G01N2333/165G01N2469/10C12N2795/00021A61K2039/505Y02A50/30
Inventor 刘剑峰徐涛张胜蓝徐小兰
Owner BIOISLAND LAB
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