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Specific antibody of novel coronavirus S protein as well as preparation method and application thereof

A specific, protein-based technology, applied in the direction of antiviral immunoglobulin, virus/bacteriophage, and microbial-based methods, can solve the problems of no practical clinical treatment value, large-scale mass production of high finished products, unfavorable large-scale mass production, etc. , to achieve the effects that are conducive to large-scale popularization and application, large-scale production, and large-scale clinical therapeutic use

Active Publication Date: 2022-07-19
BIOISLAND LAB +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the above-mentioned technology based on the immune system of animals such as mice to produce S protein antibodies and separate and extract them has the following disadvantages: (1) the specificity of antibodies is limited: Spike The protein structure is large and the spatial folding is complex. Traditional monoclonal antibodies such as mice, rabbits, and monkeys are difficult to recognize the complex spatial structure on the surface of Spike; more importantly, a large number of biological studies have shown that the S protein is in the form of homotrimers. Existing on the surface of coronavirus, if only a certain fragment (domain) of S protein is used as the antigen, the specificity of the obtained antibody is limited; (2) the application value of the antibody is limited: most of these antibodies can only be used for virus (3) The stability of the antibody is poor: the above-mentioned mouse monoclonal antibody needs to be stored, transported, tested and verified at low temperature (4 degrees Celsius) to maintain the concentration and specificity of the antibody (4) Large-scale mass production and high finished product: the above-mentioned mouse monoclonal antibody is a full-length immunoglobulin, which needs to be recombined in a relatively expensive expression system such as mammalian cells Separation, purification and extraction after expression are complicated and costly, which is not conducive to large-scale mass production

Method used

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  • Specific antibody of novel coronavirus S protein as well as preparation method and application thereof
  • Specific antibody of novel coronavirus S protein as well as preparation method and application thereof
  • Specific antibody of novel coronavirus S protein as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0126] Example 1: Preparation of antigens

[0127] The conventional practice in the prior art is to recombinantly express and purify a certain region of the S protein as an antigen.

[0128] The present invention is to prepare antigen for nearly full-length new coronavirus S protein, and the specific method is as follows:

[0129] (1) Synthesize the DNA sequence encoding the S protein (amino acids 1-1208), connect it in series with a DNA sequence encoding the T4 fibritin region, and construct it into the pFASTBAC insect cell recombinant expression vector to form the S protein trimer recombinant expression plasmid ;

[0130] The amino acid sequence of the T4 fibritin region can induce the entire peptide chain to spontaneously trimerize, and then fold to form an S protein trimer;

[0131] The amino acid sequence, nucleotide sequence of the above-mentioned S protein and the amino acid sequence of the T4 fibritin region are shown in Table 3;

[0132] table 3

[0133]

[013...

Embodiment 2

[0145] Example 2: Alpaca immunization injection

[0146] In this example, alpacas were immunized with the antigen prepared in Example 1. Specific steps are as follows:

[0147] (1) The antigens prepared in Example 1 were equally divided into 4 parts, each of about 1 mg;

[0148] (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.

[0149] 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 peripher...

Embodiment 3

[0150] Example 3: Construction of Antibody Libraries

[0151] 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:

[0152] (1) Using density gradient centrifugation to separate lymphocytes from the peripheral blood of alpaca veins;

[0153] (2) Extracting the total mRNA of lymphocytes and reverse transcribing into cDNA;

[0154] (3) using appropriate DNA primers, using the above-mentioned cDNA as a template, through polymerase chain reaction (PCR) amplification to obtain the VHH fragments of alpaca immunoglobulin IgG2 and IgG3, that is, the DNA fragments of Nanobodies;

[0155] (4) the DNA of VHH is connected to the phage surface display screening vector, constitutes the VHH-pIII fusion protein expression vector plasmid library; wherein, pill is the protein present on the phage surface fl...

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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 with S protein of SARS-Cov-2. And the S protein of the SARS-Cov-2 has a trimer structure. According to the invention, the antibody for specific recognition and combination with the new coronavirus S protein is screened, identified and prepared by relying on an immune system of a camelidae animal, and the obtained antibody is high in specificity, can be used for rapid detection of the new coronavirus, has potential clinical diagnosis and treatment values, and is easy to carry out genetic engineering modification and humanization. The antibody provided by the invention is high in stability, can avoid the requirement that a conventional antibody needs low-temperature storage and transportation, and is beneficial to large-scale popularization and application, reduction of production cost and realization of large-scale production.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to a specific antibody of the new coronavirus S protein and a preparation method and application thereof. Background technique [0002] The envelope of the new coronavirus (SARS-Cov-2, hereinafter referred to as the new coronavirus) particles is composed of the Spike protein (hereinafter referred to as the S protein), the E protein and the M protein, of which the S protein is one of the main components on the surface of all coronaviruses. The S protein on the surface of the new coronavirus has a full length of 1273 amino acids and can be divided into two parts, S1 and S2. After the virus invades the cell, the S protein is cleaved into S1 subunit and S2 subunit. S1 mainly includes the NTD region and the RBD region, of which the RBD region is called the receptor binding region, which is a necessary region for the S protein to invade host cells, but its amino acid sequence homology...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13C12N1/21C12N7/01C07K19/00C12N5/10C12N15/70C40B40/02C40B50/06G01N33/569G01N33/58G01N33/60A61K39/42A61P31/14C12R1/19C12R1/92
CPCC07K16/10C07K16/005C07K14/7051C12N7/00C12N5/0636C12N15/70C40B40/02C40B50/06G01N33/56983G01N33/581G01N33/583G01N33/60A61P31/14C07K2317/569C07K2317/565C07K2317/92C07K2317/76C07K2317/94C07K2317/24C12N2510/00C07K2319/03C07K2319/33G01N2333/165C12N2795/00021C12N2795/00043A61K2039/505
Inventor 刘剑峰徐涛张胜蓝徐小兰
Owner BIOISLAND LAB
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