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Novel coronavirus antibody with broad-spectrum neutralizing activity as well as preparation method and application of novel coronavirus antibody

A virus and antibody technology, applied in the field of new coronavirus antibodies and their preparation, can solve the problems of low antibody level, low antibody neutralization activity, and difficult to achieve, and achieve the effect of good thermal stability

Active Publication Date: 2022-02-01
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Previous studies attempted to immunize laying hens with inactivated vaccines to prepare egg yolk antibodies, but due to the low neutralizing activity of the obtained antibodies, they would be diluted by body fluids after entering the body, making it difficult to achieve the minimum antibody level required for immune protection

Method used

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  • Novel coronavirus antibody with broad-spectrum neutralizing activity as well as preparation method and application of novel coronavirus antibody
  • Novel coronavirus antibody with broad-spectrum neutralizing activity as well as preparation method and application of novel coronavirus antibody
  • Novel coronavirus antibody with broad-spectrum neutralizing activity as well as preparation method and application of novel coronavirus antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Example 1, the preparation of immunogen

[0091] First, the preparation of recombinant plasmid

[0092] 1. Construction of wild-type RBD expression plasmid

[0093] The wild-type RBD expression plasmid was prepared according to the patent number "ZL 2010,801,084,7271", inventive Name as "a COVID-19 subunit vaccine and the preparation method thereof." Specific steps are as follows:

[0094] The double-stranded DNA molecules shown in the sequence table 2 were inserted into the NOTII and Xbai enzyme somewhere of the PCDNA3.1 vector (Saimi Fei Company, the item number V79020) to obtain recombinant plasmid pcDNA / NCOV-RBDFD. The recombinant plasmid has been sequenced.

[0095] The fusion protein shown in the DNA molecular coding sequence list shown in the sequence 2 in the sequence table, from the N-terminal to C end

[0096] The following functional components are included: human interleukin 10 signal peptide (1-18 in sequence 1) is used to secrete RBD recombinant proteins int...

Embodiment 2

[0123] Example 2, Preparation of anti-champional virus yolk antibody

[0124] First, immunization program

[0125] Experimental animals: 150 days old eggs (Tianjin Challenge Biotechnology Co., Ltd.).

[0126] Experimental method: The experimental animals were randomly divided into 8 groups for muscle injection, and 5 egg chickens per group were injected once every 3 weeks (3 times in total), and each group of treatment is specifically as follows:

[0127] Wild strain immunization group (RBD-WT): Injection of RBD-WT protein prepared in Example 1, 20 μg per injection dose, with white oil adjuvant (Zhejiang Zhengxin, Item No .: C-ZS-007), each injection 1 ml.

[0128] Mutant strain immunization group (RBD-MU): Injection of RBD-Mu protein prepared in Example 1, with an injection of 20 μg, with a white oil adjuvant, and 1 ml per injection.

[0129] Hybrid immunochin group (RBD-WT + RBD-Mu): Injection of the RBD-WT protein and RBD-MU protein prepared in Example 1, each injection dose is...

Embodiment 3

[0143] Example 3, anti-champional virus oviviation antibody stability

[0144] Example 2 was placed in an oval antibody solution (mixed immunity group) in 25 ° C for 3 months, and the activity was detected every other month, and SDS-PAGE electrophoresis was performed.

[0145] Such as image 3 and Figure 4 Indicated. The results show that antibodies have good thermal stability, and 25 ° C is placed in 3 months. These and active did not change, and the protein purity and concentration did not change significantly.

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Abstract

The invention discloses a novel coronavirus antibody with broad-spectrum neutralizing activity as well as a preparation method and application of the novel coronavirus antibody. The preparation method of the novel coronavirus antibody comprises the following steps: immunizing a poultry animal by adopting an immunogen to obtain the antibody; wherein the immunogen comprises a protein A and a protein B, and the amino acid sequence of the protein A is shown as a sequence 3 in a sequence table; and the amino acid sequence of the protein B is shown as a sequence 6 in the sequence table. The novel coronavirus antibody has good cross-neutralization activity on wild strains and variant strains of the novel coronavirus, and the titer of the induced antibody is far higher than that of the reported antibody obtained by adopting an inactivated vaccine. After being inhaled by the nasal cavity of a mouse, the mouse can be protected against high-dose new coronavirus challenge, and no obvious pathological change is seen in the lung. After a big-ear white rabbit inhales in the nasal cavity, a high-titer neutralizing antibody can be detected in bronchus and lungs. The antibodies can be developed into nasal formulations for use in the prevention and / or treatment of new coronavirus infection.

Description

Technical field [0001] The present invention relates to the field of biotechnology, and more particularly to new crown viral antibodies having broad spectrum neutralization and its preparation and application thereof. Background technique [0002] The global new crown virus (SARS-COV-2) epidemic is still grim, and the continuous mutant strains have caused multiple epidemics. From the early Alpha strain, to the Delta strain, which is now traveled to hundreds, and the new OMICRON strains, the infectivity of the variant strains is gradually increased, and the protection efficacy of new crown vaccines has been reduced. Although nearly half of the population has undergone vaccines, the breakthrough infection caused by the variant strains has reduced the immunization effectiveness of the group, and the rehabilitation cases of the rehabilitation population are constantly rising, and new challenges are put forward on our epidemic prevention and control. [0003] Although there are many v...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K16/10C07K16/02C07K1/36C07K1/34C07K1/22C07K1/16C12N15/62C12N15/85A61K39/42A61P31/14
CPCC07K14/005C07K16/10C07K16/02C12N15/85A61P31/14C07K2319/02C07K2317/11C07K2317/23C07K2317/76C07K2317/33C07K2317/94C12N2770/20022A61K2039/505Y02A50/30
Inventor 杨利敏范文辉刘文军毕玉海
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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