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SARS-CoV-2 receptor bind region glycosylation modification antigen and application thereof

A coronavirus-receptor binding technology, applied in the field of peptides, can solve the problems of low immunogenicity and insufficient cross-protection, and achieve the effects of simple preparation method, good cross-protection effect, and good application prospect.

Active Publication Date: 2021-11-12
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Vaccines with RBD as the main antigen in many domestic and foreign units have been promoted to clinical practice. The results show that RBD can stimulate specific antibody and neutralizing antibody responses, but there are still shortcomings such as low immunogenicity and insufficient cross-protection. Modified and optimized to enhance its immune potency (Nature 586:572–577(2020); Cell 182:722–733(2020))

Method used

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  • SARS-CoV-2 receptor bind region glycosylation modification antigen and application thereof
  • SARS-CoV-2 receptor bind region glycosylation modification antigen and application thereof
  • SARS-CoV-2 receptor bind region glycosylation modification antigen and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Glycosylation design and expression purification of the RBD monomer in the receptor binding region of the novel coronavirus

[0036] First, according to the two important N-glycosylation sites (located at the 331st and 343rd amino acids respectively) of the reported new coronavirus RBD protein (sequence such as Genebank, YP_009724390.1, R319-K537) (Science 369:330-333 (2020)), carried out N-glycosylation modification on RBD, removed glycosyl masking and exposed antigenic epitopes to enhance its immunogenicity. The transformation method is to first obtain the full-length RBD 219 Antigen (R319-K537, due to its C-terminal C538 cysteine ​​may form a multimer due to the formation of an intermolecular disulfide bond, so it ends at K537), followed by truncating the full-length RBD 219(R319-K537) The N-glycosyl site of the region or its point mutation, design three kinds of glycosylation engineered antigen RBD 219 -N331A (mutate amino acid 331 from asparagine N to a...

Embodiment 2

[0038] Example 2 Evaluation of Immunogenicity of Glycosylated RBD Monomer Antigen

[0039] The wild-type RBD prepared above 219 Protein and RBD monomer glycosylation engineered antigen RBD 219 -N331A, RBD 219 -N343Q (mutate amino acid 343 from asparagine N to glutamine Q to destroy N2 glycosylation site), RBD 206(I332-K537) ) protein for mouse immunization experiment to compare its immunogenicity. Select 6-8 weeks of female BALB / c mice, 6 in each group, dilute the protein obtained in Example 1 to 100 μg / mL with phosphate buffer, and mix with an equal volume of AL(OH) 3 The adjuvant (Alhydrogel, 1000 μg / mL) was mixed and adsorbed at room temperature for 1 h. Then carry out group immunization, and the grouping situation is shown in Table 1. By intramuscular injection, each mouse received two vaccine immunizations on day 0 and day 14 respectively, each inoculation volume was 100 μL (5 μg antigen, 50 μg aluminum adjuvant). On the 14th and 21d days, blood was collected from t...

Embodiment 3

[0043] Example 3 Novel coronavirus glycosylation engineered antigen RBD 206 -dimer and RBD 206 -Design and expression purification of trimer

[0044] On the basis of Example 2, through structural optimization design, the monomer RBD 206 Glycoengineered antigens undergo tandem repeats to form single-chain multimeric forms, such as dimeric RBD 206 -dimer (two sequences shown in SEQ ID NO: 1 in forward tandem) and trimeric RBD 206 -trimer (three sequences shown in SEQ ID NO: 1 for forward tandem), see sequence design figure 1 , to further enhance its immunogenicity (Cell182:722–733(2020)). See Example 1 for the method of expressing RBD using the Expi293F mammalian cell expression system 206 - dimer, RBD 206 -trimer antigen protein, using GE's His-trap affinity chromatography column for protein purification. After obtaining the recombinant protein, use BCA method and UV spectrophotometer to measure the protein concentration, use SDS-PAGE experiment to identify the purified ...

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Abstract

The invention discloses an SARS-CoV-2 receptor bind region (RBD) glycosylation modification antigen, which is characterized in that the N glycosylation site of the SARS-CoV-2 receptor bind region (RBD) is truncated and connected in series to form a single-chain polymer form, and natural glycosyl shielding on the surface of the RBD is reduced so as to more favorably expose antigen epitopes. Compared with a wild RBD monomer and RBD polymer form, the antigen can excite an organism to generate a higher-level SARS-CoV-2 specific antibody and a neutralizing antibody; an immune effect obtained through one-needle immunization can be equal to the immune effect of two-needle immunization of the wild type RBD monomer; and immune serum has similar neutralizing titer by aiming at an original strain and a variant strain, and shows a good cross protection effect. The invention also provides application of the glycosylation modification antigen in preparation of SARS-CoV-2 treatment and prevention drugs or vaccines.

Description

technical field [0001] The invention relates to a novel coronavirus receptor binding region glycosylation modified antigen and its application, belonging to the technical field of polypeptides. Background technique [0002] So far, the new coronavirus (SARS-CoV-2) has caused about 180 million infections and nearly 4 million deaths, posing a great threat to human health (https: / / covid19.who.int / ). SARS-CoV-2 is mainly transmitted through the respiratory tract and close contact. Currently, there is no specific drug for treatment. Vaccines are the most effective means to prevent and control the epidemic. Hundreds of SARS-CoV-2 vaccine studies have been carried out around the world, among which mRNA vaccines, adenovirus vector vaccines, inactivated vaccines, recombinant subunit vaccines and other vaccine types have made important progress. Some vaccines have been approved for marketing, providing strong scientific and technological support for the response to the new crown viru...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/165C12N15/50C12N15/85A61K39/215A61P31/14
CPCC07K14/005C12N15/85A61K39/12A61P31/14C12N2770/20022C12N2770/20034C12N2800/107A61K2039/53Y02A50/30
Inventor 陈薇宰晓东徐俊杰周楚格于蕊张军殷瑛张跃李汭桦李耀辉赵晓帆
Owner ACADEMY OF MILITARY MEDICAL SCI
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