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Beta-coronavirus antigen as well as preparation method and application thereof

A coronavirus and antigen technology, applied in the field of biomedicine, can solve problems such as difficult control, threats to agricultural livestock and pets, and high lethality

Active Publication Date: 2020-08-28
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no specific drug yet, and there is no vaccine to prevent it. Only symptomatic and supportive treatment can be used
[0005] In addition, several other coronaviruses cause many serious animal diseases, especially for agricultural livestock and pets
For example, porcine transmissible gastroenteritis virus (TGEV) can cause severe diarrhea in pigs with high mortality; its deletion virus porcine respiratory coronavirus (PRCV) can cause severe respiratory diseases in pigs; feline peritonitis virus (FIPV) can cause Feline peritonitis and ascites gather, and the fatality rate is very high; canine coronavirus (CCoV) can cause dogs to have gastroenteritis symptoms in varying degrees, which spreads quickly and is difficult to control; porcine epidemic diarrhea virus (PEDV) causes porcine epidemic diarrhea and other enteric diseases. Taoist disease, easy to spread among pigs with high fatality rate

Method used

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  • Beta-coronavirus antigen as well as preparation method and application thereof
  • Beta-coronavirus antigen as well as preparation method and application thereof
  • Beta-coronavirus antigen as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0105] Example 1: Preparation of recombinant baculovirus expressing MERS-CoV antigen and expression and purification of RBD protein

[0106] The nucleotide sequence (as shown in SEQ ID NO: 24) encoding the amino acid RBD (E367-Y606) sequence (as shown in SEQ ID NO: 1) in the MERS-CoV S protein (sequence as GenBank: AFS88936.1) ) 3' end plus a translation stop codon and cloned between the EcoR I and Xho I restriction sites of the pFastBac vector (pFastBac-SP from Invitrogen) containing the gp67 signal peptide, so that the protein coding region is in the sequence of the signal peptide gp67 The following fusion expression is used for the secretion of the target protein, and the C-terminus of the target protein is carried with 6 histidines to obtain the vector pFastBac-SP-MERS-RBD (E367-Y606), and then transfect the cells of the expression system for expression , after expression, the cell supernatant was collected and purified.

Embodiment 2

[0108] Example 2: MERS-RBD protein immunization experiment on mice

[0109] MF59 (AddaVax used below is a MF59-like adjuvant) and aluminum adjuvant are two commonly used adjuvants approved by SFDA. We use these two adjuvants as vaccine components to have more direct guidance for subsequent clinical trials effect. In vitro neutralization test is a classic method to detect the protective effect of vaccines. Therefore, we combined different doses of antigen with AddaVax adjuvant and Imject TM Alum adjuvant was mixed for immunization. The grouping of immunizations, the type of RBD used in each group, the dose of RBD used in each immunization and the situation of adjuvants are shown in Table 1, and the blank part means "none".

[0110] Dilute the MERS-RBD antigen (dimer or monomer) obtained in Example 1 to a desired concentration in physiological saline, and emulsify in groups with an adjuvant. Then 4-6 weeks old BALB / c mice (average body weight 15-20 g, the same below) were im...

Embodiment 3

[0115] Embodiment 3: ELISA test detects the specific antibody titer induced by the vaccine

[0116] (1) The RBD monomeric protein of MERS-CoV was diluted to 3 μg / ml with ELISA coating solution (Solaibao, C1050), and 100 μl was added to each well of a 96-well ELISA plate (Coring, 3590), and placed at 4°C for 12 hours.

[0117] (2) Pour off the coating solution, add PBS, and wash again. 5% skimmed milk prepared in PBS was used as a blocking solution, added to a 96-well plate, 100 μl per well, blocked, and left at room temperature for 1 hour. Wash once with PBS solution after blocking.

[0118] (3) Dilute the mouse serum during the blocking period. Serum samples were also diluted in blocking solution. Serum samples were serially diluted from a 20-fold starting point. Afterwards, 100 μl was added to each well of the ELISA plate, and the negative control was added blocking solution, incubated at 37 degrees for 2 hours, and then washed 4 times with PBST.

[0119] (4) Add goat a...

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Abstract

The embodiment of the invention relates to a beta-coronavirus antigen as well as a preparation method and application thereof. Wherein the amino acid sequence of the beta-coronavirus antigen comprisesan amino acid sequence arranged according to a (A-B)-(A-B) style or an amino acid sequence arranged according to a (A-B)-C-(A-B) style or an amino acid sequence arranged according to a (A-B)-(A-B ')style or an amino acid sequence arranged according to a (A-B)-C-(A-B') style, and the beta-coronavirus antigen has a single-chain dimer structure. The single-chain dimer expressed by the embodiment ofthe invention is stable in content and has good immunogenicity as a beta-coronavirus antigen, and a vaccine prepared by using the single-chain dimer as a beta-coronavirus antigen can stimulate mice to generate a neutralizing antibody with very high titer.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a betacoronavirus antigen, its preparation method and application. Background technique [0002] Coronaviruses belong to the genus Coronaviridae of the family Coronaviridae. They are enveloped positive-strand RNA viruses with the largest genomes among all RNA viruses. Both animals and humans are the hosts of coronaviruses. Coronaviruses mainly infect the respiratory tract and digestive tract of mammals and birds. There are currently seven known coronaviruses that infect humans, four of which cause mild colds (HCoV-229E, HCoV-NL63, OC43 and HKU1). There are three biggest threats to global public health, namely the severe respiratory syndrome coronavirus (SARS-CoV) that broke out in 2002-2003; the Middle East respiratory syndrome coronavirus (MERS-CoV) that broke out in 2012 and continues to this day; and 2019 The new coronavirus (2019-nCoV) that broke out in 2019, all three b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/85C12N15/70C12N5/10A61K39/215A61P31/14A61P11/00
CPCC07K14/005C12N15/85C12N15/70A61K39/12A61P31/14A61P11/00C12N2770/20022C07K2319/02C12N2770/20034A61K39/001129Y02A50/30A61K39/215A61K39/39A61K2039/55505A61K2039/55566
Inventor 戴连攀严景华高福李燕郑天依徐坤刘梅安亚玲施一
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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