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Subunit coronavirus vaccine for dimerization-based receptor binding domains

A coronavirus, purpose technology, applied in the field of medicine, can solve the problems of limited immunogenicity and insufficient safety evaluation of fusion proteins

Active Publication Date: 2017-07-07
ANHUI ZHIFEI LONGCOM BIOPHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In summary, the current vaccines developed based on the RBD monomer of the coronavirus S protein have limited immunogenicity.
However, RBD-based fusion proteins (such as the Fc segment of IgG) have the defect of insufficient safety evaluation

Method used

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  • Subunit coronavirus vaccine for dimerization-based receptor binding domains
  • Subunit coronavirus vaccine for dimerization-based receptor binding domains
  • Subunit coronavirus vaccine for dimerization-based receptor binding domains

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Preparation of recombinant baculoviruses expressing MERS and SARS antigens

[0033]After the MERS-CoV RBD nucleotide sequence (as shown in SEQ ID NO: 3) and the SARS-CoV RBD (as shown in SEQ ID NO: 4) nucleotide sequence 3' end are added with a translation stop codon, they are cloned into a Between the EcoR I and Xho I restriction sites of the pFastBac vector (pFastBac-SP) of the gp67 signal peptide, the protein coding region is fused and expressed behind the signal peptide gp67 sequence for the secretion of the target protein. The vectors pFastBac-SP-MERS-RBD and pFastBac-SP-SARS-RBD were obtained respectively.

[0034] The vector used to express the MERS monomeric RBD is inserted into the EcoRI and XhoI restriction sites of pFastBac-SP by inserting the nucleic acid fragment from amino acid 379 to 589 in the S protein sequence (GenBank: AFS88936.1 on NCBI), and The 3' end is composed of 6 histidines. The vector used to express the SARS monomer RBD is mutat...

Embodiment 2

[0036] Embodiment 2: Purification of MERS-RBD and SARS-RBD

[0037] Filter the supernatant of cells expressing MERS-RBD and SARS-RBD through a 0.22 μm filter to remove cell debris. The target protein was captured by a HiTrap QHP anion exchange column, and then crudely purified by elution with a buffer containing 1M NaCl. After identification by SDS-PAGE, fractions containing the target protein were collected and pooled. Load the hydrophobic column HiTrap Phenyl HP as solution A for binding. Prepare low-salt eluent solution B (20mM Tris-HCl pH 8.0) for gradient elution. The eluted RBD was subjected to SDS gel electrophoresis to detect the target protein. The elution peaks containing the target protein obtained by crude purification by hydrophobic chromatography were combined, concentrated to 5 mL through a 10K cut-off concentration tube (Millipore), and then subjected to molecular sieve chromatography on a Superdex200 Hiload16 / 60 column (GE) for further purification of the t...

Embodiment 3

[0041] Embodiment 3: mouse immunization experiment

[0042] The MERS and SARS antigens obtained in Example 2 were diluted in physiological saline according to the method in Table 1, and emulsified in groups with adjuvants. Then 4-6 weeks old Balb / C mice were immunized in groups. immunization strategies such as image 3 , that is, through thigh muscle injection, each mouse received three vaccine immunizations on the 0th day, the 21st day, and the 42nd day respectively, with an inoculation volume of 100ul each time. On the 54th day (ie, the 12th day after the 3rd immunization), the tail blood was collected from the mice. The mouse serum was obtained by centrifuging at 3000 rpm for 10 minutes after standing for a period of time to allow the serum to separate out, and stored in a -20°C refrigerator for pseudovirus neutralization detection.

[0043] Table 1: Animal immunization groups

[0044] antigen or control Antigen content Adjuvant number of animals PB...

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Abstract

The invention discloses a subunit coronavirus vaccine for dimerization-based receptor binding domains and belongs to the technical field of medicine. A baculovirus expresses RBD (receptor binding domain (E367-Y606) of MERS-CoV (middle east respiratory syndrome coronavirus) protein and RBD (R294-F515) of SARS-CoV (severe acute respiratory syndrome coronavirus) in insect cells, the RBDs may form a dimer through cysteine residue at 603 of S (spike) protein or form a dimer through cysteine residue at 512 of the S protein, and purified RBD protein dimer and monomer are used respectively to immunize Bald / c mice. The dimerized RBDs have the advantages that the defect that RBD monomers have poor immunogenicity is overcome and the generation of neutralizing antibodies in against MERS-CoV is increased greatly.

Description

technical field [0001] The invention relates to a coronavirus vaccine based on a dimerized receptor binding region subunit, belonging to the technical field of medicine. Background technique [0002] Coronaviruses belong to the genus Coronaviridae of the family Coronaviridae and are enveloped positive-strand RNA viruses with the largest genomes among all RNA viruses. Both animals and humans are hosts of coronaviruses. Coronaviruses mainly infect the respiratory and digestive tracts of mammals and birds. Six of these coronaviruses infect humans. Among them, the severe respiratory syndrome coronavirus (SARS-CoV), which broke out in China in 2002-2003, and the Middle East respiratory syndrome coronavirus (MERS-CoV), which broke out in 2012 and continues to this day, are the greatest threat to global public health. Coronaviruses also cause a number of serious animal diseases, particularly posing a serious threat to agricultural livestock and pets. For example, porcine transm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/165C12N15/50C12N15/866C12N7/01C12N1/21C12N5/10C07K1/36C07K1/34C07K1/20C07K1/18C07K1/16A61K39/215A61P31/14
CPCA61K39/12A61K2039/55505A61K2039/55566C07K14/005C12N15/86C12N2710/14043C12N2770/20022C12N2770/20034
Inventor 高福戴连攀严景华李世华袁园
Owner ANHUI ZHIFEI LONGCOM BIOPHARM CO LTD
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