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Novel coronavirus subunit vaccine for cats as well as preparation method and application of novel coronavirus subunit vaccine

A technology of subunit vaccine and coronavirus, which is applied in the field of feline new coronavirus subunit vaccine and its preparation, can solve the problems of unreported new coronavirus vaccine of susceptible animal cats, and achieve increased protein stability, purification and recovery High efficiency and high expression effect

Pending Publication Date: 2022-01-11
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Currently, a series of SARS-CoV-2 vaccines for human use have been urgently approved for use, including inactivated vaccines, nucleic acid vaccines, viral vector vaccines, recombinant subunit vaccines, etc., but no new coronavirus vaccines for susceptible cats have yet see the report

Method used

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  • Novel coronavirus subunit vaccine for cats as well as preparation method and application of novel coronavirus subunit vaccine
  • Novel coronavirus subunit vaccine for cats as well as preparation method and application of novel coronavirus subunit vaccine
  • Novel coronavirus subunit vaccine for cats as well as preparation method and application of novel coronavirus subunit vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Construction of SARS-CoV-2-RBD-cat Fc and RBD-His recombinant plasmids

[0028] 1. Codon optimization of the RBD coding gene of the new coronavirus

[0029] In order to achieve high-efficiency expression of RBD protein (NCBI accession number of RBD-protosequence: NC_045512, shown in SEQ ID NO.13), the present invention is based on the preference of codons in the CHO eukaryotic expression system for SARS-CoV-2-RBD- The sequences of cat Fc and SARS-CoV-2-RBD-His coding genes were optimized. Codon optimization is a key technical means to achieve high-efficiency expression of heterologous proteins, and protein expression is a systematic project, so when performing codon optimization, only replacing codons with the most frequently used codons of a certain species may not be effective. For the best results, it is necessary to consider adjusting GC content, restriction sites, mRNA stability, codon coherence and codon sensitivity, etc., but even if these are conside...

Embodiment 2

[0035] Example 2: Eukaryotic expression of pCMV-RBD-cat Fc and pCMV-RBD-His recombinant plasmids and screening, cloning and domestication of stable and highly expressive cell lines

[0036] Using the CHO eukaryotic expression system, the cells were inoculated into fresh medium at a density of 2x10^6cell / mL the day before transfection, and cultured in a constant temperature shaker at 37°C, 5% CO2, 150-175rpm; transfection On the same day, samples were taken to calculate cell density and viability. The cell density should be 3-5x10^6cell / mL, and the viability should be higher than 90%. Adjust the cell density to 3x10^6cell / mL, place the cells in a 125mL cell shaker flask with a total volume of 20mL; dilute 20μg of plasmids (pCMV-RBD-catFc-GS-1#, pCMV-RBD -cat Fc-GS-2#, pCMV-RBD-cat Fc-GS-3#, pCMV-RBD-catFc-GS-4# and pCMV-RBD-His-GS-1#, pCMV-RBD-His- GS-2#, pCMV-RBD-His-GS-3#, pCMV-RBD-His-GS-4#,) to a total volume of 0.5mL, gently blow and mix with a pipette; use optim medium ...

Embodiment 3

[0040] Example 3: Purification and activity identification of SARS-CoV-2-RBD-cat Fc and SARS-CoV-2-RBD-his recombinant proteins.

[0041] 1. Expression and purification of SARS-CoV-2-RBD-cat Fc and SARS-CoV-2-RBD-his recombinant proteins

[0042] Expand the stable high-efficiency expression cell line screened in Example 2 to a 1L cell bottle with 250ml culture medium, and culture it continuously for 14 days at 37°C, 5% carbon dioxide, 120 rpm, and collect the upper Clearance was purified successively with proteinA and Ni excel filler using AKTA purifier, RBD-cat Fc recombinant protein was eluted with 0.11mol / L glycine (pH3.0), and the collected eluate was eluted with 1mol / L Tris-Hcl (pH8.8) for use after neutralization, the best imidazole concentrations for washing and eluting RBD-His are 50mmol and 300mmol respectively, and the collected eluate is desalted for use to obtain the effective concentration of purified RBD-cat Fc recombinant protein The effective concentration of ...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a novel coronavirus subunit vaccine for cats as well as a preparation method and application of the novel coronavirus subunit vaccine. The invention provides an RBD-cat Fc protein which is expressed by fusion of an RBD structural domain of a novel coronavirus S protein and a cat IgG FC structural domain, and the subunit protein has good immunogenicity, and can induce a high-level antibody after immunizing a cat; and in addition, the generated antibody has the effect of neutralizing SARS-CoV-2 live viruses, compared with the RBD-his protein of the directly expressed SARS-CoV-2 virus S protein, the RBD-cat Fc recombinant protein does not influence the activity of the RBD protein and can increase the protein stability, and the novel coronavirus subunit vaccine can be used for preventing cats from being infected with novel coronavirus and has important significance in preventing propagation among novel coronavirus species.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a feline novel coronavirus subunit vaccine and its preparation method and application. Background technique [0002] A large number of studies have found that SARS-CoV-2 has a wide range of hosts, and hamsters, ferrets, cats, dogs, and some non-human primates (rhesus monkeys, cynomolgus monkeys) can be infected by SARS-CoV-2. Laboratory studies have shown that the new coronavirus can replicate efficiently in cats and can be transmitted among cats through respiratory droplets. In addition, previous studies in our laboratory have shown that SARS-CoV-2 has infected cats. Among the samples investigated, 14.7 % of cats could be detected seropositive by indirect enzyme-linked immunosorbent assay (E LISA), and these infections may be caused by cats exposed to SARS-CoV-2 contaminated environments or patients who had close contact with cats. Although the intermediate host of SARS...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/85C12N5/10A61K39/215A61P31/14
CPCC07K14/005C12N15/85C12N5/0682A61K39/12A61P31/14C12N2770/00022C12N2770/20034C12N2800/107C12N2800/22C07K2319/21C07K2319/02C07K2319/30A61K2039/552C12N2510/02
Inventor 金梅林赵亚余世曼杨丽吴超胡长敏张强张宇飞钟鸣孙小美
Owner HUAZHONG AGRI UNIV
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