Recombinant protein highly targeting SARS-ConV-2 virus S protein extracellular BD-terminal domain and subunit vaccine thereof

A sars-conv-2, recombinant protein technology, applied in the direction of positive-sense single-stranded RNA viruses, viruses, viral peptides, etc., can solve problems such as breaking the immune balance of the body, achieve good development and application prospects, and improve immune stress ability. Effect

Active Publication Date: 2021-07-23
WUHAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies suggest that the continuous expression of mRNA vaccines in the body produces antigenic proteins, which may break the body's own immune balance and trigger immune tolerance

Method used

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  • Recombinant protein highly targeting SARS-ConV-2 virus S protein extracellular BD-terminal domain and subunit vaccine thereof
  • Recombinant protein highly targeting SARS-ConV-2 virus S protein extracellular BD-terminal domain and subunit vaccine thereof
  • Recombinant protein highly targeting SARS-ConV-2 virus S protein extracellular BD-terminal domain and subunit vaccine thereof

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Experimental program
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preparation example Construction

[0048] According to another typical implementation of the embodiments of the present invention, a method for preparing a highly targeted recombinant protein of the outer BD end domain of the SARS-ConV-2 virus S protein is provided, the method comprising:

[0049] Obtain the antigenic determinant Sc and Se of SARS-ConV-2 virus S protein membrane extramembrane BD terminal domain domain, the nucleotide sequence of described Sc is as shown in SEQ ID NO: 1, the nucleotide sequence of described Se is as SEQ ID NO: ID NO: 2 shown;

[0050] Constructing and obtaining the expression plasmids of Sc and Se, wherein the nucleotide sequence of the expression region of the Sc expression plasmid is shown in SEQ ID NO: 5, and the nucleotide sequence of the expression region of the Se expression plasmid is shown in Shown in SEQ ID NO: 6;

[0051] The expression plasmids of the Sc and Se were secreted, expressed and purified, respectively, to obtain a highly targeted recombinant protein of the...

Embodiment 1

[0057] Example 1 Analysis and design of epitope in extramembrane domain of BD end of SARS-Cov-2 virus S protein

[0058] 1. Construction of a three-dimensional model of the S protein of the SARA-Cov-2 virus

[0059] Retrieve the amino acid sequences of SARS-Cov-2 and SARS-Cov coronavirus S proteins from the database of the National Center for Bioinformatics (https: / / www.ncbi.nlm.nih.gov), and the retrieval numbers in GenBank are respectively for QHD43416 and P59594. The amino acid sequences of SARS-Cov-2 and SARS-Cov S proteins were compared by Clustal software, and their amino acid identity was 76%, showing high homology. The highly homologous sequences of the S proteins of the two viruses are mainly concentrated in the middle to the C-terminus. A three-dimensional structural model of the 2019-nCoV viral S protein was further constructed. The 2019-nCoV virus forms the spike structure on the surface of the virion envelope through the polymerization of three S proteins. Fur...

Embodiment 2

[0074] Example 2 Expression and Purification of S Protein BD-related Epitopic Determinant Peptides

[0075] 1. Expression of 2Sc and 3Se

[0076] The constructed plasmids were respectively transformed into BL21(DE3) expression competent cells, coated on LB plates containing Kanna resistance, placed in a 37°C incubator, and cultured overnight to grow single clones. Four single clones were selected respectively, and cultured in 5 ml of Kanna-resistant LB medium at 200 rpm at 37° C. for 5 h. When the OD600 reached 0.6-0.8, take out 500 μL and put it into a new 1.5mL EP tube marked as before induction. The remaining bacterial liquid was induced by adding IPTG (final concentration 1 mM), and the samples marked as pre-induction and post-induction samples were placed at 37° C. and incubated for 4 hours. Aspirate 200 μL of the bacterial solution before and after induction, centrifuge at 12000 rpm for 5 min. Remove the supernatant, add 50 μL of PBS to resuspend, add 2*loading buffer...

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Abstract

The invention provides a recombinant protein highly targeting SARS-ConV-2 virus S protein extracellular BD-terminal domain and a subunit vaccine thereof. A nucleotide sequence for coding the recombinant protein is as shown in SEQ ID NO: 5 or SEQ ID NO: 6. The novel coronavirus subunit vaccine comprises the recombinant protein and a pharmaceutically acceptable adjuvants. According to the invention, the recombinant protein highly targeting SARS-ConV-2 virus S protein extracellular BD-terminal domain is prepared by the following steps: screening S protein extracellular domain surface antigenic determinants to find Sc and Se peptide fragment gene sequences capable of stimulating cells to generate strong immune response, carrying out codon optimization, cloning into a plasmid vector containing a diphtheria toxin T structure, and carrying out secretory expression and purification. Experiments show that the purified recombinant protein can induce specific cell reaction, is safe and efficient, and has good development and application prospects.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a highly targeting recombinant protein of the outer BD end domain of SARS-ConV-2 virus S protein and a subunit vaccine thereof. Background technique [0002] SARA-Cov-2 belongs to the genus β-coronavirus of the Coronaviridae family, and is related to severe acute respiratory syndrome (severe acute respiratory syndrome, SARS) coronavirus (SARS-CoV) and Middle East respiratory syndrome (Middle East respiratory syndrome, MERS) coronavirus ( MERS-CoV) belong to the same genus. SARA-Cov-2 is a positive-strand single-stranded RNA virus with a genome of 29.9kd. For RNA viruses, the genome is relatively large, encoding a total of 9860 amino acids. The genome includes a cap structure at the 5' end and a poly A tail at the 3' end. The replicase genes encoding 16 non-structural proteins are close to the 5' end, accounting for about 2 / 3 of the entire genome, and the remaining 1 / 3 of the genome en...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/50C12N15/70C12N1/21C07K14/165A61K39/215A61K39/385A61P31/04C12R1/19
CPCC07K14/005C12N15/70A61K39/12A61K39/385A61P31/04C12N2770/20022C12N2770/20034C07K2319/55C12N2800/22A61K2039/6037Y02A50/30
Inventor 郑忠亮权春菊邓辉
Owner WUHAN UNIV
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