Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Nucleic acid molecule encoding structural protein of novel coronavirus and novel coronavirus vaccine

A coronavirus and nucleic acid molecule technology, applied in the field of genetic engineering, can solve the problems of low protein expression and difficult vaccine production of new coronary pneumonia, and achieve the effects of high immunogenicity, high yield and stable structure

Active Publication Date: 2021-03-30
WEST CHINA HOSPITAL SICHUAN UNIV +1
View PDF2 Cites 7 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, the gene sequences of the four structural proteins of the wild type of the new coronavirus, E (small envelope), M (membrane), N (nucleocapsid) and S (Spike), have very low protein expression in eukaryotic cells, making the new coronavirus Pneumonia vaccine production becomes more difficult

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Nucleic acid molecule encoding structural protein of novel coronavirus and novel coronavirus vaccine
  • Nucleic acid molecule encoding structural protein of novel coronavirus and novel coronavirus vaccine
  • Nucleic acid molecule encoding structural protein of novel coronavirus and novel coronavirus vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Find the sequences of the E gene, M gene, N gene and S gene of the wild-type novel coronavirus through NCBI, and analyze its codon bias, GC content, and CPG island and other parameters. Four genes were codon-optimized, and the optimized and wild-type sequences are as follows:

[0037] The nucleotide sequence of the optimized E gene is shown in SEQ ID NO.1; the nucleotide sequence of the wild-type E gene is shown in SEQ ID NO.5;

[0038] The nucleotide sequence of the optimized M gene is shown in SEQ ID NO.2; the nucleotide sequence of the wild-type M gene is shown in SEQ ID NO.6;

[0039] The nucleotide sequence of the optimized N gene is shown in SEQ ID NO.3; the nucleotide sequence of the wild-type N gene is shown in SEQ ID NO.7;

[0040] The nucleotide sequence of the optimized S gene is shown in SEQ ID NO.4; the nucleotide sequence of the wild-type S gene is shown in SEQ ID NO.8.

Embodiment 2

[0042] The above-mentioned target gene was obtained by gene synthesis, and it was re-cloned into the expression vector PCI (for the vector map, see Figure 6 ), the present invention takes 2 μg of the target gene and carrier respectively, uses two restriction endonucleases Kpn I and Not I to carry out enzyme digestion at 37°C for 3-4 hours, recovers the gel to obtain the target gene and carrier, and separates the target gene and carrier According to the molar mass of 3:1, use T4 ligase to connect at 16°C for 3-4 hours, then transform it into DH5α strain, pick a single clone and extract the plasmid, and finally identify it by enzyme digestion and Sanger sequencing . Then the recombinant expression plasmids PCI-coE, PCI-coM, PCI-coN and PCI-coS and wild-type PCI-wtE, PCI-wtM, PCI-wtN and PCI-wtS were transfected into HEK293T cells by calcium phosphate transfection and For transient expression, 37°C, 5% CO 2 Cultivate in the environment for 8 hours, replace with fresh DMEM (con...

Embodiment 3

[0045] Preparation of VLPs

[0046] VLPs were transfected with four recombinant expression plasmids PCI-coE, PCI-coM, PCI-coN, PCI-coS or four wild-type plasmids PCI-wtE, PCI-wtM, PCI-wtN, PCI-wtS by calcium phosphate transfection method Mass ratio 1:1:1:1 was co-transfected into HEK293T cells at 37°C, 5% CO 2 Cultivate in the environment for 8 hours, replace with fresh DMEM (containing 10% FBS, 1% double antibody), 37°C, 5% CO 2 Continue culturing for 48 hours, collect the supernatant, centrifuge at 5000rpm, 4°C for 15min, take the supernatant, filter through a 0.45μM filter membrane, ultracentrifuge at 25000rpm, 4°C for 2h 30min, discard the supernatant, and dissolve the precipitate with PBS Finally, the VLPs were concentrated 200 times, and then the optimized (coVLPs) and wild-type (wtVLPs) VLPs yields were compared by methods such as silver staining and western blot. It was found that using the optimized E (small envelope), M (membrane), The yield of VLPs packaged by N (...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a nucleic acid molecule for encoding a structural protein of a novel coronavirus and a novel coronavirus vaccine, and relates to the technical field of gene engineering. The base sequence of the nucleic acid molecule disclosed by the invention is as shown in any one of SEQ ID NO.1 to SEQ ID NO.4. The invention also discloses a preparation method of the nucleic acid molecule. The nucleic acid molecule provided by the invention can express the structural protein of the novel coronavirus more efficiently through codon optimization, and lays a foundation for development ofnovel coronapneumonia vaccines.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a nucleic acid molecule encoding a structural protein of a novel coronavirus and a novel coronavirus vaccine. Background technique [0002] The 2019 novel coronavirus was officially named 2019-nCoV by the World Health Organization on January 12, 2020, and its genome sequence was also analyzed by relevant scientists and released on NCBI. However, at present, the gene sequences of the four structural proteins of the wild type of the new coronavirus, E (small envelope), M (membrane), N (nucleocapsid) and S (Spike), have very low protein expression in eukaryotic cells, making the new coronavirus Pneumonia vaccine production has become more difficult. [0003] In view of this, the present invention is proposed. Contents of the invention [0004] The object of the present invention is to provide a nucleic acid molecule encoding a structural protein of a novel coronaviru...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N15/50C12N15/85C12N5/10C07K14/165A61K39/215A61P31/14
CPCC07K14/005C12N15/85A61K39/12A61P31/14C12N2770/20022C12N2770/20023C12N2770/20034C12N2800/107Y02A50/30
Inventor 董飚刘瑜干春梅杨荔叶静娅陈治安
Owner WEST CHINA HOSPITAL SICHUAN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com