Stable coronavirus recombinant protein dimer and expression vector thereof

A coronavirus and recombinant protein technology, applied in the field of recombinant protein, can solve the problems of poor protein stability and weak immunogenicity, and achieve good immunogenicity, avoid degradation, and broad application prospects

Active Publication Date: 2021-02-05
SOUTH CHINA UNITED VACCINE INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when the S-RBD monomer is used as an antigen, the immunogenicity is weak and the protein stability is poor

Method used

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  • Stable coronavirus recombinant protein dimer and expression vector thereof
  • Stable coronavirus recombinant protein dimer and expression vector thereof
  • Stable coronavirus recombinant protein dimer and expression vector thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059]Example 1: A vector expressing the recombinant protein of the novel coronavirus SARS-CoV-2

[0060]The structure diagram of SARS-CoV-2 recombinant protein is as followsfigure 1 Shown.

[0061]Connect the N-terminal signal peptide MFVFLVLLPLVSS of the new coronavirus SARS-CoV-2 S protein to the N-terminal of SARS-CoV-2S-RBD, and connect the CTD region (aa248-365) of the N-protein to the C-terminal of S-RBD. Use linker to connect in the middle. Put in pFastBacTM SCUVI plasmid Ppolh Under the promoter. Obtain the shuttle vector pFastBacTM SCUVI-SP-S-RBD-CTD, the vector expressing the recombinant protein of the new coronavirus SARS-CoV-2.

Embodiment 2

[0063]The preparation method of the vector expressing the recombinant protein of the novel coronavirus SARS-CoV-2 includes the following steps:

[0064]1) Gene synthesis optimization

[0065]Connect the new coronavirus SARS-CoV-2 S protein N-terminal signal peptide SP, the 306-575 region (S-RBD) and the N protein CTD region (aa248-365) (S-RBD and CTD are connected with Linker GSGSG), According to the host cellSpodoptera frugiperda The preference of Spodoptera frugiperda (Sf9) cell line is optimized for codon source, PUC57 is the carrier, andXho I,KpnI Synthesize SP-S-RBD-CTD for restriction site, and clone it into PUC57 vector to obtain recombinant vector PUC57-SP-S-RBD-CTD.

Embodiment 3

[0067]The preparation method of the vector expressing the recombinant protein of the novel coronavirus SARS-CoV-2 includes the following steps:

[0068]1) Gene synthesis optimization

[0069]Connect the new coronavirus SARS-CoV-2 S protein N-terminal signal peptide SP, the 306-575 region (S-RBD) and the N protein CTD region (aa248-365) (S-RBD and CTD are connected with Linker GSGSG), According to the preference of the host cell Spodopterafrugiperda (Sf9) cell line, the codon source is optimized, PUC57 is the vector, andXho I ,Kpn ISynthesize SP-S-RBD-CTD for restriction site, and clone it into PUC57 vector to obtain recombinant vector PUC57-SP-S-RBD-CTD.

[0070]2) Preparation of shuttle carrier

[0071]Using Xho I, Kpn I, the SP-S-RBD-CTD in the vector PUC57-SP-S-RBD-CTD was digested and connected to pFastBacTM SCUVI plasmid Ppolh Under the promoter. Obtain the shuttle vector pFastBacTM SCUVI-SP-S-RBD-CTD.

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Abstract

The invention discloses a stable coronavirus recombinant protein dimer and an expression vector thereof. Coronavirus recombinant protein is composed of coronavirus S protein S-RBD, coronavirus N protein CTD region N-CTD and a linker for coupling the coronavirus S protein S-RBD and the coronavirus N protein CTD region N-CTD. The coronavirus recombinant protein in some embodiments can form and maintain a stable dimer structure, avoids monomer S-RBD degradation, is beneficial to improving the immunogenicity of the coronavirus recombinant protein, and is expected to be used for preparing detectionreagent raw materials, vaccines, antibodies and preventive or therapeutic drugs. The coronavirus recombinant protein dimers in some embodiments have good immunogenicity and have wide application prospects in the vaccine development field. Expression vectors in some embodiments can easily express the coronavirus recombinant protein dimers and have high expression quantity.

Description

Technical field[0001]The invention relates to a recombinant protein, in particular to a stable coronavirus recombinant protein dimer and its expression vector.Background technique[0002]Coronavirus belongs to the order Nidovirales, Coronaviridae, and Coronavirus. It is an important pathogen of many domestic animals, pets, including human diseases, and causes a variety of acute and chronic diseases. According to the phylogenetic tree, coronaviruses can be divided into four genera: α, β, γ, and δ. Among them, β genus coronaviruses can be divided into four independent subgroups A, B, C and D.[0003]Coronavirus infection is extremely common all over the world. So far, about 15 different coronavirus strains have been discovered, which can infect a variety of mammals and birds, and some can cause illness in people. In addition to the recent outbreak of the new coronavirus SARS-CoV-2, there are six other coronaviruses including HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-Co...

Claims

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Application Information

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IPC IPC(8): C07K14/165C12N15/50C12N15/63A61K39/215A61P31/14G01N33/569
CPCA61K39/12A61P31/14C07K14/005C12N2770/20022C12N2770/20034G01N33/56983G01N2333/165
Inventor 彭涛许煜华冯金郭伟丽陈丽云郑燕萍王炳彰刘双标
Owner SOUTH CHINA UNITED VACCINE INST
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