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Vesicular stomatitis virus vector-based novel coronavirus chimeric recombinant vaccine as well as preparation method and application thereof

A technology for vesicular stomatitis and coronavirus, applied in the field of novel coronavirus chimeric recombinant vaccine and its preparation, to achieve good safety, strong mucosal immune response, and easy cultivation

Active Publication Date: 2021-02-19
INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Currently, vaccines and drugs against the novel coronavirus (SARS-CoV-2) are still in clinical trials or at an earlier stage, and comprehensive evaluation and further optimization are urgently needed

Method used

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  • Vesicular stomatitis virus vector-based novel coronavirus chimeric recombinant vaccine as well as preparation method and application thereof
  • Vesicular stomatitis virus vector-based novel coronavirus chimeric recombinant vaccine as well as preparation method and application thereof
  • Vesicular stomatitis virus vector-based novel coronavirus chimeric recombinant vaccine as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Embodiment 1, the preparation of recombinant virus rVSV-SARS-CoV / 2-RBD

[0071] 1. Preparation of recombinant vector VSV-SARS-CoV / 2-RBD

[0072] 1. Optimization of chimeric envelope protein S sequence

[0073] In order to develop a new coronavirus vaccine, the 1-314th and 537-1259th positions of the envelope protein S amino acid sequence (GenBank accession number AAP30030.1) of the SARS-CoV BJ01 strain (GenBank accession number AY278488.2) were The coding gene sequence of the position is the same as the 319-541th position of the envelope protein S amino acid sequence (GenBank accession number YP_009724390.1) of the SARS-CoV-2 Wuhan-Hu-1 strain (GenBank accession number NC_045512.2) After humanization, optimization and chimerization of the coding gene sequence of the chimeric envelope protein S, the coding gene sequence of the chimeric envelope protein S is obtained, and its nucleotide sequence is shown in sequence 1 in the sequence list.

[0074] The chimeric envelope...

Embodiment 2

[0092] Embodiment 2, the antigen expression identification of recombinant virus rVSV-SARS-CoV / 2-RBD

[0093] In order to identify the situation of rVSV-SARS-CoV / 2-RBD expressing the chimeric S protein, collect the Vero cells infected by the rVSV-SARS-CoV / 2-RBD prepared in Example 1 and the control recombinant virus rVSV-SARS-CoV-2 The supercentrifugation method was used to centrifuge at 39,000 rpm at 4°C for 3 hours (Beckman SW41 rotor) to obtain the virus pellet, and the concentrated virus was obtained after resuspending the virus pellet. Then use Western Blot method (anti-RBD protein antibody, Beijing Yiqiao Shenzhou Technology Co., Ltd., rabbit anti-SARS-CoV-2 RBD polyclonal antibody, catalog number 40592-T62) to detect S protein on purified virus particles and infected Vero cells The expression situation in Qing Dynasty. Uninfected Vero cells were used as controls.

[0094] The result is as image 3 shown. The results show that the expression of S protein can be detect...

Embodiment 3

[0095] Embodiment 3, the growth curve of recombinant virus rVSV-SARS-CoV / 2-RBD

[0096] The Vero cells were passaged in a 10cm cell culture dish at a ratio of 1:3. The next day when the cells grow to about 80% density, add the recombinant virus rVSV-SARS-CoV / 2-RBD prepared in Example 1 and the control recombinant virus rVSV-SARS-CoV-2, M.O.I.=0.01, and change after 2 hours of infection into DMEM medium containing 2% (volume fraction) FBS. Samples were taken every 12 hours after infection until 108 hours after infection. Virus titers in supernatants at different infection times were determined by immunofluorescence.

[0097]The operation method of measuring virus titer by immunofluorescence method is as follows: the Vero cells are subcultured in a 96-well plate at 10,000 cells per well. The next day, when the cells grow to about 80%-90% density, add the virus supernatant to be tested. Perform a 10-fold serial dilution of the virus supernatant, that is, mix 30 µl of the viru...

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Abstract

The invention discloses a vesicular stomatitis virus vector-based novel coronavirus chimeric recombinant vaccine as well as a preparation method and application thereof. The active component of the recombinant vaccine is recombinant virus rVSV-SARS-CoV / 2-RBD, and is a virus obtained by replacing glycoprotein G of the vesicular stomatitis virus with chimeric envelope protein S; the chimeric envelope protein S is a protein obtained by replacing the RBD of the SARS-CoV envelope protein S with the RBD of the SARS-CoV-2 envelope protein S; the amino acid sequence of the RBD of the SARS-CoV envelopeprotein S is the 315-536 site of the amino acid sequence of the SARS-CoV envelope protein S; and the amino acid sequence of the RBD of the SARS-CoV-2 envelope protein S is the 319-541 site of the amino acid sequence of the SARS-CoV-2 envelope protein S. The recombinant virus has great significance to vaccine development of new coronaviruses.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a novel coronavirus chimeric recombinant vaccine based on vesicular stomatitis virus vector and its preparation method and application. Background technique [0002] The novel coronavirus (SARS-CoV-2) is a newly emerged βcoronavirus. Virus genome analysis shows that the virus is very close to the same genus SARS-CoV (Severe acute respiratory syndrome-coronavirus) (89.1% nucleotide similarity), and can cause symptoms such as pneumonia in humans after infection. The novel coronavirus is a single-stranded positive-sense RNA virus with an envelope. Observed under the electron microscope, the virus particles are round or oval with radial protrusions. This radial protrusion is a characteristic structure of coronavirus, which is composed of the envelope protein Spike (S). S protein is an important structural protein of coronavirus, which plays a key role in the process of vir...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/86G01N33/68A61K39/215A61P31/14C12R1/93
CPCA61K39/12A61K2039/5256A61P31/14C07K14/005C12N7/00C12N15/86C12N2760/20221C12N2760/20243C12N2760/20252C12N2770/20022C12N2770/20034C12N2800/107G01N33/6854
Inventor 郑爱华李虹悦张毓航温丹袁菲
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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