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Novel coronavirus recombinant protein with broad-spectrum neutralizing activity and preparation method of novel coronavirus recombinant protein

A recombinant protein, coronavirus technology, applied in the preparation methods of peptides, chemical instruments and methods, microorganism-based methods, etc., can solve problems such as virus immune escape

Active Publication Date: 2021-12-17
ZHEJIANG PUKANG BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, amino acid mutations in the RBD region are most likely to cause viral immune escape

Method used

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  • Novel coronavirus recombinant protein with broad-spectrum neutralizing activity and preparation method of novel coronavirus recombinant protein
  • Novel coronavirus recombinant protein with broad-spectrum neutralizing activity and preparation method of novel coronavirus recombinant protein
  • Novel coronavirus recombinant protein with broad-spectrum neutralizing activity and preparation method of novel coronavirus recombinant protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Preparation of novel coronavirus recombinant protein with broad-spectrum neutralizing activity

[0038] (1) Insert the nucleotide sequence of SEQ ID NO: 2 into the pET28a Escherichia coli expression plasmid through two restriction sites of Xho I and Nco I to obtain a recombinant expression plasmid. The resulting recombinant expression plasmid was transformed into E. coli BL21 (DE3) competent cells by 42-degree heat activation method, and monoclonal screening was performed on LB solid medium plates to obtain recombinant E. coli monoclonal colonies expressing the target protein. Recombinant Escherichia coli monoclonal colonies were cultured overnight at 37°C in 5 ml of LB liquid medium to obtain recombinant Escherichia coli activation solution.

[0039] (2) Take the recombinant Escherichia coli activation liquid obtained in step (1), inoculate it into 5 ml of LB liquid medium at a ratio of 1:100 by volume, and cultivate overnight at 37°C. Inoculate the bacteria...

Embodiment 2

[0052] Example 2 Novel coronavirus recombinant protein with broad-spectrum neutralizing activity induces the body to produce broad-spectrum neutralizing antibodies

[0053] (1) The novel coronavirus recombinant CRM-RBD protein with broad-spectrum neutralizing activity was prepared according to the preparation method in Example 1, and it was mixed and compatible with aluminum adjuvant to prepare a protein containing 50 μg / ml protein and 0.5 mg / ml aluminum adjuvant Vaccine samples.

[0054] (2) Take 4-week-old Babl / c mice and divide them into experimental group and control group, with 6 mice in each group. The experimental group was immunized with 1 ml of vaccine samples, and the control group was immunized with 0.5 mg / ml aluminum adjuvant. A total of three vaccinations were given with an interval of 28 days. Sera were collected two weeks after the last immunization.

[0055] (4) Dilute the mouse serum (1:40-1:5120), mix it with the new coronavirus dilution (1000 half cell in...

Embodiment 3

[0058] Example 3 Novel coronavirus recombinant protein with broad-spectrum neutralizing activity induces broad-spectrum specific cellular immunity

[0059] (1) The novel coronavirus recombinant CRM-RBD protein with broad-spectrum neutralizing activity was prepared according to the preparation method in Example 1, and it was mixed and compatible with aluminum adjuvant to prepare a protein containing 50 μg / ml protein and 0.5 mg / ml aluminum adjuvant Vaccine samples.

[0060] (2) Take 4-week-old Babl / c mice and divide them into experimental group and control group, with 6 mice in each group. The experimental group was immunized with 1 ml of vaccine samples, and the control group was immunized with 0.5 mg / ml aluminum adjuvant. A total of three vaccinations were given with an interval of 28 days. Sera were collected two weeks after the last immunization.

[0061] (3) Take the spleen of Babl / c mice to prepare cell suspension, wash twice with erythrocyte lysate and PBS, resuspend w...

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Abstract

The invention relates to a novel coronavirus recombinant protein with broad-spectrum neutralizing activity and a preparation method of the novel coronavirus recombinant protein. The novel coronavirus recombinant protein is CRM-RBD recombinant protein, the amino acid sequence of the CRM-RBD recombinant protein is shown as SEQ ID NO: 1, the nucleotide sequence for encoding the CRM-RBD recombinant protein is shown as SEQ ID NO: 2, and recombinant nano-protein particles are prepared by utilizing the nucleotide sequence through the following steps: performing constructing to obtain escherichia coli recombinant expression bacteria; culturing the escherichia coli recombinant expression bacteria to obtain fermentation liquor; obtaining an inclusion body crude extract; performing denatured dissolution to obtain inclusion body denatured protein; performing purifying to obtain purified recombinant protein; performing renaturation to obtain renatured protein; and separating and purifying the renatured protein to obtain the recombinant nano-protein particles. The novel coronavirus recombinant protein disclosed by the invention has a neutralizing and protecting effect on a novel coronavirus prototype strain, a beta variant and a delta variant.

Description

technical field [0001] The invention relates to the field of molecular biology technology, in particular to a novel coronavirus recombinant protein with broad-spectrum neutralizing activity and a preparation method thereof. Background technique [0002] Since severe acute respiratory syndrome coronavirus 2 (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) broke out in the world, there is currently no specific drug against SARS-CoV-2, so can a safe drug be developed? , Effective new crown vaccine has become the key to the prevention and control of new crown pneumonia. At present, researchers around the world are accelerating the development of new crown vaccines. At present, 112 candidate vaccines have entered clinical trials, and 15 vaccines have received emergency use. Among them, 38 subunit vaccines have entered clinical trials, and 2 subunit vaccines have received emergency use. . [0003] With the increase in the number of infected people and the continuati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/165C12N15/70C12N1/21C07K1/18C07K1/34C07K1/113C07K1/14C12R1/19
CPCC07K14/165C12N15/70
Inventor 高孟沈钱通庄昉成毛子安朱赟陈刚李思奇安彤张柯欣张月徐凯陈彦霖张温阳侯云龙包佳源毛江森
Owner ZHEJIANG PUKANG BIOTECH