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Preparation method of cell for transmembrane expression of 2019-nCoV antigen, cell and application

A coronavirus and cell technology, applied in the field of genetic engineering, can solve problems such as virus infection, and achieve the effect of strong immunogenicity, high strength and outstanding effect

Active Publication Date: 2021-01-22
焦顺昌 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, using the full-length protein of the virus as an antigen for clinical use can cause a partial preventive effect on the virus, but it is also common to see clinical reports of antibody-dependent enhancement (Antibody-dependent enhancement, referred to as ADE), resulting in a higher than unused More severe viral infections in vaccine recipients

Method used

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  • Preparation method of cell for transmembrane expression of 2019-nCoV antigen, cell and application
  • Preparation method of cell for transmembrane expression of 2019-nCoV antigen, cell and application
  • Preparation method of cell for transmembrane expression of 2019-nCoV antigen, cell and application

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preparation example Construction

[0034] The invention provides a method for preparing cells expressing novel coronavirus antigens across membranes, comprising the following steps:

[0035] 1) The sequence is integrated into the PB-713 plasmid through the Bsu36I and BmgBI double restriction sites to obtain the PB-S-RBD-NGFR plasmid; the nucleotide sequence of the sequence is shown in SEQ ID No.1;

[0036] 2) The PB-S-RBD-NGFR plasmid obtained in step 1) was electrotransformed into K562 cells, screened with puromycin, and when the proportion of living cells continued to be greater than 80%, the transmembrane expression of the novel coronavirus antigen was obtained. cell.

[0037] In the present invention, the sequence is integrated into the PB-713 plasmid through the Bsu36I and BmgBI double restriction sites to obtain the PB-S-RBD-NGFR plasmid; the nucleotide sequence of the sequence is shown in SEQ ID No.1. In the present invention, the nucleotide sequence of said sequence is shown in SEQ ID No.1, specificall...

Embodiment 1

[0060] The nucleotide sequence of SEQ ID No.1 was integrated into the PB-713 plasmid (purchased from SBI Company) through the upstream Bsu36I and downstream BmgBI double restriction sites. The new plasmid was named PB-S-RBD-NGFR plasmid. The prepared plasmid was extracted and endotoxin was removed in large quantities by Jinweizhi.

[0061] Transfection of cells:

[0062] 1. K562 cells were cultured according to the instructions of ATCC, and the culture conditions were: IMDM+10% FBS. Subculture method: maintain the cell concentration at 1×10 5 ~1×10 6 / ml, centrifuge every 2-3 days to remove the old medium, and suspend the cells in fresh medium according to the density.

[0063] 2. Use the Celetrix electroporation instrument to perform plasmid electroporation on K562 cells according to the instruction manual, and take 4×10 7 K562 cells were divided into four 120μl electric shock tubes, that is, 1×10 7 Add 10 μg of PB-S-RBD-NGFR plasmid to cells / tube, electroporation condi...

Embodiment 2

[0068] 1. Purpose of the experiment

[0069] Aggregate situation of RBD-NGFR structure.

[0070] 2. Experimental materials

[0071] 1. Sample

[0072] K562-cells; K562-S-RBD-NGFR cells prepared in Example 1

[0073] 2. Reagents

[0074] Table 1 Reagents

[0075]

[0076] 3. Instrument

[0077] Table 2 Instruments

[0078]

[0079]

[0080] 2. Experimental method

[0081] (1) Extraction of protein samples

[0082] 1. Take the cell culture solution in a 15mL centrifuge tube, centrifuge at 1200rpm for 5min, discard the supernatant, add 1mL of 1×PBS solution to resuspend, centrifuge at 2000rpm for 5min, and wash twice by centrifugation;

[0083] 2. Take a 1.5mL EP tube, add 5μL 100×Phosphatase Inhibitor protein inhibitor and 495μL Pierce TM RIPABuffer protein lysate, mix thoroughly on a vortex shaker and set aside;

[0084] 3. Gently discard the supernatant, use a pipette gun to discard all the supernatant as much as possible, add 100 μL of the above mixture to re...

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Abstract

The invention provides a preparation method, of a cell for transmembrane expression of a 2019-nCoV antigen, a cell and an application, and belongs to the technical field of gene engineering. The method comprises the following steps: integrating a sequence into a PB-713 plasmid through Bsu36I and BmgBI double restriction enzyme cutting sites, so as to obtain a PB-S-RBD-NGFR plasmid; electrically transforming the PB-S-RBD-NGFR plasmid into K562 cells, performing puromycin screening, and when the proportion of living cells is continuously larger than 80%, obtaining cells for transmembrane expression of the 2019-nCoV antigen. According to the invention, the receptor binding domain of a 2019-nCoV spike glycoprotein is independently subjected to transmembrane cell expression, so that the risk ofantibody dependence enhancement caused by other S protein epitopes is avoided, and then the receptor binding domain of the 2019-nCoV spike glycoprotein is fused with a transmembrane protein domain; and a better neutralizing titer is induced in an animal body.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a preparation method, cells and applications of cells expressing novel coronavirus antigens across membranes. Background technique [0002] Novel coronavirus (2019-nCoV, SARS-CoV-2) is a type of coronavirus of the genus β. Infection with the virus can cause symptoms such as fever, dry cough, and fatigue in patients; some patients will develop severe pneumonia, and then develop Acute respiratory distress syndrome, septic shock, coagulation dysfunction, multiple organ failure, etc., and even death. The SARS-CoV-2 virus adsorbs and enters host cells through the envelope protein on its surface, thereby infecting human respiratory epithelial cells and alveolar cells. The envelope protein of SARS-CoV-2, Spike protein (S), is a glycoprotein that mainly functions in cell adhesion and cell membrane fusion. [0003] The S protein is composed of two subunits, S1 an...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/85C12N15/50A61K39/215A61P31/14
CPCA61K39/12A61P31/14C07K14/005C07K2319/02C07K2319/03C12N5/0694C12N15/85C12N2510/00C12N2770/20022C12N2770/20034
Inventor 焦顺昌张嵘袁翰张艳玲李斯慧冯向辉宋文静吕鹏敏钟宏东胡坤鲁仕豪
Owner 焦顺昌
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