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Method for preventing and treating respiratory infectious diseases by using respiratory epithelial cell membranes and application

A technology of epithelial cells and respiratory tract, applied in the fields of biotechnology and medicine, can solve the problem of undisclosed inhibition of viral infection

Pending Publication Date: 2022-01-28
SHANGHAI PUBLIC HEALTH CLINICAL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these documents do not disclose the use of receptors on the cell membrane and its surface as a means of inhibiting viral infection

Method used

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  • Method for preventing and treating respiratory infectious diseases by using respiratory epithelial cell membranes and application
  • Method for preventing and treating respiratory infectious diseases by using respiratory epithelial cell membranes and application
  • Method for preventing and treating respiratory infectious diseases by using respiratory epithelial cell membranes and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Example 1: Construction of a lung epithelial cell line ACE2-A549 stably expressing ACE2 protein

[0085] The lung epithelial cell line A549 (purchased from ATCC#CCL-185) is derived from human non-small cell lung cancer epithelial cells, and is the main in vitro cell model for studying pathogen infection of respiratory infectious diseases.

[0086] The human ACE2 gene (SEQ ID NO: 1) was synthesized in vitro by gene synthesis technology, the ACE2 gene was inserted into the lentiviral vector Phage-puro (addgene #118692), and combined with the packaging plasmid Pspax2 (addgene #12260) and the envelope plasmid PMD2 .G (addgene#12259) was co-transfected into 293T cells, and the lentiviral supernatant was collected 48 hours later. A549 cells were infected with recombinant ACE2 lentivirus, centrifuged for 2 hours, and then cultured for 24 hours to select the A549 cells expressing ACE2 with puromycin. The drug was added continuously for 14 days, and finally ACE2-A549 cells were ...

Embodiment 2

[0089] Embodiment 2: separation, preparation of cell membrane

[0090] In this example, the cell membranes of the following three cell lines were separated and prepared respectively: wild-type A549 (Wt-A549), empty vector control A549 (vector-A549), and A549 stably expressing ACE2 (ACE2-A549).

[0091] Cell membrane samples were extracted, separated, and prepared using a cell membrane separation kit (Thermo fisher scientific #89842). The brief experimental steps are as follows: 4×10 6 Cells were plated in a 10 cm cell culture dish, and a total of 5 dishes were prepared for a total of 2×10 7 number of cells. After 12 hours of conventional culture in a 37°C cell culture incubator, wash with cold PBS buffer three times, and then collect the cells with a cell scraper for future use. After washing the cells twice with the cell washing solution included in the kit, treat the cells with the permeabilization buffer for 10 minutes to release the cytoplasmic components. After centri...

Embodiment 3

[0093] Example 3: Utilize cell membrane or its components to inhibit SARS-CoV-2 infection in vitro

[0094]In this embodiment, a coronavirus pseudovirus system is used to detect pseudovirus-infected cells at the cell level. Utilize novel coronavirus (SARS-CoV-2) spike protein gene (Spike, SEQ ID NO: 4, refer to GenBank: QHD43416.1 expression plasmid pcDNA3.1-spike (produced by Shanghai Jierui Biotechnology Co., Ltd. Spike whole gene Synthesized and cloned into pcDNA3.1 expression vector) and HIV-1 backbone plasmid with luciferase reporter gene (pNL4-3-Luc-ΔEnv, NIHAIDS Reagent Program #3418) co-transfected 293T cells, harvested after 48 hours Cell culture supernatant containing coronavirus pseudoviruses.

[0095] 293T cells expressing ACE2 were infected with the supernatant containing pseudovirus (ACE2-293T cell line construction method is the same as ACE2-A549), if the pseudovirus successfully infects the target cells, by detecting the expression level of the reporter gene l...

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Abstract

The invention relates to a method for preventing and treating respiratory infectious diseases by using respiratory epithelial cell membranes and application. Specifically, the invention provides the use of respiratory epithelial cell membranes in the preparation of products for the prevention and / or treatment of respiratory infectious disease pathogen infection and corresponding products thereof, wherein the cell membranes may be unmodified or modified (e.g. modified to overexpress receptors on the surface of the cell membranes). The respiratory epithelial cell membranes and related products thereof can be widely applied to prevention and treatment of respiratory infectious disease pathogen infection, and the modified cell membranes can play a more effective role in prevention and treatment of specific pathogen infection.

Description

technical field [0001] The invention belongs to the fields of biotechnology and medicine. Specifically, the present invention relates to the method and application of using respiratory epithelial cell membranes to prevent and treat pathogen infection of respiratory infectious diseases. Background technique [0002] Respiratory tract infectious diseases refer to infectious diseases caused by the invasion of pathogens (such as viruses, bacteria, fungi, mycoplasma, chlamydia, etc.) from the nasal cavity, pharynx, larynx, trachea, bronchi and lungs of the human body. [0003] The new, sudden and severe infectious diseases caused by pathogens of respiratory infectious diseases seriously threaten people's health and social stability, and cause serious damage to regional exchanges and economic construction. The most representative ones include, for example, the coronavirus such as the 2003 Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS) in 2012, S...

Claims

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Application Information

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IPC IPC(8): A61K35/42A61P31/00A61P11/00
CPCA61K35/42A61P31/00A61P11/00
Inventor 徐建青张晓燕丁龙飞赵晨
Owner SHANGHAI PUBLIC HEALTH CLINICAL CENT
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