Preparation method of SARS-CoV-2 gene silencing immortalized lung stem cells

A virus gene, stem cell technology, applied in the field of biomedicine

Inactive Publication Date: 2021-04-13
翁炳焕
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there has been no literature report on the functional modification of the new coronavirus RNA interference on stem cells for the treatment of COVID-19

Method used

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  • Preparation method of SARS-CoV-2 gene silencing immortalized lung stem cells
  • Preparation method of SARS-CoV-2 gene silencing immortalized lung stem cells
  • Preparation method of SARS-CoV-2 gene silencing immortalized lung stem cells

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Embodiment Construction

[0028] The specific implementation methods of the present invention will be described in detail below in conjunction with the accompanying drawings, but these exemplary descriptions do not constitute any limitation to the scope of protection defined by the claims of the present invention.

[0029] 1. Collection of cell samples

[0030] 1.1. Collection of amniotic fluid fibroblasts

[0031] According to the prenatal diagnosis process, the amniocytes of pregnant women to be tested are collected, cell culture and prenatal diagnosis are carried out, and the spindle-shaped amniocytes growing on the wall are screened from the remaining amniocytes under an inverted microscope.

[0032] 1.2. Collection of lung mesenchymal stem cells

[0033] Take aseptically the lung tissue of aborted fetuses, mechanically dissociate, digest with 0.25% trypsin, filter with gauze with a pore size of 100 μm, centrifuge at 1000 r / min for 5 min, discard the supernatant, add DMEM culture medium (0.1 μmol ...

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Abstract

The invention discloses a preparation method of SARS-CoV-2 gene silencing immortalized lung stem cells, and the preparation method is characterized in that amniotic fluid fibroblast stem cells are separated from amniotic fluid cells remained after prenatal diagnosis, an immortalized amniotic fluid stem cell line is constructed by an SV40LT and / or hTERT transfection method, so that the immortalized lung stem cell line is screened out, and then the RNA interference sequences shRNA of the SARS-CoV-2 M, N, E and / or S genes are inserted into the immortalized lung stem cell line DNA to prepare the SARS-CoV-2 gene silencing immortalized lung stem cell, and the obtained SARS-CoV-2 gene silencing immortalized lung stem cell can interfere the replication of the SARS-CoV-2 in the lung stem cell by shRNA targeting, and can be industrially amplified in vitro.

Description

technical field [0001] The invention relates to a preparation method of novel coronavirus gene silenced immortal lung stem cells, which belongs to the infectious disease prevention and control technology in the field of biomedicine. Background technique [0002] The novel coronavirus (SARS-CoV-2) has posed a serious threat to global public health. The main structure of SARS-CoV-2 includes single-stranded positive-strand nucleic acid (ssRNA), spike protein (S), membrane protein (M), envelope protein (E) and nucleocapsid protein (N). Among them, the S protein is cleaved by host proteases into the N-terminal S1 subunit and the C-terminal S2 subunit during the infection process. The S1 subunit is composed of an N-terminal domain (S1-NTD) and a receptor binding domain (S1-RBD). -RBD is responsible for recognizing and binding the host cell surface receptor angiotensin-converting enzyme 2 (ACE2), and the S2 subunit mediates the fusion between the viral envelope and the host cell m...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/113
CPCC12N5/0689C12N15/113C12N2310/141C12N2310/531C12N2510/04
Inventor 翁炳焕
Owner 翁炳焕
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