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PK-15 cell line capable of stably expressing CD63-GFP and construction and application thereof

A technology of LVX-CD63-PK-15 and CD63-GFP, applied to genetically modified cells, cells modified by introducing foreign genetic material, applications, etc., can solve problems such as inability to directly observe, and achieve rapid and accurate evaluation Effect

Active Publication Date: 2019-04-16
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Fu Yuxuan constructed Hela and HT-29 cell lines capable of stably expressing CD63-luciferase fusion protein to detect exosome secretion after virus-infected cells, and the results were consistent with WB and nanoparticle size counting methods (Fu Yuxuan. Exocrine Body-mediated miR-146a promotes the replication of enterovirus 71 by inhibiting the production of type I interferon [D]; Nanjing University, 2018.), the article established Hela and HT-29 cell lines, and did not involve PK -15 cell line; in addition, the fusion expression of CD63 in this article is luciferase, which must be detected with a specific instrument and cannot be directly observed

Method used

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  • PK-15 cell line capable of stably expressing CD63-GFP and construction and application thereof
  • PK-15 cell line capable of stably expressing CD63-GFP and construction and application thereof
  • PK-15 cell line capable of stably expressing CD63-GFP and construction and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] The construction of embodiment 1 lentiviral vector and virus packaging specifically include the following contents:

[0040] (1) Referring to the porcine CD63 gene (XM_005663878.2) published in Genebank, design the porcine CD63 gene with restriction endonuclease EcoR I and BamH I recognition sites added at both ends, and biosynthesize it by Nanjing Jingbai . The porcine CD63 gene sequence used in the present invention is consistent with the sequence whose accession number is XM_013980209.2 in Genebank.

[0041] (2) Use EcoR I and BamH I to double-digest the target gene synthesized in step (1) and the pLVX-AcGFP1-N1 (Lv-pLVX) lentiviral vector, respectively.

[0042] (3) The target gene and the digested plasmid vector were ligated using T4 DNA ligase to construct a recombinant plasmid and named it Lv-CD63; the recombinant plasmid was identified by double digestion and sequencing.

[0043] (4) Inoculate 293T cells on a 10cm plate (the culture medium in the plate is DMEM...

Embodiment 2

[0044] Example 2 The screening and identification of PK-15 cell lines stably expressing CD63-GFP specifically include the following:

[0045] Lentivirus infection of PK-15 cells:

[0046] The lentiviruses Lv-pLVX and Lv-CD63 obtained in Example 1 were respectively infected with 6-well plate PK-15 cells, the volume of the virus in the infection system was 200 μl, and the number of viruses was about 2×10 7 , about 80% of the PK-15 cells in the 6-well plate were infected, and the infection system was replenished to 2 ml with complete medium, and 2 μL of the transfection enhancer polybrene was added to the infection system, and terminated after 8 hours.

[0047] Observation of virus infection by indirect immunofluorescence microscopy:

[0048] After 48 hours, use indirect immunofluorescence microscopy to observe, such as figure 1 As shown, the two constructed cell lines had strong green fluorescent signals, which indicated that the packaged lentiviruses Lv-pLVX and Lv-CD63 had s...

Embodiment 3

[0060] Example 3 Verification of extracellular exosome western blotting under the action of exosome inhibitors

[0061] LVX-PK-15 and PK-15-CD63-GFP cells were cultured in plates containing DMEM culture medium (the culture medium contained 10% fetal calf serum), and 10 μM GW4869 was added when the cells grew to about 80%, and at the same time The same amount of DMSO was added to the control group. After 36 h, the cell supernatant was taken. The cell supernatant was subjected to differential centrifugation and ultracentrifugation, the pellet was resuspended in PBS, and the denatured sample was used for western blotting verification. Western blotting verification steps are as follows: Prepare 10% polyacrylamide gel protein gel; take an appropriate amount of protein sample and add protein pre-stained marker as molecular weight indicator; voltage 80V until the end of electrophoresis; after electrophoresis, 200mA, transfer to membrane for 2h After transfer, block with 5% skimmed ...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a PK-15 cell line capable of stably expressing CD63-GFP and construction and application thereof, wherein the preservation number of the cell line is CCTCC NO: C2018253. The construction method comprises the following steps: target gene synthesis, target gene and lentivirus carrier double enzyme digestion, recombinant plasmid construction and identification, transfection of 293T cells, infection of PK-15 cells, and cell line screening verification. The PK-15 cell strain capable of stably expressing CD63-GFP can be used for evaluating a content of exudates inside and outside cells. The PK-15 cell line capable of stably expressing CD63-GFP fusion protein can be used for observing green fluorescent protein byfluorescence or conveniently evaluating the content of exudates by a flow cytometry. The establishment of the cell line provides a reliable material for researching the application of the exudates invirus infection of PK-15 cells.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a PK-15 cell line stably expressing CD63-GFP and its construction and application. Background technique [0002] Exosomes are a type of double-layer membrane vesicles with a diameter of 30-150 nm that are produced in cells and secreted to the outside of cells, and their main components are proteins, lipids, and nucleotides. Exosomes were originally considered as carriers for the excretion of cellular waste from cells, however, increasing evidence supports that exosomes affect the fate of recipient cells by transporting various substances such as nucleic acids, lipids, and proteins. function in intercommunication. In 2007, the first study found that exosomes can be used as messengers for intercellular information exchange. Subsequent studies have found that exosomes play an important role in host immune response, pathogenic infection, and tumor development. Exosomes carr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/867C12N15/66C12N5/10G01N21/64C12R1/91
CPCC07K14/70596C07K2319/60C12N15/66C12N15/86C12N2510/00C12N2740/15043G01N21/6428
Inventor 郑海学张克山徐守兴田宏茹毅李丹朱紫祥杨帆刘湘涛
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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