Porcine endothelium-derived IL-8 overexpression stably transfected cell line, preparation method and applications thereof

A technique of endothelial cells and cell lines, applied in the field of genetic engineering

Inactive Publication Date: 2017-10-13
BEIJING UNIV OF AGRI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

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  • Porcine endothelium-derived IL-8 overexpression stably transfected cell line, preparation method and applications thereof
  • Porcine endothelium-derived IL-8 overexpression stably transfected cell line, preparation method and applications thereof
  • Porcine endothelium-derived IL-8 overexpression stably transfected cell line, preparation method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Establishment of porcine endothelial-derived IL-8 overexpression stable cell line

[0033] 1. Materials and methods

[0034] 1.1 cells

[0035] Porcine hip arterial endothelial cell line (PIEC): purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences Shanghai Life Science Research Cell Resource Center, catalog number is GN015. 293T cells (human kidney epithelial cell line): purchased from Nanjing Ke Bai Biotechnology Co., Ltd.

[0036] 1.2 Reagents

[0037] XbaI and NotI restriction endonucleases and quick ligation kits were purchased from Bao Biological Engineering (Dalian) Co., Ltd.; gel recovery kits, endotoxin-free plasmid extraction kits, and lentivirus concentrates were purchased from Biomiga; lipofectamine2000 transfection reagents Purchased from Invitrogen; Lenti-OE carrier was purchased from Quanshijin Biotechnology Co., Ltd. PE-labeled CD1a mouse anti-pig monoclonal antibody, FITC-labeled SWC3a mon...

Embodiment 2

[0059] Example 2 Gene overexpression identification of porcine endothelial-derived IL-8 overexpression stable cell line

[0060] When the cells were digested and passaged after virus infection, 250uL of cell suspension was collected, RNA was extracted according to conventional methods, and RT-PCR was performed to identify the expression effect (RNA level) of IL-8 gene.

[0061] By extracting the RNA in PIEC after lentivirus infection and detecting by RT-PCR, it was found that the level of IL-8 gene in the PIEC cell line infected with lentivirus was significantly increased ( Figure 4 ).

Embodiment 3

[0062] Example 3 Identification of protein overexpression in porcine endothelial-derived IL-8 overexpression stable cell line

[0063] PIEC stably transfected cells containing Lenti-OE and Lenti-OE-IL8 viruses were planted in a 6-well plate (2×105 / well). After 24 hours, the cells were congested, and the supernatant was collected for ELISA detection to identify the expression of IL-8 protein Level.

[0064] The expression of IL-8 protein in the supernatant of the IL-8 stably transfected PIEC cell line was detected by ELISA, and it was found that the protein expression of IL-8 in the stably transfected cell line was significantly increased compared with the normal PIEC cells (P Figure 5 shown.

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Abstract

This application discloses a porcine endothelial-derived IL-8 overexpression stable cell line, which was preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee on July 7, 2016, and the preservation registration number is CGMCC NO. 12682. The application also discloses a preparation method and application of a porcine endothelial-derived IL-8 overexpression stable cell line. The porcine endothelial-derived IL-8 overexpression stably transfected cell line of the present application has good application in the porcine virus immunopathogenic mechanism and the porcine endothelial-derived IL-8 action mechanism, etc. value. The porcine endothelial IL-8 overexpression stable cell line of the present invention also has the effect of anti-inflammation.

Description

technical field [0001] The application belongs to the field of genetic engineering, and in particular relates to a porcine endothelial-derived IL-8 overexpressed stably transfected cell line and its preparation method and application. Background technique [0002] Interleukin 8 (IL-8), also known as neutrophil-activating protein (NAP-1), has a molecular mass of 8.3 kDa and can be produced by a variety of cells, including monocytes, macrophages, endothelial cells, fibroblasts cells and epidermal cells. IL-8 is the most important neutrophil-activating chemokine, which can promote the exudation and aggregation of neutrophils and lymphocytes, and act on different cells to promote the process of inflammatory response, stimulate the formation of blood vessels, Promote mitosis, regulate host immune function, etc. Studies have found that endothelial-derived IL-8 is distinguished from monokaryotic IL-8 due to the presence of an extension of AVLPR at the N-terminus, which makes endo...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/85C12N5/09C12N7/00A61K35/44A61P29/00C12R1/93
CPCC12N5/069A61K35/44C12N5/0694C12N7/00C12N15/85C12N2501/2308C12N2502/99C12N2510/00C12N2750/10051C12N2800/107
Inventor 李焕荣周双海张永红崔德凤
Owner BEIJING UNIV OF AGRI
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