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Pig kidney cell line and application thereof

A porcine kidney cell and cell technology, applied in the field of biology, can solve the problems of high cost, troublesome PAM cell preparation process, and inability to guarantee the stability of different batches, and achieve the effect of expanding the range of host cells

Inactive Publication Date: 2014-01-22
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the cumbersome and expensive preparation process of PAM cells, and the inability to guarantee the stability between different batches; the source of Marc-145 cells is derived from monkeys, not pigs, which will lead to the limitation of PRRSV in vitro research on cells that cannot respond to natural hosts infection, and the current patent of the cell is not in our country

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Example 1 Cloning of the full-length porcine CD163 cDNA

[0014] By preparing porcine PAM cells, extracting total cellular RNA, and using CD163 specific primers to amplify porcine CD163 fragments by RT-PCR, the CD163 cDNA is 3400 bp in length, and the open reading frame is 3333 bp in length. At present, the CD163cDNA sequence has been submitted to GenBank, the accession number is: JX292263.

Embodiment 2

[0015] Example 2 Construction of PiggyBac expression vector for CD163

[0016] According to the sequence of the multi-cloning restriction site of the PiggyBac expression vector, XbaI and EcoRI were selected to linearize the vector, and the CD163cDNA was ligated to the expression vector by In-fusion homologous recombination. After digestion and sequencing were correct, the vector was named PB-CMV-CD163-EF1α-GFP-Puro.

Embodiment 3

[0017] Example 3 Establishment of PK-15 cell line stably expressing CD163

[0018] The PB-CMV-CD163-EF1α-GFP-Puro vector and the piggybac transposase-expressing vector (PA) were transfected into a PK-15 cell line with a confluence of 90% at a mass ratio of 5:1. After 48 hours of transfection, select the transfected cells with puromycin selective medium with a concentration of 5-10μg / mL. Observe the expression of green fluorescence in the cells under a fluorescence microscope. The selective medium was replaced every 48h, and the concentration of puromycin increased appropriately as the proportion of positive cells increased. After about 10 days of puromycin screening, cell clones expressing green fluorescence can be seen, and cell clones expressing a higher amount of green fluorescence are selected for further subcloning. After another week of culturing, a cell line stably expressing CD163 was obtained.

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PUM

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Abstract

The invention discloses a pig kidney cell line and application thereof. The preservation number of the pig kidney cell line is CCTCC (China Center for Type Culture Collection) C201324. A PK-15 cell line for stably expressing CD163 built by the pig kidney cell line can be applied to separation and cultivation of a porcine reproductive and respiratory syndrome (PRRSV) virus, and can also be applied to research of a PRRSV cell receptor. By building of a pig cell line susceptible to the PRRSV, an alternative cell line is provided for cultivation of the PRRSV; the range of a host cell for researching the PRRSV is enlarged; the pig kidney cell line has important significance on production of a PRRSV vaccine.

Description

Technical field [0001] The invention belongs to the technical field of biology, and relates to a pig kidney cell line and its application. Specifically, it relates to the establishment of a pig kidney cell line that stably expresses a PRRSV cell receptor and the separation and culture of PRRSV derived from pig cells. Background technique [0002] Porcine reproductive and respiratory syndrome (PRRS), also known as "pig blue ear disease", is an acute infectious disease of pigs caused by PRRS virus (PRRSV) infection. PRRSV has a strong host cell tropism. The cell lines currently used to isolate and culture PRRSV are mainly porcine alveolar macrophages (PAM) and African green monkey kidney cells (Marc-145). Studies have shown that the cell tropism of PRRSV is determined by the virus receptor on the surface of the host cell. The current study found that CD163 molecule can be used as a cell receptor of PRRSV and can mediate the non-susceptible cell line of PRRSV into the susceptible c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N7/00C12R1/91
Inventor 周恩民王向鹏李爽
Owner NORTHWEST A & F UNIV
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