Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Pig kidney cell line and application thereof

A technology of pig kidney cells, CCTCCC201324, applied in the field of biology, can solve the problems of high cost, inability to guarantee the stability of different batches, troublesome preparation process of PAM cells, etc., and achieve the effect of expanding the range of host cells

Inactive Publication Date: 2015-06-17
NORTHWEST A & F UNIV
View PDF4 Cites 0 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Cloning of full-length porcine CD163cDNA in embodiment 1

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

Embodiment 2

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

[0016] According to the sequence of the multiple cloning enzyme cutting site of the PiggyBac expression vector, XbaI and EcoRI were used to linearize the vector, and the CD163cDNA was connected to the expression vector by the method of In-fusion homologous recombination. After the digestion and sequencing were correct, the vector was named as PB-CMV-CD163-EF1α-GFP-Puro.

Embodiment 3

[0017] Example 3 Establishment of a 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 the PK-15 cell line with a confluence of 90% at a mass ratio of 5:1. After 48 hours of transfection, the transfected cells were selected under pressure using puromycin selective medium with a concentration of 5-10 μg / mL. The expression of green fluorescence in the cells was observed under a fluorescence microscope. The selective medium was replaced every 48 hours, and the concentration of puromycin was appropriately increased 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 relatively high amount of green fluorescence can be selected for further subcloning. After another week of culturing, a cell line stably expressing CD163 was obtained.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

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 porcine kidney cell line and its application, in particular to the establishment of a porcine kidney cell line stably expressing PRRSV cell receptors and the isolation and cultivation of PRRSV derived from pig-derived 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 strong host cell tropism, and 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 cellular tropism of PRRSV is determined by the viral receptors on the surface of host cells. Current studies have found that the CD163 molecule can act as a cell receptor for PRRSV, which can mediate PRRSV non-susceptible cell lines into PRRS...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N7/00C12R1/91
Inventor 周恩民王向鹏李爽
Owner NORTHWEST A & F UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com