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Gene knockout cell line capable of replicating IBV virus QX subtype strain as well as construction method and application of gene knockout cell line

A gene knockout and construction method technology, applied in the field of biotechnology genetic engineering, can solve the problems of cell mechanism research lagging behind, achieve strong adaptability, broad market application prospects, and reduce cultivation costs

Pending Publication Date: 2022-01-28
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the lack of replicating cell lines of this subtype, the virus is still mainly propagated through chicken embryos in the world and domestically, which makes the research on the reverse genetics and cellular mechanism of the QX subtype of chicken infectious bronchitis lag behind.

Method used

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  • Gene knockout cell line capable of replicating IBV virus QX subtype strain as well as construction method and application of gene knockout cell line
  • Gene knockout cell line capable of replicating IBV virus QX subtype strain as well as construction method and application of gene knockout cell line
  • Gene knockout cell line capable of replicating IBV virus QX subtype strain as well as construction method and application of gene knockout cell line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Isolation and acquisition of chicken infectious bronchitis Qx subtype virus

[0055] Avian infectious bronchitis virus is a new strain of IBV (Qx strain). Chicken infectious bronchitis virus was obtained by the pathology teaching and research group of the College of Veterinary Medicine, Nanjing Agricultural University, from yellow feather and white feather diseased dead chickens submitted for inspection, and the mixed samples of chicken trachea, liver, spleen, intestine, lung and pancreas were homogenized to obtain supernatant, allantoic After cavity inoculation of 9-day-old SPF chicken embryos, the allantoic fluid was collected and subjected to RT-PCR, and the PCR product (S1 gene) was recovered and sent to a sequencing company for sequencing. The sequence was compared with the existing IBV sequence in the National Center for Biotechnology Information database , the isolated IBV belongs to QX type. Then the whole sequence was amplified by our laboratory in s...

Embodiment 2

[0056] Example 2 The difference in the tissue tropism of the virus with the preservation number CCTCC NO: V202172 for chickens

[0057] Virus inoculation and isolation: select 2-week-old white-feathered chickens of SPF level, and give the chickens a chicken infectious bronchitis QX subtype virus (preservation number is CCTCC NO: V202172) nasal drip, and the EID50 of the virus=10 -5.8 / 0.1ml, and a negative control group was set at the same time; and tissue samples such as trachea, spleen, kidney, and glandular stomach were sequentially collected at 1h, 4h, 12h, and 24h time points. Wash the tissue with a PBS solution containing 2% penicillin-streptomycin mixed antibiotics, then take an equal weight tissue into a grinding tube, add Trizol and grind it thoroughly in a homogenizer. Transfer the grinding solution to a centrifuge tube, add chloroform, mix thoroughly and centrifuge; absorb the supernatant and add an equal volume of isopropanol, mix overnight to precipitate RNA; cent...

Embodiment 3

[0064] Example 3 Screening of virus replication amount in different cells with deposit number CCTCC NO: V202172

[0065] Screening of cells: According to the tissue tropism of chicken infectious bronchitis QX subtype virus, several corresponding passage cell lines were screened out, namely DF-1 (chicken fibroblasts), HEK293T, Vero cells And three human gastric cancer cell lines AGS, NCI-N87 and MGC803 (all purchased from ATCC cell bank). Resuscitate and subculture all the cells until the cell doubling rate is stable, digest them with 0.05% and 0.25% trypsin respectively, and spread them in 12-well plates. When the cell density reaches 70%-80%, replace with Serum culture medium, drop 5×10 per well 4 The PFU / mL virus was placed in a cell incubator and incubated with the cells for 2 hours, and a negative control was set. Wash 3 times with serum-free medium, add virus maintenance solution containing 2% serum and 2% penicillin-streptomycin antibiotics to continue culturing. The ...

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Abstract

The invention discloses an IBV virus QX subtype strain, and discloses a gene knockout cell line capable of replicating the IBV virus QX subtype strain as well as a construction method and application of the gene knockout cell line. The method comprises the steps of: selecting a plurality of passage cell lines for infection of the IBV virus, detecting the replication load and titer of the IBV virus, and preliminarily screening cell lines capable of replicating the IBV virus; and interfering and / or knocking out related genes for inhibiting virus replication on the screened cell lines by using an siRNA interference method and / or a gene knockout method, and screening to obtain a stable passage cell line for massively replicating the virus. According to the invention, a stable passage cell line for knocking out related enzymes in the miRNA generation process is constructed, so that the replication amount of the infectious bronchitis virus QX subtype can be remarkably increased. The gene knockout cell line is expected to be developed into a tool cell line for replacing traditional chick embryo virus propagation, poultry viruses propagated by using the cell line are high in purity, few in impurities and simple and convenient to purify, and the propagation cost of the viruses can be remarkably reduced.

Description

technical field [0001] The invention belongs to the field of biotechnology genetic engineering, and in particular relates to a gene knockout cell line capable of replicating IBV virus QX subtype strain, a construction method and application thereof. Background technique [0002] Chicken infectious bronchitis is an acute, highly contagious infectious disease caused by infectious bronchitis virus (IBV). The disease is widely prevalent in the world and has brought huge economic losses to the poultry industry. Chicken infectious bronchitis virus (Infectious Bronchitis Virus, IBV) belongs to Coronaviridae, γ-coronavirus genus, is one of the main pathogens affecting the poultry industry in the world. IBV is a single-stranded positive-sense RNA virus with a genome length of about 27.6kb and at least 10 open reading frames (ORFs), encoding small membrane proteins (E), membrane proteins (M), nucleocapsid proteins (N) and fiber Spike protein (S) four main structural proteins. [00...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N15/85C12N15/113C12N5/10C12N15/50A61K39/215A61P31/14
CPCC12N7/00C12N15/85C12N5/0693C12N5/0679C12N15/113C07K14/005A61K39/12A61P31/14C12N2770/20021C12N2310/141C12N2800/107C12N2510/00Y02A50/30
Inventor 林建左金娇陈天欣陈雨涵杨倩
Owner NANJING AGRICULTURAL UNIVERSITY
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