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The mdck-komavs cell line suitable for the propagation of canine distemper virus and avian influenza virus and its application

A technology of avian influenza virus and canine distemper virus, applied in the field of molecular biology, can solve the problems of exogenous virus pollution, gene variation, chicken embryo source and insufficient quantity, etc., and achieve the effect of high proliferation titer and rapid disease of CDV virus

Active Publication Date: 2021-06-01
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, most avian influenza virus antigens are prepared from non-immunized chicken embryos. The passage of the virus in chicken embryos containing antibodies will cause gene mutations. At the same time, there are problems of insufficient source and quantity of chicken embryos in large-scale production. Possibility of source virus contamination

Method used

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  • The mdck-komavs cell line suitable for the propagation of canine distemper virus and avian influenza virus and its application
  • The mdck-komavs cell line suitable for the propagation of canine distemper virus and avian influenza virus and its application
  • The mdck-komavs cell line suitable for the propagation of canine distemper virus and avian influenza virus and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] Embodiment 1 is suitable for the construction of the MDCK-KOmavs cell line of canine distemper virus isolation and propagation

[0100]1. Construction of replication-defective lentivirus for expressing Cas9

[0101] Spread 293FT cells into a 6-well plate, transfect cells with lentiviral packaged plasmids pMD2.G, psPAX2, and lentiCas9-Blast at a ratio of 2:3:5, and the total amount of plasmids at 3 μg, among which pMD2.G can form a viral envelope , psPAX2 can express gag and pol for virus packaging. The lentiCas9-Blast plasmid not only contains packaging signals and long terminal repeats that are packaged into pseudovirions, but also contains Cas9 protein and Blasticidin resistance selection genes; change the medium after 8 hours of transfection, The cell culture supernatants of transfection 48h and transfection 72h were collected, centrifuged at 4°C, 12,000 rpm for 10 minutes, and the supernatant was obtained as a solution containing lentiviral particles.

[0102] 2. D...

Embodiment 2

[0159] Example 2 Using the above-mentioned MDCK-KOmavs cell line to isolate CDV

[0160] Collect the eye and nose swabs and blood of the canine distemper cases tested positive by the colloidal gold test strip, centrifuge at 4°C, 3000rpm, 10min to get the supernatant, centrifuge again at 4°C, 8000rpm, 5min, and take the supernatant in the ultra-clean bench After filtering and sterilizing with a 0.22um filter, the supernatant samples were divided into sterile 1.5ml EP tubes and stored at -80°C for later use.

[0161] After the MDCK-KOmavs and MDCK are cultured until the monolayer is covered, discard the old medium, wash it with trypsin, add an appropriate amount of trypsin, put it in a 37°C incubator for about 5 minutes, discard the trypsin, and add an appropriate amount of The serum-free DMEM medium was resuspended, and the cell resuspension and the processed CDV positive sample were made in a ratio of 1000:1, and each was repeated in triplicate on a 12-well cell culture plate ...

Embodiment 3

[0163] Example 3 Using the above-mentioned MDCK-KOmavs cell line to isolate AIV

[0164] Collect eye and nose swabs and blood from avian influenza cases tested positive by colloidal gold test strips, centrifuge at 4°C, 3000rpm, 10min to get the supernatant, centrifuge again at 4°C, 8000rpm, 5min, and take the supernatant in an ultra-clean bench After filtering and sterilizing with a 0.22um filter, the supernatant samples were divided into sterile 1.5ml EP tubes and stored at -80°C for later use.

[0165] After MDCK-KOmavs and MDCK were cultured in a 12-well cell culture plate until the monolayer was confluent, the old medium was discarded, washed twice with Hank's solution to remove residual serum, and serum-free DMEM medium was added to each well 1mL, the culture medium and the treated AIV positive samples according to the ratio of 100:1, 1000:1, 10000:1, each doing three replicates, connected to a 12-well cell culture plate, placed in a cell culture incubator for 2h, poured ...

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Abstract

The invention discloses a MDCK-KOmavs cell line suitable for the propagation of canine distemper virus and avian influenza virus and its application. In the whole genome of the cell line, the mavs gene is located on chromosome No. 24, and a chromosome of the No. 24 chromosome Two bases GC are inserted in the second exon sequence of the mavs gene, and the insertion site is located at the 17520583 site of chromosome 24; the second exon of the mavs gene on another chromosome of chromosome 24 Two bases GC are inserted into the exon sequence, and the insertion site is located at position 17520583 of chromosome 24; at the same time, the base of the second exon of the mavs gene is at position 17520587 of chromosome 24 C is mutated to the base T. The MDCK-KOmavs cell line constructed by the present invention has the characteristics of rapid cell pathology after infection with canine distemper virus and avian influenza virus, high virus proliferation titer, and is more suitable for the proliferation of canine distemper virus and avian influenza virus.

Description

technical field [0001] The invention relates to molecular biology, in particular to a MDCK-KOmavs cell line suitable for the propagation of canine distemper virus and avian influenza virus and its application. Background technique [0002] Canine distemper (CD) is an acute, febrile and highly contagious infectious disease caused by canine distemper virus (CDV) of the genus Morbillivirus in the Paramyxoviridae family. The main clinical features are biphasic fever, mucous membrane inflammation of the eyes, nose, and digestive tract, and catarrhal pneumonia, dermatitis, and neurological symptoms. Since it was first reported in 1809, the disease has become a worldwide epidemic, seriously affecting companion animals and wildlife. CDV is divided into six types: Asia-I, Asia-II, Europe, America-II, Arctic and Vaccine genotype, but only one serotype. [0003] The isolation and identification of viruses is of great significance for the diagnosis of epidemic diseases and mastering ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N7/00C12R1/93
CPCC07K14/4702C12N5/0686C12N7/00C12N2510/00C12N2760/16151C12N2760/18451
Inventor 韩丽蔡聪张安定王晓萍赵亚靳泽华
Owner HUAZHONG AGRI UNIV
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