MDCK-KOmavs cell line suitable for proliferating canine distemper virus and avian influenza virus, and application thereof

A technology of avian influenza virus and canine distemper virus, which is applied in the field of molecular biology, can solve the problems of insufficient source and quantity of chicken embryos, foreign virus contamination, gene variation, etc.

Active Publication Date: 2019-02-01
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 g

Method used

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  • MDCK-KOmavs cell line suitable for proliferating canine distemper virus and avian influenza virus, and application thereof
  • MDCK-KOmavs cell line suitable for proliferating canine distemper virus and avian influenza virus, and application thereof
  • MDCK-KOmavs cell line suitable for proliferating canine distemper virus and avian influenza virus, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] Example 1 Construction of MDCK-KOmavs cell line suitable for canine distemper virus isolation and propagation

[0100] 1. Construction of replication-deficient lentivirus used to express Cas9

[0101] The 293FT cells were plated into a 6-well plate, and the lentivirus-packaged plasmids pMD2.G, psPAX2, lentiCas9-Blast were transfected into the cells according to 2:3:5, and the total amount of plasmids was 3μg. Among them, 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 pseudoviral particles, but also contains Cas9 protein and blasticidin resistance screening genes; the solution is changed 8 hours after transfection, Collect the cell culture supernatant after 48h of transfection and 72h of transfection. After centrifugation for 10 minutes at 4°C, 12,000 rpm, the supernatant is the solution containing lentiviral particles.

[010...

Embodiment 2

[0159] Example 2 Isolation of CDV using the above MDCK-KOmavs cell line

[0160] Collect the eye and nose swabs and blood of the canine distemper cases that are positive by the colloidal gold test strip, centrifuge at 4℃, 3000rpm, 10min to take the supernatant, 4℃, 8000rpm, 5min, centrifuge again, and take the supernatant in the ultraclean table After filtering and sterilizing through a 0.22um filter, the supernatant samples were aliquoted into sterilized 1.5ml EP tubes and placed at -80°C for later use.

[0161] After the MDCK-KOmavs and MDCK are cultured to cover the monolayer, discard the old medium, wash it with pancreatin, add appropriate amount of pancreatin and put it in a 37℃ incubator for digestion for about 5 minutes, discard the pancreatin, and add an appropriate amount Resuspend in the serum-free DMEM medium, and connect the prepared cell resuspension and the processed CDV positive sample at a ratio of 1000:1, each of which is connected to a 12-well cell culture plate a...

Embodiment 3

[0163] Example 3 Isolation of AIV using the above MDCK-KOmavs cell line

[0164] Collect the eye and nose swabs and blood of avian influenza cases that are positive by the colloidal gold test strip, centrifuge at 4℃, 3000rpm, 10min to take the supernatant, 4℃, 8000rpm, 5min again, take the supernatant in the ultra-clean table After filtering and sterilizing with a 0.22um filter, the supernatant sample is aliquoted into a sterilized 1.5ml EP tube and placed at -80°C for use.

[0165] After culturing MDCK-KOmavs and MDCK in a 12-well cell culture plate to cover the monolayer, discard the old medium, wash twice with Hank's solution to remove residual serum, and add serum-free DMEM medium to each well 1mL, with medium and processed AIV positive samples in the ratio of 100:1, 1000:1, and 10000:1, each is connected to a 12-well cell culture plate and placed in a cell incubator for 2 hours. The infection solution was removed, washed twice with Hank's solution, and DMEM maintenance medium...

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Abstract

The invention discloses a MDCK-KOmavs cell line suitable for proliferating a canine distemper virus and an avian influenza virus. An mavs gene in the whole genome of the cell line is located on chromosome 24, two bases GC are inserted to a second exon sequence of the mavs gene on the chromosome 24, and the insertion site is located at the 17520583 site position of one of the chromosomes of the chromosome 24; two bases GC are inserted to the second exon sequence of the mavs gene on the remaining chromosome of the chromosome 24, and the insertion site is located at the 17520583 site position ofthe chromosome 24; and the base C of the second exon of the mavs gene at the 17520587 site of the chromosome is mutated to a base T. The MDCK-KOmavs cell line constructed in the invention has the characteristics of rapid cytopathy after canine distemper virus and avian influenza virus infection, high virus proliferation titer, and suitableness for the proliferation of the canine distemper virus and the avian influenza virus.

Description

Technical field [0001] The invention relates to molecular biology, in particular to a MDCK-KOmavs cell line suitable for the proliferation 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 measles virus genus of the Paramyxoviridae family. The main clinical features are biphasic fever, inflammation of the mucous membranes of the eyes, nose, and digestive tract, as well as catarrhal pneumonia, dermatitis, and neurological symptoms. Since it was first reported in 1809, the disease has shown a global epidemic trend, causing serious effects on companion animals and wild animals. CDV is divided into 6 types: Asia Ⅰ (Asia-Ⅰ), Asia Ⅱ (Asia-Ⅱ), Europe (Europe), America Ⅱ (America-Ⅱ), Arctic (Arctic) and Vaccine (Vaccine) Genotype, but only one serotype. [0003] The isolation and identification of the virus...

Claims

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Application Information

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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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