Canine distemper virus (CDV) sensitive cell line and establishment method and application thereof
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A technology of canine distemper virus and sensitive cells, applied in the biological field, can solve the problems of decreased CDV virulence, unobvious virus cell lesions, and biological safety hazards
Inactive Publication Date: 2012-12-05
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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When MDCK is used to isolate CDV wild strains, the cytopathy formed by the virus is not obvious and takes a long time to form, while Vero can cause CDV virulence to decrease
Although B95a has a high success rate in isolating CDV, the virus strains isolated from it can not only interact with canine SLAM (SALM, the known receptor of CDV), but also use human and marmoset SLAM. Artificially expanding the host range of CDV has brought serious hidden dangers to biological safety
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Embodiment 1
[0093] Example 1 Establishment of lentivirus-mediated expression of canine SLAM MDCK-CSL cell line
[0094] 1. Method
[0095] 1 Construction of canine SLAM recombinant lentiviral expression vector
[0096] 1.1 Lentiviral expression vector information
[0097] Select the lentiviral transfer plasmid pCDH-CMV-MCS-EF1-GFP-T2A-Puro (SBI: CD513B-1) (Transfer Vector) as the vector (the vector map is as follows figure 1 shown). The canine SLAM target gene was constructed into the vector, and then co-transfected with the packaging plasmid and envelope plasmid into 293T cells to package the lentiviral particles.
[0098] 1.2 pCDH-CMV-MCS-EF1-GFP-T2A-Puro-SLAM vector construction
[0099] 1) Digestion of pCDH-CMV-MCS-EF1-GFP-T2A-Puro vector:
[0100] pCDH-CMV-MCS-EF1-GFP-T2A-Puro was digested with Saw I, the enzyme digestion reaction system:
[0101]
[0102] Mix well and place in a 37°C water bath for 4 hours, and recover the target fragment by 1% agarose gel electrophoresis. ...
Embodiment 2
[0282] Application of embodiment 2 CDV sensitive cell line
[0283] 2.1 Collection and processing of suspected CD disease materials
[0284] Feces and blood were collected from a dog suspected of canine distemper infection with clinical manifestations of thick eye and nose secretions, body temperature above 40°C, vomiting, and diarrhea in a pet hospital. Colloidal gold detection result, PCR detection result are all positive. CAV, CPV test results were negative. The mixture was resuspended in PBS with pH 7.4 and mixed evenly, centrifuged at 6000 rpm at 4°C for 20 min, and the supernatant was sterilized by a 0.45 μM filter membrane and then aliquoted at -70°C for later use.
[0285] 2.2 Value-added of viruses in MDCK-CSL
[0286] After the MDCK-CSL cell line was cultured until the cells grew to 80% monolayer, the culture medium was discarded and washed twice with PBS at pH 7.4. Replace the maintenance solution after the supernatant at 37°C, 5% CO 2 The virus was harvested a...
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Abstract
The invention discloses a canine distemper virus (CDV) sensitive cell line and an establishment method and application thereof, and belongs to the technical field of biology. According to the hundestaupe virus sensitive cell line, lentivirus four-plasmid packaging system is adopted to obtain a strain of stable cell line madin-darby canine kidney-cell isolate (MDCK-CSL) which expresses canine source signal lymphocyte activating molecules (SLAM), wherein the conservation number of strains is CGMCC No. 5881. The comparison of the propagation conditions of a CDV standard strain on the MDCK-CSL and MDCK cells indicates that after the CDV-Snyder Hill standard strain infects the MDCK-CSL cell line for 24 hours, cytopathy of the fusion, rounding contraction, obvious death and the like of cells can be observed; and the MDCK cells infected for 6 days continuously grow slowly, and the cytopathy does not occur. The CDV sensitive cell line MDCK-CSL provides a technical platform for the separation of CDV wild strains and the complete study of biological properties of the CDV wild strains, and also establishes a foundation for the prevention and control of canine distemper.
Description
technical field [0001] The invention relates to a sensitive cell line used for virus culture, in particular to a sensitive cell line used for canine distemper virus culture. The invention also relates to the establishment method and application of the cell line, belonging to the field of biotechnology. Background technique [0002] Canine distemper (CD) is an acute, highly contagious infectious disease caused by canine distemper virus (CDV) infection in dogs. Since it was first reported in 1809, the disease has been distributed worldwide, causing great harm to the dog industry, fur animal breeding industry and wildlife protection industry. CDV belongs to the genus Morbillivirus of the family Paramyxoviridae. Although CDV is divided into six types: Asia-I, Asia-II, Europe, USA-II, Arctic and Vaccine Different genotypes, but only one serotype is recognized. CDV is not easy to survive in the environment, and the success rate of virus isolation on site is low, which limits th...
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