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GFP (Green Fluorescent Protein) tracing system of mouse hepatitis coronavirus and application of tracing system

A coronavirus and hepatitis technology, applied in the field of mouse hepatitis coronavirus GFP tracking system, to achieve the effect of stable fluorescence quality

Active Publication Date: 2015-02-11
INST OF PLA FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] There is no report on the GFP tracer of murine hepatitis coronavirus

Method used

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  • GFP (Green Fluorescent Protein) tracing system of mouse hepatitis coronavirus and application of tracing system
  • GFP (Green Fluorescent Protein) tracing system of mouse hepatitis coronavirus and application of tracing system
  • GFP (Green Fluorescent Protein) tracing system of mouse hepatitis coronavirus and application of tracing system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] The construction of the recombinant murine hepatitis coronavirus of embodiment 1, GFP mark

[0067] 1. Construction of Homologous Recombination Plasmids

[0068] 1. Construction and identification of recombinant plasmid pGPT-IN-ORF4

[0069] No. 27500-27966 (corresponding to No. 1-467 of Sequence 1) of the genomic cDNA sequence of wild-type murine hepatitis coronavirus A59 strain (GenBank number: NC_001846.1, Up date: 2012-8-23) was named upstream homology arm) and positions 28265-28700 (corresponding to positions 766-1201 of sequence 1, named downstream homology arms) were cloned into the upstream and downstream of the gpt gene of plasmid pSV2-gpt, respectively, to obtain the recombinant plasmid pGPT-IN- ORF4. The specific operation is as follows:

[0070] (1) Extraction of vaccinia virus inf-1 genomic DNA

[0071] Since the vaccinia virus vector vaccinia virus inf-1 (referred to as v.v.-inf-1) contains the full-length cDNA of the genome of the wild-type mouse hepa...

Embodiment 2

[0184] Embodiment 2, the biological activity detection of MHV-GFP recombinant virus

[0185] 1. Determination of one-step growth curve of MHV-GFP recombinant virus

[0186]The MHV-GFP recombinant virus prepared in Example 1 was used to infect (MOI=1) 17Cl-1 cells, and after adsorption at 37°C for 1 hour, the virus liquid was removed, and the cells were washed with DMEM for 3 times to remove residual uninfected virus, and then in Cells were cultured at 33°C with DMEM containing 10% (volume percent) FBS. The virus was collected at different time points (0, 2, 4, 6, 8, 10, 12, 24 hours) within 0-24 hours after infection, and the collected virus was subjected to plaque analysis at 33°C to determine the virus titer (PFU / ml ), and then draw a one-step growth curve. At the same time, the wild-type MHV A59 strain was used as a control to detect the difference between the MHV-GFP recombinant virus and its one-step growth curve. Experiments were repeated three times.

[0187] The re...

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Abstract

The invention discloses a GFP (Green Fluorescent Protein) tracing system of mouse hepatitis coronavirus and an application of the tracing system. The GFP tracing system of the mouse hepatitis coronavirus is used for recombining the mouse hepatitis coronavirus by replacing or inserting wild mouse hepatitis coronavirus genome RNA (Ribose Nucleic Acid); in the replacing process, any segment in a segment a in the wild mouse hepatitis coronavirus genome RNA is replaced with a segment b; in the inserting process, any locus in the segment a in the wild mouse hepatitis coronavirus genome RNA is inserted into the segment b; the segment a is RNA coded by a sequence l in a sequence table; and the segment b is RNA coded by a DNA (Deoxyribonucleic Acid) segment containing a green fluorescent protein coding gene. According to the GFP tracing system of the house hepatitis coronavirus and application of the tracing system disclosed by the invention, the MHV (Mouse Hepatitis Virus)-GFP (Green Fluorescent Protein) and the wild virus can be used for research at the same time. Besides, the MHV-GFP can be used for greatly improving the virus monitoring flexibility due to a GFP signal amplifying effect, so that a new idea is provided for establishment of a low-dose virus subclinical infected animal model, and a new technical platform is provided for the application, research and the like of the coronavirus resistant drug screening.

Description

technical field [0001] The invention relates to a GFP tracer system of murine hepatitis coronavirus and its application, in particular to a recombinant murine hepatitis coronavirus obtained by inserting GFP into the genome of wild type murine hepatitis coronavirus A59 strain. Background technique [0002] Coronavirus is the single-stranded positive-sense RNA virus with the largest genome known so far. It mainly causes diseases of the respiratory tract and digestive tract. It is the pathogen of various economic animal infectious diseases and often causes huge social and economic losses. Since a new SARS-CoV was identified as the causative agent of severe infectious disease SARS in 2003, coronavirus (Coronavirus) has become one of the hotspots of global life science research. [0003] Green fluorescent protein (GFP) is a protein with unique functions derived from luminescent jellyfish (aequorea victoria), with a molecular weight of 27kD and composed of 238 amino acids. Since ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/01C12N5/10C12N1/15C12N1/19C12N1/21C12N15/63A01K67/027C12R1/93
Inventor 刘京梅常国辉孙走南柳洪涛户义吴晓燕张雨杨益苏文莉何湘
Owner INST OF PLA FOR DISEASE CONTROL & PREVENTION
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