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Screening non-infective virus recombinant gene SARS-Cov-EGFP for medicine of anti-SARS coronavirus

A recombinant gene, non-infectious technology, applied in the field of medicine, can solve the problems of strict protection requirements, restricted experiment development, restricted personnel access, etc., and achieve the effect of convenient work

Inactive Publication Date: 2005-07-27
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the high construction and maintenance costs of BSL3 laboratories, strict protection requirements, and limited personnel access, the number of such laboratories in my country is currently limited, which brings great inconvenience to large-scale drug screening and basic virology research. restricts the development of many experiments
[0003] It is well known that positive-strand RNA viruses replicate in the form of RNA-RNA and do not form DNA intermediates, so genome manipulation cannot be performed directly

Method used

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  • Screening non-infective virus recombinant gene SARS-Cov-EGFP for medicine of anti-SARS coronavirus
  • Screening non-infective virus recombinant gene SARS-Cov-EGFP for medicine of anti-SARS coronavirus
  • Screening non-infective virus recombinant gene SARS-Cov-EGFP for medicine of anti-SARS coronavirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0036]1. Construction of recombinant plasmid pUC19 / SARS-CoV-EGFP

[0037] 1. Restriction fragmentation of the whole genome of SARS-CoV TOR2:

[0038] The whole genome DNA of SARS-CoV TOR2 was first digested with BamH I, and the reaction conditions were: 6 μl whole genome (1 μg / μl), 5 μl 10× buffer [150 mmol sodium chloride, 10 mmol tris(hydroxymethyl)aminomethane·hydrochloric acid (pH7 .9), 10 mmol magnesium chloride, 1 mmol dithiothreitol], 1 μl BamH I (10 units / μl), filled with sterile water to 50 μl, and digested in a water bath at 37°C for 4 hours. The DNA fragments were separated by electrophoresis with 0.6% low-melting point agarose, and two DNA fragments of 26044bp and 3707bp were purified and recovered respectively with the gel recovery kit of QIAGEN Company.

[0039] The 26044bp fragment was then digested with Cla I. The reaction system was: 3 μl 26044bp fragment (800ng / μl), 3 μl 10× buffer [50mmol potassium acetate, 20mmol tris(hydroxymethyl)aminomethane·acetic acid...

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Abstract

The present invention relates to a noninfectious virus recombinant gene SARS-CoV-EGFP for screening medicine for resisting SARS virus and making basic research of virology. In 5' end of total genome of SARS coronate virus the promotor sequence T7 Pro. is introduced, the coding sequence of external structural domain related to viral pathogenicity and toxicity in S protein can be sustituted with EGFD gene of code enhanced green fluorescin, and in the 3' end of total genome HDV antisense genome ribozyme sequence HDV R2 and T7 transcription terminator sequence T7 Ter are successively introduced to create recombinant gene SARS-CoV-EGFP, then it is inserted into carrier to construct recombinant plasmid pUC19 / SARS-CoV-EGFP, after cell transfection, through replication, transcription, traslation, processing and packaging to obtain recombinant virus capable of making transfected cell give out green fluorescence under the fluorescent microscope.

Description

Technical field: [0001] The invention relates to the field of medical technology, and is a non-infectious virus recombinant gene SARS-CoV-EGFP that can be used for screening anti-SARS coronavirus drugs and basic virology research. Background technique: [0002] The pathogen of severe acute respiratory syndrome (Severe Acute Respiratory Syndrome, SARS) is a newly isolated coronavirus—SARS coronavirus (SARS-CoV), which is highly infectious and harmful. Many experiments involving screening of antiviral drugs and basic research of virology are required to be completed in BSL3 level laboratories. However, due to the high cost and maintenance costs of BSL3 level laboratories, strict protection requirements, and limited personnel access, the number of such laboratories in my country is currently limited, which brings great inconvenience to large-scale drug screening and basic virology research. Many experiments were limited. [0003] As we all know, positive-strand RNA viruses rep...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10C12N15/33C12N15/52C12N15/63C12Q1/68
CPCY02A50/30
Inventor 戚中田潘欣
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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