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

A non-infectious, recombinant gene 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: 2003-11-12
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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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

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Experimental program
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Effect test

Embodiment Construction

[0030] 1. Construction of recombinant plasmid pUC19 / SARS-CoV-EGFP

[0031]1. Restriction digestion and segmentation of the whole genome of SARS-CoV TOR2 strain: The whole genome DNA of SARS-CoV TOR2 strain is digested with BamHI first, and the reaction conditions are: 6μl whole genome (1μg / μl), 5μl 10× buffer [150mmol chloride Sodium, 10mmol tris(hydroxymethyl)aminomethane·hydrochloric acid (pH7.9), 10mmol magnesium chloride, 1mmol dithiothreitol], 1μl BamHI (10 units / μl), make up sterile water to 50μl, 37℃ water bath Digestion for 4 hours. The DNA fragments were separated by 0.6% low-melting agarose electrophoresis, and two DNA fragments of 26044bp and 3707bp were purified and recovered using QIAGEN's gel recovery kit.

[0032] The 26044bp fragment was then digested with ClaI, the reaction system was: 3μl 26044bp fragment (800ng / μl), 3μl 10× buffer [50mmol potassium acetate, 20mmol tris(hydroxymethyl)aminomethane·acetic acid (pH7.9), 10mmol magnesium acetate , 1mmol dithiothreito...

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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 technical field of medicine, and is a non-infectious viral recombinant gene SARS-CoV-EGFP that can be used for screening anti-SARS coronavirus drugs and basic virological research. Background technique: [0002] The pathogen of severe acute respiratory syndrome (Severe Acute Respiratory Syndrome, SARS) is a newly isolated coronavirus-SARS coronavirus (SARS coronavirus, SARS-CoV), which is extremely contagious and harmful. Many experiments involving the screening of antiviral drugs and basic virological research are required to be completed in a BSL3 laboratory. However, due to the high cost and maintenance cost of BSL3 laboratories, strict protection requirements, and restricted personnel access, my country currently has a limited number of such laboratories, which brings great inconvenience to large-scale drug screening and basic virological research. Limits the development of many experiments. [0003] As we all know, the rep...

Claims

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Application Information

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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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