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High-throughput screening method and application of respiratory syncytial virus resisting drug

A high-throughput, drug technology, applied in the fields of molecular biology and biomedicine, can solve problems such as serious toxicity and teratogenic effects, and achieve the effects of simple operation, accurate reflection, and accurate virus status.

Inactive Publication Date: 2017-08-18
BEIJING JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] There is currently no authorized vaccine to prevent RSV, and the only drugs used to treat RSV are ribavirin and palivizumab. However, palivizumab is an expensive humanized monoclonal antibody that can only be used For preventive treatment, ribavirin is a nucleoside antimetabolite with severe toxicity and teratogenic effects

Method used

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  • High-throughput screening method and application of respiratory syncytial virus resisting drug
  • High-throughput screening method and application of respiratory syncytial virus resisting drug
  • High-throughput screening method and application of respiratory syncytial virus resisting drug

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Construction of rRSV-EGFP plasmid

[0056] 1. Insert the synthetic linker ClaI-PmeI-XhoI-AsuII-XmaI-NheI-MluI-NruI into the pBR322 vector, transform it into a pBRAT vector, and use XhoI and NotI to digest the transformed vector, and obtain it at 3466bp destination strip ( figure 1 ), the vector fragment.

[0057] 2. Connect the synthetic fragment of NheⅠLMluⅠ to the vector pMD18-T to obtain the pMD18-T-L plasmid; take the synthetic fragment of A1 (PmeⅠNS1NS2NPMSHXhoⅠ), pBR322B vector, digest with PmeⅠ and XhoⅠ, and connect to obtain the pBR322B-RSV A1 plasmid; take A2 (SacISHGAsuⅡ) Synthetic fragment, pBR322B vector, SacI and AsuII digestion, ligation, to obtain pBR322B-RSV A2 plasmid.

[0058] 3. Antigenomic rRSV-EGFP plasmid construction

[0059] Take the pMD18-T-L plasmid, pBRAT vector, digest with NheI and MluI, and connect to obtain the pBRAT-L plasmid. Take the AsuIIGEGFPFM2LNheI synthetic plasmid, pBRAT-L plasmid, digest with AsuII and NheI, and conn...

Embodiment 2

[0060] Example 2 Rescue of recombinant virus

[0061] BHK T7 / 9 cells were inoculated in 6-well plates, and after 24 hours, the rRSV-EGFP plasmid and four kinds of auxiliary protein plasmids (pCDNA3.1-N, P, L, M2-1) were transfected into host cells (BHK T7 / 9) cultured at 37°C (experimental group), and set up a positive control group and a negative control group mock at the same time, wherein the positive control group was transfected with the RSV mini-replicon pBR322B-RSV-EGFP and four kinds of auxiliary protein particles (pCDNA3. 1-N, P, L, M2-1), the negative control mock was only transfected with four auxiliary protein plasmids (pCDNA3.1-N, P, L, M2-1), and blindly passed to the host cells after 96 hours of transfection HEp-2 continued to be cultured at 37°C and transfected ( image 3 ) and blind pass ( Figure 4 ) and observe the fluorescence expression under the microscope every day. According to the fluorescence of the cells, it can be judged that the experimental grou...

Embodiment 3

[0062] Example 3 Recombinant Virus Stability Identification

[0063] Fluorescent expression of rRSV-EGFP after serial passage was observed by fluorescence microscope ( Figure 5 ) and the plaque forming units (PFU / mL) of the recombinant virus after serial passage were detected by immunoenzyme method ( Image 6 ), it can be seen from the fluorescent expression of continuous passage that the rescued recombinant virus is stable, and the results of immunoenzyme detection show that the amount of rescued recombinant virus tends to be stable after 3 generations of continuous passage (P<0.05).

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Abstract

The invention discloses a recombinant RSV (Respiratory Syncytial Virus) antigenome plasmid rRSV-EGFP. The sequence of the recombinant RSV antigenome plasmid rRSV-EGFP is shown as SEQ ID No.1. The invention also discloses a recombinant RSV obtained by rescuing the plasmid and application of the recombinant RSV in screening of an RSV resisting drug. A high-throughput screening method for screening the RSV resisting drug comprises the following steps: inoculating a host cell, and adding the recombinant RSV and to-be-screened drugs; detecting the fluorescence intensity of GFP (Green Fluorescence Protein); evaluating an RSV resisting effect of the to-be-screened drugs. According to the recombinant RSV antigenome plasmid rRSV-EGFP disclosed by the invention, interaction of the recombinant RSV for expressing the GFP and the host cell and to-be-screened compounds is utilized; as the expression level of the GFP is closely related to the replication level of a virus, the antivirus effect of the compound can be analyzed by detecting the fluorescence intensity, further a high-throughput drug screening model for the RSV is established, a large-scale drug screening is realized, and a foundation is laid for research and development of the RSV-resisting drug.

Description

technical field [0001] The present invention relates to the fields of molecular biology and biomedicine. More specifically, it relates to the establishment and application of a high-throughput screening method for anti-respiratory syncytial virus drugs based on recombinant viruses. Background technique [0002] Respiratory syncytial virus (RSV) is one of the most common viral pathogens causing severe respiratory disease and bronchiolitis in children and infants under 5 years of age worldwide. Seasonal infections caused by RSV usually last 1-2 weeks and present with mild, cold-like symptoms in most healthy adults. However, RSV infection predisposes to severe lower respiratory tract infections in vulnerable populations—infants, the elderly, and the immunocompromised. It is estimated that in 2005, approximately 338 million children under the age of 5 were infected with RSV worldwide. Of these, about 3.4 million required hospitalization for severe lower respiratory infections...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N7/01C12Q1/70
CPCC12N7/00C12N15/63C12N2760/18521C12Q1/70
Inventor 何金生付远辉彭向雷郑妍鹏姜男许敏
Owner BEIJING JIAOTONG UNIV
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