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Replicon for transient transfection of duck Tembusu virus carrying Renilla luciferase and its construction method and application

A technology of renilla luciferase and duck tambusu virus, which is applied in the field of transient transfection replicon construction, can solve the problem that the tambusu virus replicon has not been successfully constructed, and achieves the effects of excellent sensitivity and convenient use.

Active Publication Date: 2021-03-16
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Replicons have been used to great effect on other flaviviruses, such as yellow fever virus (YFV), dengue virus (DEGV), Japanese encephalitis virus (JEV), Kun virus (KUN), West Nile virus (WNV), there is no relevant report on the construction of a successful Tambusu virus replicon at home and abroad at present.

Method used

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  • Replicon for transient transfection of duck Tembusu virus carrying Renilla luciferase and its construction method and application
  • Replicon for transient transfection of duck Tembusu virus carrying Renilla luciferase and its construction method and application
  • Replicon for transient transfection of duck Tembusu virus carrying Renilla luciferase and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1, the construction of the Tembusu virus replicon vector carrying Renilla luciferase

[0033] 1. Construction of pACYC177B plasmid

[0034] Using pEGFP-C2 plasmid as template, SEQ ID No.3 (CMV-F: 5'-gccagtatacactccgctagcgtgatgcggttttggcagtac-3') and SEQ ID No.4 (CMV-R: 5'-ttccggctcgagcggactagtagctctgcttatatagacctccc-3') as primers, PCR amplification CMV was obtained by using SEQ ID No.5 (SV40-F: 5'-atatcaaggcaaaaagcggccgcaacttgtttattgcagcttataat-3') and SEQ ID No.6 (SV40-R: 5'-cgggcttcccatacaatcgattaagatacattgatgagtttggac-3') as primers, PCR amplification to obtain SV40, Amplified products were recovered and purified.

[0035] The pACYC 177A plasmid ( figure 1 ) for linearization by double enzyme digestion, the pACYC 177A plasmid sequence is shown in SEQ ID No.7. Using the ClonExpress II One Step Cloning Kit, the linearized pACYC 177A was ligated with the amplified CMV and transformed into SURE Escherichia coli. After the colonies grew out, the bacteria we...

Embodiment 2

[0052] Embodiment 2, RLuc reporter gene activity detection

[0053] A method for detecting RLuc reporter gene activity, comprising the steps of:

[0054] 1) Inoculate BHK21 cells in good growth state into 48-well cell culture plates;

[0055] 2) Grow for 12-24 hours, and when the cells grow to a confluence of 70-90%, transfect the pACYC-Rep-TMUV-Rluc plasmid according to the instructions of TransIntro ELTransfection Reagent.

[0056] 3) 4h, 8h, 12h, 24h, 36h, 48h, 72h, 96h, and 120h after transfection, measure the activity of the reporter gene Rluc according to the instructions of the TransDetect Single-Lucifersase (Renilla) Reporter Assay Kit. The specific operations are as follows:

[0057] a) Discard the cell culture medium, carefully rinse the cells twice with PBS, add 60 μl Cell Lysis solution, lyse at room temperature for 10 minutes, scrape the cells into an EP tube, centrifuge at 12000g at 4°C for 10 minutes, and take the supernatant for later use;

[0058] b) Add the...

Embodiment 3

[0059] Embodiment 3, RLuc activity detection

[0060] Using mMESSAGE mMACHINE TM T7Transcription Kit performs in vitro transcription, strictly following the supplier’s recommended procedures, using RNase-free pipette tips and EP tubes:

[0061] (1) In order to prevent the transcription of an excessively long chain, NotI single enzyme digestion fully linearized 10 μg of pACYC-Rep-TMUV-Rluc plasmid to terminate transcription;

[0062] (2) Preparation of in vitro transcription system:

[0063]

[0064] Then incubate at 37°C for 3h;

[0065] (3) After the transcription is completed, add 1 μl TuRBO DNase, mix well, and incubate at 37°C for 15 minutes;

[0066] (4) Lithium chloride precipitation to recover RNA:

[0067] a) Add 30μl Nuclease-free ddH 2 O and 30 μl LiCl precipitation solution, mix gently, and place at -20°C for at least 30 minutes;

[0068] b) Centrifuge at 4°C for 15 minutes, ≥12000 rpm / min;

[0069] c) Aspirate and remove the supernatant, add 1ml of 70% e...

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Abstract

The invention relates to a duck tembusu virus transient transfection replicon carrying ranilla luciferase as well as a construction method and application of the duck tembusu virus transient transfection replicon. The replicon is characterized in that a ranilla luciferase reporter gene is linked after a tembusu virus deletes an encoding structure gene and the sequences of 38 amino acids in front of N-terminal of C protein are encoded; a sequence FMDV2A for encoding self-splicing peptide of a foot and mouth disease virus is inserted behind Rluc; an eukaryotic promoter CMV and a prokaryote promoter T7 are added to the upstream of the 5' UTR sequence of the tembusu virus; hepatitis delta virus ribozyme sequence HDVr and SV40poly(A) sequence are added to the downstream of 3' UTR; the obtainedreplicon can accurately and quantitatively reflect replication conditions of the replicon in cells and can be conveniently applied to research of replication mechanisms of the tembusu virus or even other flaviviruses, antiviral drug screening, virus and protein interaction and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a transient transfection replicon of duck Tembusu virus carrying renilla luciferase, and also relates to a construction method and application of the transient transfection replicon. Background technique [0002] my country is a big poultry raising country in the world, and it is also the largest duck raising country in the world. Duck Tembusu virus (TMUV) is a new type of flavivirus that can cause a sharp drop in feed intake and laying rate of laying ducks, and even stop production until death. Since the first outbreak of duck Tembusu virus disease in 2010, it has caused huge economic losses to the duck industry in my country. Almost all breeds of ducks can be infected with Duck Tembusu virus, including Cherry Valley duck, Peking duck, Shelduck duck, etc. The main clinical symptoms are: increased body temperature, significantly decreased appetite, weakness of limbs, paralysis; Grass...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/66
CPCC12N15/66C12N15/85
Inventor 陈舜王涛程安春汪铭书
Owner SICHUAN AGRI UNIV
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