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A kind of preparation method of duck Tembusu reporter virus and its product and application

A duck Tembusu virus and reporter virus technology, applied in biochemical equipment and methods, viruses, virus/bacteriophage, etc., can solve the problems of reporter virus instability, long report virus cycle, unfavorable research development, etc., to improve the passage of time The effect of stability, convenient operation and simplified process

Active Publication Date: 2021-11-26
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it has been found in practice that the DTMUV reporter virus obtained by this patent method is extremely unstable, and the reporter gene has been detected to be obviously lost in the second or third generation; within the fifth generation, the reporter gene is almost completely lost and returns to the wild type virus, which is very unfavorable for the development of follow-up research
Moreover, the patented method is relatively complicated, involving the design and use of multiple pairs of primers, the amplification of multiple gene fragments, and the cloning of multiple recombinant plasmids, and finally the plasmid vector of the reporter virus can be obtained. The operation is complicated and the cycle of obtaining the reporter virus is relatively long.

Method used

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  • A kind of preparation method of duck Tembusu reporter virus and its product and application
  • A kind of preparation method of duck Tembusu reporter virus and its product and application
  • A kind of preparation method of duck Tembusu reporter virus and its product and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] 1. Transformation of the 1-2646 nucleic acid fragment of the duck Tembusu virus genome

[0037] Using pACYC FL-TMUV plasmid as template, using SpeI-F and C38-NLuc-R as primers, fragment A was amplified by PCR; using pJET-NanoLuc plasmid as template, using NLuc-F and NLuc-R as primers, PCR amplified Fragment B was obtained by amplification; fragment C was obtained by PCR amplification with pACYCTMUV-RLuc plasmid as template and FMDV 2A-F and XhoI-R as primers.

[0038] Using SpeI-F and NLuc-R as primers, and fragment A and fragment B as templates, fusion PCR amplification was performed to obtain fragment AB; then SpeI-F and XhoI-R were used as primers, fragment AB and fragment C were used as templates, A second round of fusion PCR amplification was performed to obtain fragment ABC.

[0039] The reaction system of PCR is:

[0040]

[0041]

[0042] The conditions of PCR amplification were: pre-denaturation at 98°C for 1 min; denaturation at 98°C for 10 s, annealin...

Embodiment 2

[0048] 1. In vitro transcription and rescue of reporter virus

[0049] (1) Pick a single colony of the reporter virus plasmid with correct sequencing, shake at room temperature (25°C) at 120rpm / min until the turbidity is appropriate. Use the endotoxin-free plasmid extraction kit to extract the plasmid for later use. Using mMESSAGE mMACHINE TM The T7Transcription Kit was used for in vitro transcription, operated strictly in accordance with the supplier's recommended procedures, and used RNase-free pipette tips and EP tubes.

[0050] (2) In order to prevent the transcription of an overly long chain, 10 μg of the pACYC TMUV-NLuc plasmid was fully linearized with SmaI single enzyme digestion to terminate the transcription;

[0051] (3) Preparation of in vitro transcription system:

[0052] RNase-free ddH2O Up to 20μl 2xNTP / CAP 10μl 10xReaction buffer 2μl GTP 1μl Linearized pACYC TMUV-NLuc 5μl Enzyme Mix 2μl

[0053] (4) Then ...

Embodiment 3

[0065] Assessing passage stability of reporter virus TMUV-NLuc

[0066] The F0-generation reporter virus obtained in Example 2 was continuously passaged in BHK21 cells for 5 times, and the five-generation passaged viruses of F1, F2, F3, F4, and F5 were successively obtained, and each generation of viral RNA was extracted for RT-PCR, and the PCR results were subjected to electrophoresis , to detect whether the reporter gene still exists with the viral genome and whether it is lost. For electrophoresis results, see Figure 6 . The results showed that the reporter gene was not lost at least within 5 generations, indicating that the stability of TMUV-NLuc was significantly better than that of the previously reported duck Tembusu reporter virus.

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Abstract

The present invention discloses a method for preparing a duck Tembusu reporter virus and its products and applications. Through the reverse genetic operating system of the CQW1 strain (GenBank: KM233707.1), the first sequence of the duck Tembusu virus genome The ‑2646 nucleic acid fragment was modified, and the NanoLuc reporter gene was inserted between the 5'UTR and the structural gene of the TMUV virus to construct a full-length cDNA infectious clone of the reporter virus. The present invention optimizes the construction of the reporter virus and replaces the reporter gene, thereby effectively improving the passage stability of the reporter virus, and the rescued reporter virus will not lose the reporter gene within 5 generations; at the same time, it reduces the primers used in the construction of recombinant plasmids and the number of amplified gene fragments, reducing reaction steps, simplifying procedures, facilitating operation, effectively shortening the cycle for obtaining reporter viruses, and reducing production costs.

Description

technical field [0001] The invention relates to the field of molecular biology technology, in particular to a method for preparing a duck Tembusu reporter virus and its product and application. Background technique [0002] Since the first outbreak of Duck Tembusu virus (DTMUV) 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-green loose stools and foul-smelling odors; ducklings are characterized by paralysis and head and neck tremors after infection. After the breeding ducks are infected, the characteristic lesion is hemorrhage of the follicular membrane, deformation or even rupture of the follicles, so the disease was once called "hemorrhagic oophoritis". Death, the mortality ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/86C12N7/01C12Q1/70
CPCC12N7/00C12N15/86C12N2770/24043C12Q1/70
Inventor 陈舜贺煜程安春汪铭书
Owner SICHUAN AGRI UNIV
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