Preparation method of reporter duck tembusu virus and product and application thereof

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 achieve convenient quantification Detection, excellent sensitivity, and stable performance in passage

Active Publication Date: 2019-03-01
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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  • Preparation method of reporter duck tembusu virus and product and application thereof
  • Preparation method of reporter duck tembusu virus and product and application thereof
  • Preparation method of reporter duck tembusu virus and product and application thereof

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 ...

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 invention discloses a preparation method of a reporter duck tembusu virus and a product and application thereof. The method includes: modifying 1st -2646th nucleic acid segments of a duck tembusuvirus genome through a reverse genetics operation system of a CQW1 strain (GenBank: KM233707.1), and inserting NanoLuc reporter gene to space between 5'UTR of TMUV virus and structural gene to build full-length cDNA infectious clone of reporter virus. By optimizing building of the reporter virus and replacing the reporter gene, passage stability of the reporter virus is improved effectively, and the reporter virus after being saved does not lose the reporter gene within 5 generations; the number of primers and amplified gene segments used for building recombinant plasmid is reduced, reaction steps are reduced, process is simplified, convenience is brought to operation, period for obtaining the reporter virus is shortened effectively, and production cost is lowered.

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 Applications(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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