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Duplex attenuated vaccine capable of resisting cucumber mosaic virus and potato virus X and application of duplex attenuated vaccine

A cucumber mosaic virus and attenuated vaccine technology, applied in the field of plant virology and molecular biology, to achieve high controllability

Inactive Publication Date: 2021-02-26
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

And take cucumber mosaic virus as the base carrier, the dual attenuated vaccine that can hold concurrently anti-cucumber mosaic virus and potato X virus has not yet been reported.

Method used

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  • Duplex attenuated vaccine capable of resisting cucumber mosaic virus and potato virus X and application of duplex attenuated vaccine
  • Duplex attenuated vaccine capable of resisting cucumber mosaic virus and potato virus X and application of duplex attenuated vaccine
  • Duplex attenuated vaccine capable of resisting cucumber mosaic virus and potato virus X and application of duplex attenuated vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1. Intermediate vector pCC F Construction of R2-2bPTII:

[0045] in CMV Fny Infectious Cloning Plasmid pCB301-CMV Fny -R2 is the template ((CMV Fny The construction of the invasive cloning plasmid pCB301-Fny2 refers to "Agrobacterium-mediated CMV invasive cloning and construction of 2b deletion mutants", Yao Min et al., Chinese Agricultural Sciences, 2011, 44(14): 3060-3068) , under the action of PrimeSTAR HS DNA Polymerase (Takara), carry out reverse PCR amplification, the primer pair is Fny2-2bPT 3'-BSS-2662-F and Fny2-2bPT 3'-BT-2661-R; PCR conditions: 98 Denaturation at ℃ for 10s, annealing at 55℃ for 5s, extension at 72℃ for 8min, 7 cycles; denaturation at 98℃ for 10s, extension at 68℃ for 8min, 25 cycles; storage at 4℃; the amplified PCR product is the linearized intermediate carrier pCC F R2-2bPTII.

[0046] Using DpnI(NEB) to Degrade Plasmid Template pCB301-CMV in PCR Reaction Fny -R2, the reaction conditions are: 37°C, 1h; 80°C, 20min; the product...

Embodiment 2

[0049] Cloning of embodiment 2.PVX partial fragments:

[0050] Using TransZol Up (TransGen) to extract the total RNA of Nicotiana benthamiana plants infected by PVX (EU571480), under the action of Reverse Transcriptase M-MLV (Takara), the reverse transcription reaction was performed, and the reverse primer was PVX-SmaI-2166- R, reaction conditions: denature at 80°C for 3 minutes without adding enzymes; react at 42°C for 1.5 hours after adding reverse transcriptase;

[0051] Then use the reverse transcription product cDNA as a template, under the action of 2×Es Taq MasterMix (Dye) (CWBIO), carry out PCR reaction, the primer pair is PVX-BamHI-1967-F and PVX-SmaI-2166-R; PCR conditions : Pre-denaturation at 94°C for 2min; denaturation at 94°C for 30s, annealing at 53°C for 30s, extension at 72°C for 10s, 30 cycles; extension at 72°C for 2min; storage at 4°C; the PCR product is a 200bp conserved sequence fragment in PVX. The PCR product was recovered by a DNA recovery kit for use...

Embodiment 3

[0053] Example 3. pCC F R2-2bPTII-PX 1967-2166 Construct:

[0054] pCC F R2-2bPTII was double-digested with BamH I and SmaI, and the digested product was recovered with a DNA recovery kit; the 200bp conserved sequence fragment in PVXRdRp was double-digested with BamH I and SmaI, and the digested product was recovered with a DNA recovery kit. The intermediate vector pCC F R2-2bPTⅡ and PVX fragments in T 4 Under the action of DNA ligase, ligate overnight at 16°C; transform the competent cells of Escherichia coli DH5α with the ligated products, coat LB plates with 100 μg / mL kanamycin, and confirm positive clones by colony PCR screening, enzyme digestion identification and DNA sequencing pCC F R2-2bPTII-PX 1967-2166 .

[0055] Result analysis: After the transformation of the ligated product, the product was first screened by colony PCR, and the plasmid was extracted for enzyme digestion identification. BamH I and SmaI double enzyme digestion was used for identification. Th...

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Abstract

The invention discloses a duplex attenuated vaccine capable of resisting cucumber mosaic virus and potato virus X and application of the duplex attenuated vaccine. The duplex attenuated vaccine is a cucumber mosaic virus RNA2 attenuated mutant plasmid vector pCCFR2-2bPTII-PX1967-2166 inserted with a PVX gene segment; the nucleotide sequence of the PVX gene segment is shown as SEQ ID NO. 1, and thenucleotide sequence of the duplex attenuated vaccine is shown as SEQ ID NO. 2. The duplex attenuated vaccine has a cross protection effect on CMV and PVX; and a vaccine material and an effective prevention and treatment measure are provided for preventing and treating plant virus diseases caused by CMV and PVX in the field.

Description

technical field [0001] The invention relates to the technical fields of plant virology and molecular biology, in particular to a dual attenuated vaccine against cucumber mosaic virus and potato X virus and its application. Background technique [0002] Viral diseases are the second largest type of plant diseases after fungal diseases, causing varying degrees of damage to most crops. The prevention and control of plant viral diseases is a research hotspot and difficult issue. Cucumber mosaic virus (CMV) is one of the plant viruses with the widest host range known at present, which can infect more than 1200 kinds of plants in more than 100 families, causing a variety of plants with important economic value (crops, horticulture, etc.) Crops, etc.) appear symptoms such as yellowing, dwarfing, and deformity, which seriously affect their yield and quality (Mochizuki et al., 2012). Potato virus X (PVX) has a wide range of hosts and can infect 240 species of plants in 16 families. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/40C12N15/82
CPCC07K14/005C12N15/8283C12N2770/14022C12N2770/00022
Inventor 原雪峰刘珊珊葛玉倩于成明耿国伟
Owner SHANDONG AGRICULTURAL UNIVERSITY
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