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Mutant plasmid vector containing cucumber mosaic virus Fny isolate RNA2 and application thereof

A technology of cucumber mosaic virus and plasmid vector, which is applied in the fields of plant virology and molecular biology, and can solve problems such as length limitation

Active Publication Date: 2021-09-14
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because the virus has packaging restrictions on its own size, when constructing a multivalent attenuated vaccine, the inserted foreign virus fragment has a length limit

Method used

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  • Mutant plasmid vector containing cucumber mosaic virus Fny isolate RNA2 and application thereof
  • Mutant plasmid vector containing cucumber mosaic virus Fny isolate RNA2 and application thereof
  • Mutant plasmid vector containing cucumber mosaic virus Fny isolate RNA2 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1. Vector pCC F Construction of R2-2bPTIII:

[0039] in CMV Fny Infectious Cloning Plasmid pCB301-CMV Fny -R2 is template (CMV FnyThe 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, primer pair Fny2-del2b-BSS-2752-F and Fny2-del2b-2661-R; PCR conditions: denaturation at 98°C for 10s, extension at 68°C 7min 30s, 30 cycles; store at 4°C; the amplified PCR product is the linearized basic vector pCC F R2-2bPTIII.

[0040] 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 is recovered by ethanol precipitation: add the product to ddH 2 From 0 to 450 μl, add 50 μl of pH5.2, 3.0 mM sodium acetate, 1 m...

Embodiment 2

[0044] Example 2. Plasmid vector pCC F Determination of biological effects and cross-protective effects of R2-2bPTⅢ:

[0045] Agrobacterium infiltrates Nicotiana benthamiana: first will contain wild-type CMV Fny RNA1, pCC F R2-2bPTⅢ, wild-type CMV Fny The RNA3 plasmid was transformed into competent cells of Agrobacterium GV3101, spread evenly on LB solid medium (50 μg / ml kanamycin, 100 μg / ml rifampicin), at 28°C, after 48 hours of culture, pick a single spot for colony PCR After verification, a single spot was picked and cultured in 2ml LB medium (50μg / ml kanamycin, 100μg / ml rifampicin) at 28°C with shaking at 200rpm for 24h.

[0046] Take 100 μl of bacterial liquid in 5ml LB culture medium (50 μg / ml kanamycin, 100 μg / ml rifampicin), 28 ° C, 200 rpm shaking culture to OD 600 1.0-2.0.

[0047] Centrifuge the bacterial solution in a 10ml centrifuge tube at 6000rpm for 10min at room temperature; collect the bacterial cells and resuspend them in 1ml of Agrobacterium resuspen...

Embodiment 3

[0053] Example 3. Plasmid vector pCC F Determination of the limit of R2-2bPTⅢ to accommodate foreign fragments:

[0054] For the construction of plasmid vectors containing PDS sequences of different lengths, please refer to the plasmid construction method in the previous research (patent number: 202011259238.8), extract the total RNA of Nicotiana benthamiana plants at the 6-leaf stage, and use the reverse primer PDS-BamHI-1132-R for reverse Recording reaction; then use reverse transcription product cDNA as template, forward primer PDS-SmaI-333-F, reverse primer PDS-BamHI-531-R, PDS-BamHI-632-R, PDS-BamHI-682- R, PDS-BamHI-732-R, PDS-BamHI-932-R, and PDS-BamHI-1132-R perform PCR reactions respectively, and amplify partial PDS sequences with lengths of 200bp, 300bp, 350bp, 400bp, 600bp, and 800bp; vector pCC F The R2-2bPTⅢ and PDS fragments were digested by BamHI and SmaI and recovered by DNA recovery kit; the vector pCC after the same digestion F R2-2bPTⅢ and PDS fragments o...

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Abstract

The invention discloses a mutant vector pCCFR2-2bPTIII containing cucumber mosaic virus Fny isolate RNA2. The CMVFny mutant RNA2 nucleotide sequence of the vector is shown as SEQ ID NO. 1. The mutant is mixed and inoculated with CMVFnyRNA1 and CMVFnyRNA3, and has a cross protection effect on CMV; and the mutant vector pCCFR2-2bPTIII at least can accommodate 350bp exogenous fragments and maintain the genetic stability, provides a more optimized basic vector for researching and developing multivalent attenuated vaccines, and provides vaccine materials and effective prevention and treatment measures for preventing and treating CMV and other viruses in the field.

Description

technical field [0001] The invention relates to the technical fields of plant virology and molecular biology, in particular to a mutant plasmid vector pCC containing cucumber mosaic virus Fny isolate RNA2 F Construction and application of R2-2bPTⅢ. Background technique [0002] Plant virus pathogens are called "cancer" of plants and are one of the important diseases of crops. They cause different degrees of harm to crops all over the world every year. In my country alone, the annual loss due to viruses is as high as 30 billion yuan (Xie Kunlun et al., 2020), the prevention and treatment of plant virus diseases has always been a difficult problem in agricultural production. Cucumber mosaicvirus (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, horticultural crops, etc.) etc.) have symptoms such as yellowing,...

Claims

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Application Information

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IPC IPC(8): C12N15/84C12N15/83C12N15/40
CPCC12N15/8205C12N15/8203C12N15/8283C07K14/005C12N2770/14022C12N2770/14034C12N2770/14043Y02A50/30
Inventor 原雪峰刘珊珊于成明耿国伟
Owner SHANDONG AGRICULTURAL UNIVERSITY
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