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A kind of attenuated vaccine against cucumber mosaic virus, preparation method and application thereof

A technology of cucumber mosaic virus and attenuated vaccine, which is applied in the field of plant anti-virus genetic engineering, can solve the problems of few types of attenuated strains, inconvenient application, mutation of strong virus strains, etc. Easier to restore mutations and reduce damage

Active Publication Date: 2022-04-12
SHANDONG AGRICULTURAL UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The key factors that limit the wide application of cross-protection are firstly that there are not many types of attenuated strains available at present, and the application is inconvenient; is an amino acid mutation
These attenuated strains or mutants have the risk of mutating into virulent strains when used to prevent and treat viral diseases

Method used

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  • A kind of attenuated vaccine against cucumber mosaic virus, preparation method and application thereof
  • A kind of attenuated vaccine against cucumber mosaic virus, preparation method and application thereof
  • A kind of attenuated vaccine against cucumber mosaic virus, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Construction of embodiment one multi-site TVBMV attenuated mutant

[0044] 1. Construction of an infectious clone of tobacco vein mosaic virus

[0045]Tobacco vein mosaic virus RNA was used as a template for reverse transcription with random primers. According to the existing restriction enzyme map of the whole genome of tobacco vein mosaic virus, it can be amplified in three parts, and assembled into a TVBMV full-length cDNA clone after enzyme digestion. First, the 35S promoter was fused to the upstream of the fragment from the 5' untranslated region of TVBMV to the Nru I restriction site of the HC-pro gene by Overlap-PCR, and the inventor named the fragment p35S-HC; PCR amplified HC- The fragment between the pro gene Nru I restriction site and the 6K2 Xho I restriction site, the inventor named the fragment pHC-6K2; PCR amplification of the 6K2 Xho I restriction site to the poly(A) tail This fragment, the inventor named the fragment p6K2-polyA (such as figure 1 show...

Embodiment 2

[0063] Embodiment 2 Amplification of related gene fragments of cucumber mosaic virus and construction of attenuated vaccine

[0064] 1. Amplification of gene fragments related to cucumber mosaic virus

[0065] Using CMV-QZ genome as a template, each gene fragment was amplified by RT-PCR. The examples provided by the present invention are all according to conventional experimental conditions, wherein the primer sequences used are as follows:

[0066] Table 2 Sequences of primers for amplification of CMV gene fragments

[0067]

[0068]

[0069] Among them, primers 1 and 2 were used to amplify the 1a region of CMV, primers 3 and 4 were used to amplify CMV 2a region, primers 5 and 6 were used to amplify CMV 2b region, and primers 7 and 8 were used to amplify CMV In the MP region, primers 9 and 10 were used to amplify the CP region of CMV. The nucleotide sequence of the amplified 1a gene is shown in Seq ID No.17, the nucleotide sequence of the 2a gene is shown in Seq ID N...

Embodiment 3

[0082] Embodiment three: Chimera virus inoculation plant

[0083] The chimeric virus obtained in Example 2 was transformed into Agrobacterium GV3101. After colony PCR verification, the recombinant bacteria were obtained. Then single spots were inoculated in liquid LB medium containing kanamycin (50 μg / mL), rifamycin (50 μg / mL) and tetracycline (50 μg / mL). Add 500 μL of bacterial liquid to 5 mL of LB medium containing 10 mmol / L 2-(N-morpholine)-ethanesulfonic acid (MES), 20 μmol / L acetosyringone (AS) and the above three antibiotics, at 28 °C Shake culture to logarithmic growth phase.

[0084] Collect the bacteria by centrifugation and resuspend in 10mmol / L MgCl 2 , 10mmol / L MES, 150μmol / L AS, adjust the concentration to make it OD 600 It is about 0.5, and it is allowed to stand at room temperature for 3 hours. Take a 5mL disposable syringe, remove the needle to absorb the Agrobacterium liquid, and infiltrate from the back of the leaves of common tobacco (5-6 weeks old or 4...

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Abstract

The invention relates to the field of plant anti-virus genetic engineering, and discloses an anti-cucumber mosaic virus attenuated vaccine, a preparation method and an application thereof. The attenuated vaccine against cucumber mosaic virus is based on the TVBMV attenuated mutant, and an effective gene fragment that can induce cross-protection to cucumber mosaic virus is embedded in the TVBMV attenuated mutant, and the effective gene fragment includes the cucumber mosaic virus. 2b gene. The nucleotide sequence of the 2b gene is shown in Seq ID No.19. The anti-cucumber mosaic virus attenuated vaccine of the invention has a stable effect, can play an effective cross-protective role, significantly reduces the damage of plants infected by the virulent strain of the cucumber mosaic virus, delays the onset of plants, and greatly reduces losses.

Description

technical field [0001] The invention belongs to the field of plant anti-virus genetic engineering, and in particular relates to an anti-cucumber mosaic virus attenuated vaccine, a preparation method and application thereof. Background technique [0002] Viral diseases are important diseases on crops, causing huge losses to agricultural production. Because there are many kinds of crop viral diseases, the transmission routes are complicated, there are no varieties with immunity or high resistance to viral diseases in production, and there are no effective medicaments for viral diseases on the market, so the prevention and treatment of viral diseases is very difficult. [0003] Tobacco virus disease has always been an important factor restricting tobacco production in my country. At present, the main tobacco varieties in production are not ideal for the resistance to viral diseases, and the prevention and treatment of tobacco viral diseases mainly depends on agricultural contr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/01C12N1/21A01N63/40A01P1/00C12R1/94
CPCC12N7/00C07K14/005A01N63/00C12N2770/00021C12N2770/14022C12N2770/14031
Inventor 李向东刘锦李现道耿超田延平刘茜郭兆奎万秀清乔婵李若
Owner SHANDONG AGRICULTURAL UNIVERSITY
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