Potato X virus attenuated vaccine and preparation method and application thereof

A technology of attenuated vaccines and potatoes, applied in the fields of botanical equipment and methods, biochemical equipment and methods, applications, etc., can solve the problems of easy reversible mutation, limited protected varieties, etc., and achieve the effect of good application potential

Active Publication Date: 2019-01-18
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Aiming at the problem that the protected varieties of the attenuated potato virus X virus (potexviruses) vaccine prepared by the prior art ...

Method used

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  • Potato X virus attenuated vaccine and preparation method and application thereof
  • Potato X virus attenuated vaccine and preparation method and application thereof
  • Potato X virus attenuated vaccine and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1: Preparation of Potato virus X attenuated vaccine.

[0066] The attenuated potato virus X vaccine described in this embodiment is an infectious clone or recombinant bacteria containing the transformed potato virus X cDNA, and the GGXYXDGTK motif of the three-gene box protein 2 is encoded in the transformed potato virus X cDNA. All nucleotide sequences are knocked out, X in the GGXYXDGTK motif sequence is any amino acid, and the host bacteria of the recombinant bacteria is Agrobacterium.

[0067] The specific preparation method is as follows:

[0068] 1. Obtain the full-length cDNA of the Potato virus X genome; the full-length cDNA of the Potato virus X genome in this example comes from the infectious clone pGR107 (GenBank accession number: AY297842) containing the full-length cDNA of the Potato virus X.

[0069] 2. Construction of Potato virus X virus attenuated vaccine:

[0070] a. According to the sequence of the potato X virus infectious clone pGR107, desig...

Embodiment 2

[0085] Example 2: Detection of infection effect of attenuated potato virus X vaccine.

[0086] For plants, mild toxicity usually means that no or only mild virus symptoms are caused, and there is no or only very small impact on the yield of plants. The infection effect of the potato X virus attenuated vaccine prepared by the present invention is investigated by experiment below.

[0087] Step 1: Potato virus X attenuated vaccination

[0088] Potato virus X mutant PVX-TGBp2 was transformed by electroporation Δ52-60 Introduce and transform Agrobacterium GV3101 with pSOUP helper plasmid, use LB solid medium containing Tetra (final concentration 5mg / L), Rif (final concentration 10mg / L) and Kana (final concentration 50mg / L) three kinds of antibiotics in Cultivate at 28 degrees, then use primer 1 and primer 5: 5'-GTTGGTCTCGAAATCGAAGC-3' (potato virus X genome 4883-4902nt reverse complement) to carry out PCR reaction to screen positive clones.

[0089] The PCR system is 50 μL, inc...

Embodiment 3

[0098] Example 3: Verification of cross-protection of attenuated potato virus X vaccine.

[0099] Step 1: Inoculate wild-type virulent potato X virus.

[0100]The leaves of Nicotiana benthamiana with virus symptoms after inoculation with pGR107 were ground into tissue homogenate in a mortar with inoculation buffer (0.01M sodium phosphate, pH7.0) at the ratio of 1g plant tissue to 1mL inoculation buffer, and the tissue was The homogenate was inoculated mechanically into the leaves of Nicotiana benthamiana benthamiana benthamiana benthamiana benthamiana benthamiana benthamiana benthamiana benthamiana benthamiana benthamiana benthamiana benthamiana benthamiana plant growth box observation symptoms after inoculation. The culture conditions of Nicotiana benthamiana were as follows: the photoperiod was 18 hours of light and 6 hours of darkness; the temperature was 25°C.

[0101] Step 2: Analyze the effect of cross-protection.

[0102] The typical virus symptoms ( Figure 4 The mi...

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Abstract

The invention relates to a potato X virus attenuated vaccine and a preparation method and application thereof, and relates to the technical field of plant virus vaccine. The invention aims at the limitation of the protective varieties existing in the attenuated vaccine of potato X virus at present, Reversible mutation problem, A nucleotide sequence encoding "GGXYXDGTK" amino acid motif (where X isany amino acid) of potato virus X is completely knocked out by molecular biological technology, A modified attenuated vaccine was obtained. The recombinant virus lacking the peptide had the ability to infect plants systematically and did not cause distinguishable viral symptoms compared with healthy control plants. The invention is applicable to the development and utilization of plant attenuatedvirus vaccine.

Description

technical field [0001] The invention relates to the technical field of plant virus vaccines, and provides a potato X virus attenuated vaccine and its preparation method and application. Background technique [0002] The use of low pathogenicity (attenuated) plant viruses to control high pathogenicity strains has important application value in agriculture. At present, attenuated strains are mainly obtained through the following methods: [0003] 1) Screen and obtain attenuated strains from the natural environment; for example: REGULES Miguel ARAND A Sempere Raquel Pedro Jesús Use of two isolates of pepino mosaic virus to protect against infection by the same virus (EP3309256A1); Schenk MF, Hamelink R, van derVlugt RAA, Vermunt AMW, Kaarsenmaker RC, Stijger, ICCMM.2010, The use of attenuated isolates for pepino mosaicoss virus -protection. European journal of plant pathology, 127:249-261. [0004] 2) Attenuated strains are obtained by artificially modifying viru...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/40C12R1/01
CPCC07K14/005C12N2770/00022
Inventor 程晓非武晓云刘佳慧
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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