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Fusion protein as well as amino acid sequence, coding nucleotide sequence, preparation method and application thereof

A technology of nucleotide sequence and fusion protein, applied in botanical equipment and methods, applications, fusion polypeptides, etc., can solve the problems of difficult to make scientific and effective guidance, high concentration of use, unclear receptors, etc., to achieve Reduce the ability of pathogenic microorganisms to infect, use low concentration, and reduce the effect of agricultural production costs

Pending Publication Date: 2021-06-11
SICHUAN AGRI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these products have considerable limitations: first, their receptors are not yet clear, and it is difficult to make scientific and effective guidance on their use. Only experimental experience can be used to judge their scope of application and concentration; second, plant The sensitivity to these plant immune pesticides is low, the use concentration is high and the cost is high
Axiom Harpin Protein is a biological protein pesticide widely used in the world. However, the unit price of its pure protein product is higher than 5,000 RMB per gram, and the price of its 1% protein pesticide product is also more than 140 RMB per gram. The high cost is extremely high. Greatly limit its application in agricultural production

Method used

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  • Fusion protein as well as amino acid sequence, coding nucleotide sequence, preparation method and application thereof
  • Fusion protein as well as amino acid sequence, coding nucleotide sequence, preparation method and application thereof
  • Fusion protein as well as amino acid sequence, coding nucleotide sequence, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1 7

[0145] Example 1 Molecular Design and Nucleotide Sequence Acquisition of Heptapeptide Fusion Protein His-MP7

[0146] (1) Among 11 different PAMP molecular polypeptides: flg22, nlp20, elf18, pip1, pep1, csp22, flgII-28, elf18, pep13, ralf17, hrp15, sys18 and their mutants, select 7 different PAMP molecules Polypeptides: flg22, nlp20, elf18, pip1, pep1, csp22, flgII-28 form a fusion protein, and the linkers are AGA and GAG;

[0147] (2) Use the online analysis platform ExPASy (https: / / www.expasy.org) to conduct basic property analysis (protscale) such as molecular mass, amino acid composition (protparam) and hydrophobicity of the above protein sequence; use Phyre2 online platform (http : / / www.sbg.bio.ic.ac.uk / phyre2 / html / page.cgi?id=index) to model the above protein structure;

[0148] (3) Based on the analysis results, one design scheme was screened out, and the fusion protein was named MP7, with the amino acid sequence shown in SEQID NO:15;

[0149] (4) Utilize bioinformati...

Embodiment 2 7

[0150] Example 2 Expression and purification of the heptapeptide fusion protein His-MP7

[0151] (1) Cloning the nucleotide sequence shown in SEQ ID NO:16 into the HindIII and XholI sites of the pET-28b (+) expression vector (Novagen), heat-shocked transformation into Escherichia coli DH5α, and picking positive clones, Shake the bacteria and extract the plasmid, after enzyme digestion and sequencing to verify correctness, transform into Escherichia coli BL21(DE3) by heat shock, and the Escherichia coli containing the recombinant plasmid pET-28b-MP7 is named BL21(DE3) / pET-28b-MP7 ;

[0152] (2) Inducing the expression of BL21(DE3) / pET-28b-MP7, including the following steps: the expression strain was inoculated in LB liquid medium, 37 ° C, 200 rpm / min shaking culture overnight, to obtain the first bacterial liquid; overnight The bacterial solution was transferred to LB liquid medium containing 100 μg / mL kanamycin at a volume ratio of 1:100, and continued to shake at 37°C and 20...

Embodiment 3 7

[0155] Example 3 Study on the Potency of Immune Activation of Heptapeptide Fusion Protein His-MP7

[0156]The accumulation of callose will occur in the immune response of plant cells. Callose can strengthen the mechanical strength of plant cell walls and block the channels for pathogens to spread between cells, thereby limiting the invasion of pathogenic microorganisms. Using the model plant Arabidopsis thaliana as the material and callose accumulation as the immune indicator, the immune activation ability of the fusion protein His-MP7 at different concentrations was analyzed, and compared with a single PAMP molecule polypeptide. The specific experimental operation is as follows:

[0157] 1. Comparison of callose accumulation in Arabidopsis thaliana caused by different concentrations of fusion protein His-MP7

[0158] Adjust the final concentrations of the fusion protein His-MP7 and polypeptide flg22 solutions to 1 μM, 100 nM, and 10 nM, respectively, and use water as a bla...

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Abstract

The invention provides a fusion protein as well as an amino acid sequence, a coding nucleotide sequence, a preparation method and an application thereof, which relate to the field of agricultural biotechnology. The fusion protein comprises or consists of at least three, four, five, six, seven or eight same and / or different PAMP molecular polypeptides, and optionally, at least one connexon is arranged between two adjacent PAMP molecular polypeptides or no connexon is arranged between the two adjacent PAMP molecular polypeptides. A plurality of PAMP molecular polypeptides are assembled into the fusion protein with a plurality of and / or a plurality of immune epitopes, so that immune response defended by plants can be quickly induced, the infection ability of pathogenic microorganisms is reduced, and the disease resistance of the plants is obviously improved. According to the preparation method of the fusion protein, a PTI immune mechanism and a genetic engineering technology are jointly applied, the fusion protein with multiple immune epitopes which does not exist in the nature is obtained, and the problems that the production cost is high and the plant immune PAMP molecular polypeptide cannot be applied to agricultural production for a long time in preparation of the plant immune PAMP molecular polypeptide are effectively solved.

Description

technical field [0001] The invention relates to the field of agricultural biotechnology, in particular to a fusion protein, its amino acid sequence, encoding nucleotide sequence, preparation method and application. Background technique [0002] In agricultural ecosystems, compared with chemical pesticides, biopesticide products have the advantages of low toxicity, short degradation cycle, and high environmental compatibility, and have been widely used in crop production. However, the research and development of traditional biopesticides is mainly to screen antibacterial and insecticidal substances from biological sources, and then develop biopesticides to fight against pests and diseases, often ignoring the role of plant immunity in the process of resisting pests and diseases. In recent years, plant immune induction technology has become a new bright spot in the development of biopesticides. Plant immune inducers are also called "plant vaccines", which stimulate the plant's...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62A01N47/44A01P21/00
CPCC07K14/415C07K14/195C07K14/37A01N47/44C07K2319/40C07K2319/21C12N15/62C07K19/00A01H3/04C07K2319/00C12R2001/38C12R2001/19C12R2001/01C12R2001/125C12R2001/645C12R2001/77C12N15/82
Inventor 蔡易李琦郭晋雅李雍杨苹苹周鑫琼张丽梅张怀渝汪向荣
Owner SICHUAN AGRI UNIV
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