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Method for improving antibacterial activity of antifungal protein

A protein, antifungal technology, applied in the field of plant protection and genetic engineering, can solve the problem of no antibacterial activity, and achieve the effect of increasing the effective concentration

Inactive Publication Date: 2010-12-22
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chitin-binding domain (CBD) is a polypeptide, which is mostly found in chitinase, and can help proteins bind to chitin substrates, but the chitin-binding domain itself has no antibacterial activity

Method used

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  • Method for improving antibacterial activity of antifungal protein
  • Method for improving antibacterial activity of antifungal protein
  • Method for improving antibacterial activity of antifungal protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Construction of chitin-binding domain and antifungal protein fusion gene

[0026] 1. Synthetic CBD chiA Fragment (SEQ ID NO.1, the coding nucleic acid sequence derived from the chitin-binding domain of chitinase Al, which can encode a polypeptide whose amino acid sequence is shown in SEQ ID NO.2), it should be noted that this experiment only uses CBD chiAFragments are taken as an example, and the selected chitin-binding domain fragments are not limited thereto. Design primers CBDchiA-F and CBDchiA-R, respectively introduce restriction endonuclease recognition sites at both ends, and obtain fragments with restriction endonuclease sites after PCR (depending on the carrier to be selected, this example The introduced enzyme cutting sites are BglII and KpnI), and the primer sequence is shown in SEQ ID NO.5.

[0027] 2. Extract the total RNA in the onion seeds, and obtain the cDNA fragment of the antifungal protein Ace-AMP1 (SEQ ID NO.3, a kind of antifungal prote...

Embodiment 2

[0029] The acquisition of embodiment 2 fusion antifungal protein

[0030] 1. the CBD-containing product that embodiment 1 obtains chiA Transform the genetically engineered bacteria Origami (DE3) with the fusion protein expression vector of Ace-AMP1. It should be noted that this experiment only uses CBD chiA As a representative of chitin-binding domain, Ace-AMP1 protein is used as a representative of antifungal protein, and is not intended to limit the scope of application of the present invention.

[0031] 2. After the transformed colony grows, inoculate the colony on the plate into 300ml LB liquid medium containing kanamycin, ampicillin and tetracycline, and shake at 37°C until OD 600 =0.6-0.8, add IPTG at a final concentration of 0.05 mM, and culture at 30°C for 4 hours or at 20°C overnight.

[0032] 3. Centrifuge the Escherichia coli liquid obtained in step 2 to remove the supernatant, wash the bacteria with 1×PBS, resuspend in the 1× initial buffer required for His-tag f...

Embodiment 3

[0033] Embodiment 3 Chitin-binding ability test of fusion antifungal protein

[0034] 1. Express the fusion protein according to the method of steps 1 and 2 in Example 2, it should be noted that only CBD chiA As a representative of chitin-binding domain, Ace-AMP1 protein as a representative of antifungal protein, not intended to limit the scope of application of the present invention.

[0035] 2. After centrifuging the obtained bacterial solution, wash with 1×PBS, and resuspend in 1× binding buffer required for chitin binding. After adding PMSF with a final concentration of 1 mM, the bacteria were ultrasonically disrupted, and the supernatant protein obtained after centrifugation was filtered through a filter membrane with a pore size of 0.45 μm. Add chitin beads and incubate the supernatant protein for 1 hour, wash the miscellaneous protein with a large amount of buffer solution for several times, and finally absorb a small amount of chitin beads and mix with 2×SDS loading b...

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Abstract

The invention belongs to the technical field of genetic engineering, and in particular relates to a method for improving the antibacterial activity of antifungal protein. The method comprises the following steps of: connecting a DNA (deoxyribonucleic acid) sequence of a coding chitin binding domain with a cDNA (complementary DNA) sequence of coded antifungal protein by a correct reading frame; then cloning to a protein expression vector; expressing fused protein by using engineering bacteria; carrying out protein purification by further utilizing a commercialized medium; and detecting the chitin binding capacity of the purified protein and analyzing the antibacterial activity. The invention can improve the antibacterial efficiency of the antifungal protein. By applying the invention, the activity of the traditional antifungal protein and the targeted binding capacity of the protein on fungal cell walls are improved.

Description

technical field [0001] The invention belongs to the technical fields of plant protection and genetic engineering, and specifically relates to a method for endowing antibacterial proteins with the ability to target and bind fungal cell walls. Background technique [0002] Antifungal protein is a kind of protein that can inhibit the growth of fungi. Generally, it has a small molecular weight and stable structure. After binding to the surface of fungal cells, it can inhibit the growth of fungi. The protein against phytopathogenic fungi can be used as a biopesticide because it is not easy to make the fungi resistant to drugs, is friendly to the environment, and is safe for humans and animals. The antibacterial mechanism of antifungal proteins mainly includes: forming pores on the cell wall and cell membrane, causing ion leakage, and destroying fungi; or having a function similar to detergents, which destroys the cell wall and cell membrane of fungi (Zasloff et al, 2002). Antif...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/70C07K19/00
Inventor 葛晓春吴殷何玥
Owner FUDAN UNIV
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