Recombinant vector, strain, and expression and purification method of Rhizoctonia solani effector protein

A rice sheath blight bacteria and effector protein technology, applied in the biological field, can solve the problems of protein non-expression and insolubility

Pending Publication Date: 2020-03-24
SHENYANG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, proteins expressed in E. coli are often not expressed or insoluble due to factors such as rare codon restrictions and hindered disulfide bond formation.

Method used

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  • Recombinant vector, strain, and expression and purification method of Rhizoctonia solani effector protein
  • Recombinant vector, strain, and expression and purification method of Rhizoctonia solani effector protein
  • Recombinant vector, strain, and expression and purification method of Rhizoctonia solani effector protein

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Experimental program
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Embodiment 1

[0029] This embodiment provides a recombinant vector and a bacterial strain containing the recombinant vector, and the recombinant vector and bacterial strain are used to express the rice sheath blight effector protein; wherein, the recombinant vector is obtained by introducing the rice sheath blight The gene encoding the effector protein is inserted between the EcoRI and XhoI restriction sites of the pET-32a vector. The map of the pET-32a vector is attached figure 1 As shown; the rice sheath blight effector protein is named AGLIP1, and its amino acid sequence is shown in the sequence listing SEQID NO:1; the nucleotide sequence of the gene encoding the rice sheath blight effector protein is as in the sequence listing Shown in SEQ ID NO:2.

[0030] In addition, the bacterial strain comprising the above-mentioned recombinant vector is obtained through the following steps:

[0031] (1) Take 50 μL of Escherichia coli BL21 competent cell suspension from the -80°C refrigerator, tha...

Embodiment 2

[0036] This embodiment provides a method for expression and purification of rice sheath blight effector protein, which comprises the following steps:

[0037] (1) Place 1 mL of the bacterial strain containing the recombinant vector provided in Example 1 above into 5 mL of LB liquid medium, add ampicillin antibiotic, and then place it at 37°C and 200 rpm for overnight shake flask activation culture to obtain cultured liquid.

[0038] (2) Add all the above-mentioned culture solution to 100mL LB liquid medium, add ampicillin, and then place it at 37°C and 200rpm for expanded culture until OD600=0.4, then add 100μL with a concentration of 0.1mmol / L isopropyl-β-D-thiogalactopyranoside (IPTG), and placed under the conditions of 20°C and 140rpm to induce expression for 4h, to obtain the induced bacterial liquid.

[0039] (3) Put all the above-mentioned induced bacteria liquid into a 250mL centrifuge bottle, centrifuge at 10000rpm for 5min, discard the supernatant, resuspend the bact...

Embodiment 3

[0043] This embodiment provides a method for expression and purification of rice sheath blight effector protein, which comprises the following steps:

[0044] (1) Place 1 mL of the bacterial strain containing the recombinant vector provided in Example 1 above into 5 mL of LB liquid medium, add ampicillin antibiotic, and then place it at 37°C and 200 rpm for overnight shake flask activation culture to obtain cultured liquid.

[0045] (2) Add all the above-mentioned culture solution to 100mL LB liquid medium, add ampicillin, and then place it at 37°C and 200rpm for expanded culture until OD600=0.8, then add 100μL with a concentration of 0.2mmol / L isopropyl-β-D-thiogalactopyranoside (IPTG), and induced expression under the conditions of 20-25°C and 140rpm for 4h to obtain the induced bacterial liquid.

[0046] (3) Put all the above-mentioned induced bacteria liquid into a 250mL centrifuge bottle, centrifuge at 10000rpm for 5min, discard the supernatant, resuspend the bacteria wi...

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Abstract

The invention discloses a recombinant vector, a strain, and an expression and purification method of a Rhizoctonia solani effector protein, and belongs to the biotechnical field. The recombinant vector is used for expressing the Rhizoctonia solani effector protein; the recombinant vector is obtained by inserting a coding gene of the Rhizoctonia solani effector protein into a position between EcoRIand XhoI restriction enzyme cutting site of a pET-32a vector; the amino acid sequence of the Rhizoctonia solani effector protein is represented by SEQ ID NO:1 in a sequence table; and the nucleotidesequence of the coding gene of the Rhizoctonia solani effector protein is represented by SEQ ID NO:2 in the sequence table. The strain with the recombinant vector is subjected to activation culture, isopropyl-beta-D-thiogalactoside is added to induce the expression of the target protein, the target protein is dissolved by using a protein dissolving buffer solution, and elution and dialysis treatments are carried out multiple times with different buffer solutions to obtain a high-concentration Rhizoctonia solani effector protein solution.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for expressing and purifying recombinant vectors, bacterial strains and rice sheath blight effector proteins. Background technique [0002] Prokaryotic expression of protein in vitro is of great significance for studying the biological function and crystal structure of the protein. However, proteins expressed in Escherichia coli are often not expressed or insoluble due to factors such as rare codon restrictions and hindered formation of disulfide bonds. [0003] At present, rice sheath blight effector protein AGLIP1 has been confirmed and reported that it can improve plant disease resistance. However, how to express and purify rice sheath blight effector protein in the form of soluble protein in Escherichia coli is a difficult problem in this field. Contents of the invention [0004] The purpose of the present invention is to provide a recombinant vector to solve the pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N1/21C07K14/375C07K1/22C07K1/14C12R1/19
CPCC07K14/375C12N15/70
Inventor 李帅魏松红周建铭向世博海樱凡王应玲邢帆
Owner SHENYANG AGRI UNIV
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