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Ribosome toxin and encoding gene, and application thereof

A gene and encoding technology, applied in the field of ribosomal toxin (HtA) recombinant gene and its preparation of recombinant protein, can solve the problems of inactive inclusion body, purification of less than 1 mg, difficulty in biological activity and insecticidal properties of the toxin, and achieve Significant insecticidal activity, mass production, rapid and efficient purification

Active Publication Date: 2018-07-10
HUAIHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although HtA has important potential value in controlling agricultural pests, the content of natural ribosomal toxins is low and difficult to extract, and often only a very small amount of pure product can be obtained. Currently, less than 1 mg can be purified from 1 liter of natural fermentation broth protein, and the use of exogenous gene expression system can also obtain milligram-level recombinant protein from 1 liter of culture, which brings great difficulties for us to further study the biological activity and insecticidal properties of the toxin
Therefore, establishing an efficient exogenous gene expression system and its protein preparation method will help to solve the current bottleneck of not being able to obtain a large amount of protein
[0003] However, the current Escherichia coli and yeast expression systems express this protein and there are obvious deficiencies: the prior art constructs an expression vector with ribosomal toxin, and then transforms it into E. coli BL21(DE3) was expressed, but all of the obtained inclusion bodies were inactive. Through dissolution, denaturation, renaturation and purification under appropriate conditions in vitro, only a trace amount of soluble protein could be obtained from each liter of medium. Very low; the method of expressing the constructed expression vector in yeast, but its expression level is low and separation and purification are very difficult

Method used

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  • Ribosome toxin and encoding gene, and application thereof
  • Ribosome toxin and encoding gene, and application thereof
  • Ribosome toxin and encoding gene, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] This embodiment provides an optimized artificially synthesized HtA gene, the specific sequence of which is shown in sequence 1 in the sequence listing, and the protein sequence corresponding to the gene is shown in sequence 2 in the sequence listing. The sequence before optimization in this example is based on the DNA sequence provided by the NCBI database, which is the natural DNA for synthesizing HtA, and then optimizes and synthesizes the optimized DNA according to the expression characteristics of the toxin gene and Pichia pastoris codons. The optimized DNA sequence was compared with the natural DNA sequence of HtA (GenBank accession number M27706) by NCBI, and there was no obvious similarity.

[0062] The natural DNA of HtA before optimization and the optimized DNA were connected to the Pichia pastoris secretory expression vector pPICZαA to obtain the recombinant vector, and then the recombinant vector was transformed into the Pichia pichia using the lithium chlorid...

Embodiment 2

[0064] This embodiment provides a method for preparing protein, which specifically includes the following steps:

[0065] S1: Construction of expression vector and transformation: Link the optimized artificially synthesized DNA in Example 1 to the secretory expression vector pPICZαA of Pichia pastoris to obtain the recombinant vector pPICZαA-HtA. The vector construction is as follows figure 1 as shown, figure 1 A schematic diagram of the construction of the eukaryotic expression vector pPICZαA-HtA in the embodiment of the present invention. The main vector construction steps are preferably as follows:

[0066] (1) Digest the plasmid containing the optimized and synthesized HtA gene with XhoI and XbaI to obtain the target fragment. The reaction system is as follows (endonucleases and buffers used are purchased from Dalian TAKARA Company):

[0067] Plasmid containing synthetic HtA gene 15 μL

[0068] 5 μL of 10×M buffer

[0069] Xho I 5 U

[0070] Xba I 5 U

[0071] Ster...

Embodiment 3

[0092] This embodiment provides a method for purifying HtA protein, which specifically includes the following steps after step S3:

[0093] S4: Centrifuge the supernatant obtained by culturing S3 for 3 days at 4°C, adjust the pH of the obtained supernatant to 8.0-8.5 with NaOH, and centrifuge at a speed greater than or equal to 15,000 g for 10-20 minutes, and the obtained supernatant Load the sample onto the CM cation exchange chromatography column equilibrated with 20 mM Tris-HCl buffer solution of pH 8.0~8.5, and use 5~10 times the column volume of pH 8.0~8.5 containing 20 mM Tris-HCl and 50~100 The buffer solution of mM NaCl rinses the CM cation exchange chromatography column;

[0094] S5: Elute the CM cation exchange chromatography column with a pH 8.0-8.5 buffer solution containing 20 mM Tris-HCl and 400-800 mM NaCl, and use a dialysis bag with a molecular weight of 10 kDa to elute the obtained eluate Dialyzed against 10 mM Tris-Cl buffer, then concentrated by ultrafiltr...

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Abstract

The invention relates to a ribosome toxin and an encoding gene, and an application thereof. The gene provided by the invention is any one DNA molecule of the following (1)-(3): (1) a DNA molecule shown in a sequence 1 in a sequence table; (2) a DNA molecule hybridized with the DNA sequence defined by the (1) under strict conditions and encoding a protein having the activity of poisoning and killing insects; and (3) a DNA sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology with the DNA sequence defined by the (1) and encoding a protein having the activity of poisoning and killing insects. The DNA sequence of HtA is optimized, the method for high-efficiency expression and quick purification of the HtA recombinant protein is provided, is of important value for the field of insect resistance, especially for breeding of insect-resistant plant varieties, and is of important significance for increasing the yield of crops.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a ribosomal toxin (HtA) recombinant gene derived from Polychaete thompsonii and a method and application for preparing the recombinant protein. Background technique [0002] Thompson's Polychaete ( Hirsutella thompsonii ) is a fungal acaricide that has been used to control mites and other pests as early as the 1970s. Studies in recent years have found that there is a close relationship between the glycotoxin (HtA) produced by Thompsonia polychaetes and its insecticidal activity, and its fermentation broth also has insecticidal activity, and the ribosomal toxin purified from the fermentation broth can also kill Insect pests. Although HtA has important potential value in controlling agricultural pests, the content of natural ribosomal toxins is low and difficult to extract, and often only a very small amount of pure product can be obtained. Currently, less than 1 mg can be purified f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/37C12N15/31C12N15/81C07K1/18C07K1/34A01N47/44A01N37/46A01P7/04C12R1/84
Inventor 李洪波夏玉先
Owner HUAIHUA UNIV
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