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Artificially modified HtA protein and coding gene and application thereof

A protein and gene technology, applied in the field of ribosomal toxin HtA recombinant gene and its preparation protein, can solve problems such as low production volume and inactive inclusion bodies, and achieve the effects of preventing degradation, realizing mass production, and enhancing insecticidal activity

Active Publication Date: 2018-06-29
HUAIHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] However, the current Escherichia coli expression of this protein also has obvious shortcomings: the existing technology constructs an expression vector with the 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. Low

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  • Artificially modified HtA protein and coding gene and application thereof
  • Artificially modified HtA protein and coding gene and application thereof
  • Artificially modified HtA protein and coding gene and application thereof

Examples

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Embodiment 1

[0055] This embodiment provides an artificially modified HtA protein, which is selected from dozens of modified HtA proteins. These proteins are chemically synthesized polypeptides. The specific human sequence of an artificially modified HtA protein is shown as sequence 1 in the sequence list. Compared with the natural sequence HtA protein, the artificially modified HtA protein has significantly higher biological activity at the cellular level and at the level of insect animals.

[0056] In this example, the modified HtA protein and the natural sequence HtA protein were respectively obtained by using the method of polypeptide chemical synthesis, and the biological activity of the above synthesized protein was analyzed. The specific analysis methods and results are as follows.

[0057] Experimental method 1: Insect cell SF9 was cultured to 90% growth density in insect cell culture medium in a 10 cm petri dish, trypsinized and the cell concentration was adjusted to 5×10 5 cel...

Embodiment 2

[0070] This embodiment provides an optimized chemically synthesized artificially modified HtA gene, the specific sequence of which is shown in sequence 2 in the sequence listing, and the protein sequence corresponding to the gene is shown in sequence 1 in the sequence listing. The optimized DNA sequence was compared by NCBI, and no DNA sequence with obvious similarity was found.

[0071] The DNA corresponding to the artificially modified HtA protein was connected to the Pichia pastoris secretory expression vector pPICZαA to obtain the recombinant vector, and then the recombinant vector was transformed into the Pichia pastoris host strain using the lithium chloride transformation method provided by the Invitrogen company operation manual. In X-33, after transformation, use YPD plates containing 100 µg / mL Zeocin antibiotics to screen, use PCR to verify the transformants, and inoculate the PCR-verified Pichia transformants into cells containing different concentrations of Zeocin. ...

Embodiment 3

[0073] This embodiment provides a method for preparing protein, which specifically includes the following steps:

[0074] 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-RHtA. The vector construction is as follows: figure 1 as shown, figure 1 A schematic diagram of the construction of the eukaryotic expression vector pPICZαA-RHtA in the embodiment of the present invention. The main vector construction steps are preferably as follows:

[0075] (1) Digest the plasmid containing the 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):

[0076] Plasmid containing synthetic RHtA gene 15 μL

[0077] 5 μL of 10×M buffer

[0078] Xho I 5 U

[0079] Xba I 5 U

[0080] Sterile water ...

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Abstract

The invention relates to an artificially modified HtA protein and a coding gene and application thereof. The protein provided is a ribosomal toxin protein molecule that is shown in a sequence table 1,undergoes molecular modification and originates from Hirsutella thompsonii. The gene corresponding to the protein is any one DNA molecule as shown in (1)-(3): (1) a DNA molecule shown in sequence 2 in the sequence table; (2) a DNA molecule of protein that is hybridized with a DNA sequence defined by (1) in a strict condition, and encoded and has insecticidal activity; and (3) a DNA molecule of protein that has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of homology to the DNA sequence defined in (1), is encoded and has insecticidal activity. The DNA sequence of theinsect-killing protein is optimized. A method capable of highly expressing and purifying HtA recombined protein is provided, has high value for the insect resisting field, particularly cultivation ofinsect-resistant plant varieties, and has great significance for increase of crop yield.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a molecularly modified ribosomal toxin HtA recombinant gene derived from Polychaete thompsonii and a method and application for preparing 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 the toxin shows strong insecticidal activity, the insecticidal activity of the natural toxin still needs to be improved in order to increase its application value, especially its oral insecticidal activity. One of the most feasible ways to improve t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/37C12N15/31C12N15/81A01N63/04A01N61/00A01N37/46A01P7/04
CPCA01N37/46A01N61/00A01N63/30C07K14/37C12N15/815
Inventor 李洪波康陆鸿蒋鹰
Owner HUAIHUA UNIV
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