Antrodia camphorata immunomodulatory protein ACA1 and a coding gene thereof and application thereof

An immunomodulatory activity, protein technology, applied in application, genetic engineering, plant genetic improvement, etc.

Inactive Publication Date: 2018-11-06
HUAIHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no method to efficiently produce ACA1 recombinant protein by modifying the DNA sequence of ACA1 and optimizing the ACA1 expression and purification method

Method used

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  • Antrodia camphorata immunomodulatory protein ACA1 and a coding gene thereof and application thereof
  • Antrodia camphorata immunomodulatory protein ACA1 and a coding gene thereof and application thereof
  • Antrodia camphorata immunomodulatory protein ACA1 and a coding gene thereof and application thereof

Examples

Experimental program
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Embodiment 1

[0052] This embodiment provides an optimized artificially synthesized ACA1 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 the synthesis of ACA1, and the optimized DNA was optimized and synthesized according to the expression characteristics of Escherichia coli. Compared with the natural polynucleotide of ACA1 (GenBank: AY569691.1), the optimized DNA sequence has little obvious homology.

[0053] The genes before and after optimization were connected to the E. coli expression vector pET28 to obtain recombinant vectors. The above recombinant vectors verified by sequencing were transformed into competent cells of E. coli expression strains by heat shock, and the corresponding resistant LB plates were coated. 37...

Embodiment 2

[0056] This embodiment provides a method for preparing protein, which specifically includes the following steps:

[0057] S1: Optimizing the gene, constructing the prokaryotic expression vector and transforming: artificially synthesize the optimized mature ACA1 gene, and connect it to the expression vector pET28 to obtain the recombinant expression vector pET28 / ACA1. The vector construction is as follows: figure 1 shown. The main vector construction steps are as follows:

[0058] (1) Digest the recombinant vector with BamH I and Hind III to obtain the target fragment. The reaction system is as follows (all endonucleases and buffers used are purchased from Dalian TAKARA Company):

[0059] Vector ACA1 / pUC 15 μL

[0060] 10×H buffer 5 μL

[0061] BamH I 5U

[0062] Hind III 5U

[0063] Sterile water to 50μL

[0064] (2) Digest pET28 with BamH I and Hind III to obtain vector fragments. The reaction system is as follows (endonucleases and buffers used are purchased from Dalia...

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Abstract

The present invention relates to Antrodia camphorata immunomodulatory protein ACA1 and a coding gene thereof and application thereof, and the gene provided by the present invention is any one DNA molecule of the following (1) to (3): (1) a DNA molecule as shown in sequence 1 of a sequence table; (2) a DNA molecule that hybridizes with the DNA sequence defined in the (1) under stringent conditionsto code an immunomodulatory protein; (3) a DNA molecule that at least 90%- homologous, or at least 95%-homologous, at least 96%, at least 97%-homologous, at least 98%-homologous, or at least 99%-homologous to the DNA sequence defined in the (1) to code an immunomodulatory protein. The DNA sequence of the ACA1 is optimized, and a method for preparing a biologically active ACA1 recombinant protein is provided.

Description

technical field [0001] The invention relates to the field of utilization of biotechnology, in particular to the immune regulation protein ACA1 of Antrodia camphorata and its coding gene and application. Background technique [0002] Antrodia camphorata (also known as Antrodia cinnamomea) is a unique fungus with important medicinal value in Taiwan. It has the functions of detoxification, anti-tumor, and strengthening immunity. Antrodia camphorata has been isolated and identified nearly 100 kinds of active ingredients, among which polysaccharides and terpenoids are the main active substances, but there is still little research on active proteins. The newly discovered immunoregulatory protein of Antrodia camphorata has attracted much attention. Antrodia camphorata immunomodulatory protein can activate immune cells and has anti-tumor activity. At present, many fungal immunomodulatory proteins have been isolated from medicinal and edible fungi. In 2003, three immunoregulatory ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/70C12N1/21C07K14/375
CPCC07K14/375C12N15/70
Inventor 李洪波罗海燕张树琴
Owner HUAIHUA UNIV
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