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Agrocybe aegerita lectin AAL-2 and coding gene thereof, preparation method and application thereof

A technology of poplar mushroom lectin and AAL-2, which is applied to the preparation method of peptides, botany equipment and methods, biochemical equipment and methods, etc., can solve the problems of high recurrence rate and metastasis rate of liver cancer, and achieve low production cost , simple process and high sample purity

Active Publication Date: 2014-06-18
武汉华扬天乐生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The currently accepted methods for the diagnosis and treatment of liver cancer include ultrasonic diagnosis, subcutaneous ethanol injection, radiotherapy and chemotherapy, and surgical resection, but the recurrence rate and metastasis rate of liver cancer are relatively high.

Method used

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  • Agrocybe aegerita lectin AAL-2 and coding gene thereof, preparation method and application thereof
  • Agrocybe aegerita lectin AAL-2 and coding gene thereof, preparation method and application thereof
  • Agrocybe aegerita lectin AAL-2 and coding gene thereof, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Preparation of Poplar mushroom lectin AAL-2 protein

[0052] (1), preparation of poplar mushroom total protein

[0053] Take the sun-dried poplar mushroom fruiting bodies (purchased from Fujian Sanming Fungi Research Institute), grind them into powder with a pulverizer, add double distilled water to soak in a ratio of 1:10 (w / v), soak for 3 times in total, each time After 4 hours or overnight, take the supernatant of each soaking, add ammonium sulfate to 80%, and let it stand overnight. Centrifuge (10,000rpm / min, 20min) to collect the precipitate, dissolve the precipitate in an appropriate amount of double-distilled water, dialyze to remove salt, and freeze-dry to obtain the total protein of Poplaria edodes.

[0054] (2), affinity column separation and purification

[0055] Dissolve 400mg of poplar mushroom total protein in 90mL PBS buffer, centrifuge to remove the denatured protein, take the supernatant and pass it through an affinity chromatography column,...

Embodiment 2

[0057] Example 2 Determination of the Nucleotide Coding Region of Populus lectin AAL-2 Protein and the Amino Acid Code of AAL-2 Protein

[0058] The AAL-2 band obtained by SDS-PAGE in Example 1 was excised and identified by mass spectrometry to obtain 6 peptides. At the same time, the present invention constructed the transcriptome library (NCBI code SRA026731) of the hyphae and fruiting bodies of Populus edulis, and compared the amino acid sequences of the six peptides with the transcriptome library through local BLASTX. The comparison found that the nucleotide coding region of the AAL-2 protein is located on the complementary strand of the gene Unigene4198 in the transcriptome library. By confirming the open reading frame, a protein coding region was obtained, and the amino acid sequence translated from the protein coding region can be highly Match the 6 peptides of AAL-2 obtained by mass spectrometry ( figure 2 ), and the protein molecular weight predicted by ExPASy softw...

experiment example 1

[0061] Experimental Example 1 Homology Analysis of the Poplar Agaricus Lectin AAL-2 Isolated in the Present Invention and the Existing Poplar Agaricus Lectin AAL

[0062] 1. For the results of comparative analysis of the nucleotide homology between the agaricus lectin AAL-2 separated by the present invention and the published agaricus lectin AAL, see image 3 , It can be seen from the comparison results that the nucleotide homology between the two is 40.63% (193 / 475), Gap=61.22% (750 / 1225).

[0063] 2. For the results of amino acid homology analysis between the Agaricus lectin AAL-2 separated by the present invention and the published lectin AAL, see Figure 4 , From the comparison results, it can be seen that the amino acid homology between the two is 22.78% (36 / 158), the amino acid residue similarity is 47.47% (75 / 158), and Gap=61.18% (249 / 407).

[0064] 3. According to the results of SDS-PAGE of Agaricus lectin AAL-2 and published AAL, the molecular weight of AAL is 15.8kD...

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Abstract

The present invention provides the Agrocybe aegerita lectin AAL-2, its encoding gene, the preparation methods and its uses. The invention uses the affinity chromatography separated the lectin protein AAL-2 from the Agrocybe aegerita total protein, of which the amino acid sequence is set forth in SEQ ID NO: 2 and the nucleotide sequence encoding the said protein is set forth in SEQ ID NO: 1. Cell tests indicate that the Agrocybe aegerita lectin AAL-2 isolated in the invention has excellent inhibition activity to the tumor cells, which can induce apoptosis of hepatoma cells, significantly. Animal experiments show that AAL-2 has a better therapeutic effect on tumor. The glycochip technology test results show that the AAL-2 preferentially binds to the carbohydrate or glycoproteins with the N-Acetylglucosamine as the terminal, which can be used as a reagent for testing the N-Acetylglucosamine relevant diseases or the N-Acetylglucosamine relevant carbohydrate structure.

Description

technical field [0001] The present invention relates to a lectin isolated from fungi and its coding gene, in particular to the lectin AAL-2 and its coding gene isolated from poplar mushroom (Agrocybe aegerita). The separation method of AAL-2 and its application in anti-tumor or detection of diseases related to N-acetylglucosamine and sugar structures related to N-acetylglucosamine belong to the field of separation and application of Agaricus lectin. Background technique [0002] Medicinal fungi have a history of more than 2,000 years in the East because of their unique medicinal value (especially its antitumor activity). Until the 1950s, with the maturity of technology and the separation and identification of active ingredients such as polysaccharides, the medicinal value of medicinal fungi was gradually valued by European and American researchers. More and more active ingredients of medicinal fungi were tested in animals and clinical trials. Screened and verified in the te...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/375C07K1/22C12N15/31C12N15/63C12N1/19C12N5/10A61K38/16A61K48/00A61P35/00G01N33/68C12Q1/68
CPCC07K14/375A61K38/00G01N2333/4724G01N33/57438A61P35/00A61P35/02
Inventor 孙慧姜帅谷变利陈义杰余国军尹亚琳
Owner 武汉华扬天乐生物科技有限公司
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