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Polypeptide, its coding sequence and preparation method and application of fungus galactose agglutinin protein activity

A technology of galectin and protein activity, applied in the fields of botanical equipment and methods, biochemical equipment and methods, applications, etc., can solve problems such as physiological functions without any research

Inactive Publication Date: 2005-11-09
WUHAN ALPHA OMEGA BIOLOGY TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no research on other pharmacological and physiological functions of the 3 fungal galectins

Method used

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  • Polypeptide, its coding sequence and preparation method and application of fungus galactose agglutinin protein activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Cloning and sequencing of cDNA of Populus galectin: In this example, a new cDNA sequence was obtained, which has not been reported before.

[0047] 1. Primer amplification

[0048] Using the total RNA of Poplaria spp. as a template, use oligonucleotide A: 5'gac cac gcg tat cga tgt cga ctl6(a / c / g)3' as a reverse transcription primer to perform reverse transcription to obtain the first strand of cDNA, Oligonucleotide B (degenerate primer designed according to N-terminal amino acid): 5'ccc aag ctt ca(a / g)ggc gtc aac at(t / c)ta(t / c)aa(t / c) at3' is the forward primer, oligonucleotide C: 5'gac cac gcg tat cga tgt cga c3' is the reverse primer, PCR is carried out, the PCR condition is 95°C for 10min, and enters into three-step cycle (95°C for 1min, 53°C for 1min, 72°C for 2min, a total of 30 cycles), and finally 72°C for 10min. 20μL reaction system contains: 1μg cDNA, 0.2mMdNTP, 20pM forward primer and reverse primer, 2μL 10×buffer, 1μL Taq enzyme and 1.5mM MgCl 2 . The PCR...

Embodiment 2

[0053] Expression and purification of poplar mushroom galectin in Escherichia coli: In this example, a large number of expressed and purified protein samples can be obtained.

[0054] Firstly, EcoRI single-enzyme digested pGEM-Agaricus galectin vector to release the galectin fragment of Agaricus edulis, and at the same time digested the pET28b plasmid with EcoRI, treated the linearized plasmid pET28b with CIAP enzyme for half an hour, and recovered the poplar Mushroom galectin fragments and target plasmids were ligated according to the recovered amount to construct the prokaryotic expression vector pET28b-poplar mushroom galectin, such as figure 1 shown.

[0055] According to the aforementioned prokaryotic expression method, according to the results of inducing and expressing the recombinant poplar agaricus galectin at different times, the optimized time was selected and expressed in large quantities. The recombinant galectin was purified by Ni column, and the expression leve...

Embodiment 3

[0057] Preparation of the anti-galectin antibody of the poplar mushroom: In this embodiment, the polyclonal antibody of the galectin of the poplar mushroom can be obtained, which is used to detect the galectin of the poplar mushroom.

[0058] The natural protein and the recombinant protein obtained in Example 2 were used to immunize animals to produce antibodies, the specific method is as follows. The recombinant protein can be separated by chromatography and then used for later use. It can also be separated by SDS-PAGE gel electrophoresis. The electrophoresis band is excised from the gel and emulsified with an equal volume of complete Freund's adjuvant. Mice were injected intraperitoneally with 50-100ug / 0.2mL emulsified protein. After 14 days, the same antigen was emulsified with incomplete Freund's adjuvant, and the mice were injected intraperitoneally with a dose of 50-100ug / 0.2mL to boost immunization. A booster immunization was given at least 3 times every 14 days. The ...

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Abstract

An active polypeptide of mycogalactose agglutimin protein, its coding sequence with at least 70% humology to the SEQ ID No.1 sequece and at least 80% homology to the SEQ ID No.2 sequence, its preparing process, and its application in preparing the medicines for preventing and treating tumor and the viral diseases of plant are disclosed.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular, the invention relates to the fungus Agrocybeaegerita galectin (Agrocybeaegerita) gene sequence. The present invention also relates to the polypeptide encoded by the nucleotide sequence and its preparation method, and also relates to the application of the polypeptide in medicine, anti-tumor, agriculture and anti-plant virus. Background technique [0002] Poplar tree mushroom (Agrocybe aegerita) belongs to the Phylum Fungi, Class Basidiomycetes, Order Agaricaceae, Agrocybeaceae, and the genus Agrocybe. Also known as columnar field head mushroom, poplar mushroom, tea salary mushroom, willow matsutake and so on. It is rich in eight essential amino acids, of which lysine is as high as 1.75%. Its nutritional value exceeds other edible fungi such as shiitake mushrooms and enoki mushrooms. It also has unique medicinal value. It is often used in folks for anti-ca...

Claims

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Application Information

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IPC IPC(8): C12N15/31
Inventor 孙慧齐义鹏仝鑫赵辰光梁一
Owner WUHAN ALPHA OMEGA BIOLOGY TECH CO LTD
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