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Favolus arcularius Ames immunomodulatory protein Fip-Par1 and preparation method and application thereof

An immunomodulatory protein, fip-par1 technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, applications, etc., can solve the problem of finding fungal immunomodulatory proteins, etc., to promote the proliferation of spleen lymphocytes, promote expression, The effect of prevention and treatment of weakened immunity

Active Publication Date: 2017-03-15
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, Ganoderma lucidum (G.lucidium), Flammulina velutipes (Flammulina velutipes), Straw mushroom (Volvariella volvacea), Pine fir Ganoderma (G.tsugae), Zizhi (G.japoncium), Microspore Ganoderma (G.microsporum), sweet Fungal immunomodulatory proteins have been found in G. sinense, G. fornicatum, Postiaplacenta and Nectria haematococca, but not yet in A. Related reports on the discovery of fungal immunomodulatory proteins in (Amanita muscaria)

Method used

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  • Favolus arcularius Ames immunomodulatory protein Fip-Par1 and preparation method and application thereof
  • Favolus arcularius Ames immunomodulatory protein Fip-Par1 and preparation method and application thereof
  • Favolus arcularius Ames immunomodulatory protein Fip-Par1 and preparation method and application thereof

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Experimental program
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Embodiment 1

[0041] Sequence information and homology analysis of the immunoregulatory protein Fip-par1 gene of Macroporosa funnelina:

[0042] The Fip-par1 cDNA sequence of the macroporus funnelii immunoregulatory protein is 339bp, and the detailed sequence is shown in SEQ ID NO.2. According to the cDNA sequence, the amino acid sequence of Fip-par1 was deduced, with a total of 112 amino acid residues, a molecular weight of 12.34kDa, a theoretical isoelectric point (pI) of 7.85, an instability coefficient of 32.78, and an aliphatic coefficient of 73.12. For the detailed sequence, see SEQ ID NO .1 sequence shown.

[0043] The amino acid sequence of the immunomodulatory protein Fip-par1 from Macroporus funnelina was searched for protein homology in the Non-redundantGenBank database with the BLAST program, and it was found that it was similar to the immunomodulatory protein from G. microsporum. 62%, 63% similarity with immunomodulatory protein from Ganoderma lucidum (G.applanatum), 57% simil...

Embodiment 2

[0045] Synthesis and expression of the immunoregulatory protein Fip-par1 gene of Macroporosa funnelina:

[0046] Entrust Shanghai Sangon Bioengineering Co., Ltd. to complete the synthesis of the base sequence of the gene, construct the synthesized sequence on the cloning vector pUC57, transform the cloned strain DH5a, extract the plasmid, digest it with double enzymes (BamHI and XhoI), and agarose gel After electrophoresis, the Fip-par1 gene fragment was recovered, connected to the pET32a vector digested with BamHI and XhoI with ligase (T4ligase), and the expression vector pET-Fip-par1 was transformed into the expression strain Rosetta. Pick a single clone and culture it in LB liquid medium. On the next day, expand the strain at 1:50 to 800 mL, culture at 37°C until OD600=0.4-0.6, add 0.5mM IPTG, and induce expression at 37°C for 5h. The expression was induced by 0.5mM IPTG at 37°C for 5h. like figure 2 As shown, the Fip-par1 gene fragment was constructed on the expression ...

Embodiment 3

[0048] Isolation and purification of the immunoregulatory protein Fip-par1 from the recombinant Macroporus funnelensis:

[0049] Centrifuge at 8000rpm at 4°C for 5min to collect the induced expression strains, add 100mL disruption solution for ultrasonic lysis. Cracking conditions: temperature ice bath, power 60%, ultrasonic 2s, interval 2s, time 15min. Centrifuge at 12000rpm, 4°C for 15min, collect the supernatant and precipitate. SDS-PAGE detection to determine the expression form of the target protein. The target protein Fip-par1 exists in soluble form. Purify through NI column, collect the flow-through and eluate, and check the protein purification effect by SDS-PAGE. like image 3 , 4 As shown, the induced protein was identified by SDS-PAGE, and the molecular weight was correct.

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Abstract

The invention relates to Favolus arcularius Ames immunomodulatory protein Fip-Par1 and a preparation method and application thereof. The amino acid sequence of the immunomodulatory protein is defined as SEQ ID NO.1. The preparation method comprises the steps: (1) connecting the cDNA sequence of the Favolus arcularius Ames immunomodulatory protein into a carrier, and obtaining a recombinant vector; (2) converting the recombinant vector to a host cell, and obtaining a recombination strain; (3) cultivating the recombination strain, inducing and recombining the expression of the Favolus arcularius Ames immunomodulatory protein Fip-Par1; and (4), obtaining through the NI column purification and the dialysis segregation. The immunomodulatory protein is used for preparing health food. The Favolus arcularius Ames immunomodulatory protein Fip-Par1 obtained by recombination in vitro is capable of remarkably promoting the immunomodulatory effects, such as spleen lymphocyte proliferation and expression of tumor necrosis factor TNF-a, and has application potential for improving the body immunity.

Description

technical field [0001] The invention belongs to the field of fungal immunoregulatory proteins, and in particular relates to a macroporus funnel fungus immunoregulatory protein Fip-par1 and its preparation method and application. Background technique [0002] Fungal immunomodulatory protein (FIP) is a kind of small molecular protein isolated from higher basidiomycetes with immunomodulatory activity. In 1989, Japanese scholar Kino isolated the first fungal immunomodulatory protein from the fruiting body of Ganoderma lucidum, named Ling Zhi-8 (LZ-8), and carried out a study on its gene sequence, amino acid sequence and immunophysiological activity. determined (Kino K, 1989, J BiolChem). Through in vitro experiments, it was found that fungal immunomodulatory proteins can promote the proliferation of mouse splenocytes and human peripheral blood lymphocytes (Hsieh KY, 2003, Clin Exp Allergy), and promote the secretion and expression of adhesion molecule ICAM by T lymphocytes (WuC...

Claims

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Application Information

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IPC IPC(8): C07K14/375C12N15/31C12N15/70C12N1/21A23L33/195
CPCA23V2002/00C07K14/375A23V2200/30A23V2250/546
Inventor 鲍大鹏汪滢高英女王莹李燕唐利华茅文俊周陈力龚明万佳宁杨瑞恒吴莹莹
Owner SHANGHAI ACAD OF AGRI SCI
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