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Lentinus tigrinus immunomodulatory protein Fip-lti2 and preparation method and application thereof

An immunomodulatory protein, a technology of shiitake mushroom, applied in the directions of botanical equipment and method, biochemical equipment and method, application, etc., can solve the problem of discovering fungal immunomodulatory protein, etc., and achieve the promotion of spleen lymphocyte proliferation, expression, The effect of improving human immunity

Active Publication Date: 2017-02-22
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 Amanita muscaria Related reports on the discovery of fungal immunomodulatory proteins in (Amanita muscaria)

Method used

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  • Lentinus tigrinus immunomodulatory protein Fip-lti2 and preparation method and application thereof
  • Lentinus tigrinus immunomodulatory protein Fip-lti2 and preparation method and application thereof
  • Lentinus tigrinus immunomodulatory protein Fip-lti2 and preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Sequence information and homology analysis of the immune regulatory protein Fip-lti2 gene of Lentinus edodes:

[0042] The cDNA sequence of the immunoregulatory protein Fip-lti2 of shiitake mushrooms is 339bp, and the detailed sequence is shown in SEQ ID NO.2. According to the cDNA sequence, the amino acid sequence of Fip-lti2 was deduced, with a total of 114 amino acid residues, a molecular weight of 12.8kDa, a theoretical isoelectric point (pI) of 5.49, an instability coefficient of 16.16, and an aliphatic coefficient of 89.65. For the detailed sequence, see SEQ ID NO .1 sequence shown.

[0043] The amino acid sequence of the immunomodulatory protein Fip-lti2 of Lentinus edodes was searched for protein homology in the Non-redundantGenBank database with the BLAST program, and it was found that it had 59% similarity with the immunomodulatory protein from G. microsporum. %, 54% similarity to immunomodulatory protein from G. applanatum, 57% similarity to immunomodulatory...

Embodiment 2

[0045] Synthesis and expression of the immune regulatory protein Fip-lti2 gene of Lentinus edodes:

[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-lti2 gene fragment was recovered, connected to the pET32a vector digested with BamHI and XhoI with ligase (T4ligase), and the expression vector pET-Fip-lti2 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. Such as figure 2 As shown, the Fip-lti2 gene fragment was constructed on the expression ve...

Embodiment 3

[0048] Isolation and purification of the recombinant immunoregulatory protein Fip-lti2 from Lentinus edodes:

[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-lti2 exists in soluble form. Purify through NI column, collect the flow-through and eluate, and check the protein purification effect by SDS-PAGE. Such as image 3 , 4 As shown, the induced protein was identified by SDS-PAGE, and the size was correct.

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Abstract

The invention relates to a Lentinus tigrinus immunomodulatory protein Fip-lti2 and a preparation method and application thereof. The amino acid sequence of the immunomodulatory protein is as shown in SEQ ID NO. 1. The preparation method comprises the following steps: (1) connecting a cDNA sequence of the Lentinus tigrinus immunomodulatory protein into a vector to obtain a recombinant vector; (2) transforming host cells of the recombinant vector to obtain a recombinant strain; (3) culturing the recombinant strain, and inducing expression of the Lentinus tigrinus immunomodulatory protein Fip-lti2; and (4) and carrying out NI column purification and dialysis to obtain the final product. The immunomodulatory protein is used for preparing health food. The Lentinus tigrinus immunomodulatory protein Fip-lti2 obtained by in-vitro recombination has immunomodulatory effects of obviously promoting proliferation of splenic lymphocyte, promoting expression of tumor necrosis factors TNF-alpha and the like, and therefore, has utilization potentiality on the aspect of improvement of immunity of the organism.

Description

technical field [0001] The invention belongs to the field of fungal immunoregulatory proteins, and in particular relates to a tiger skin shiitake mushroom immunoregulatory protein Fip-lti2 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 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/375C12N15/31C12N15/70C12N1/21A23L33/195
CPCA23V2002/00C07K14/375A23V2200/30A23V2200/308A23V2200/324A23V2250/546
Inventor 汪滢高英女王莹鲍大鹏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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