Lentinus-tigrinus immunomodulatory protein Fip-lti1 as well as preparation method and application thereof

The technology of immunomodulatory protein and shiitake mushroom is applied in the directions of botanical equipment and method, biochemical equipment and method, application, etc., can solve the problem of finding fungal immunomodulatory protein, etc., achieves the promotion of spleen lymphocyte proliferation, and has a good application prospect. , the effect of improving 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-lti1 as well as preparation method and application thereof
  • Lentinus-tigrinus immunomodulatory protein Fip-lti1 as well as preparation method and application thereof
  • Lentinus-tigrinus immunomodulatory protein Fip-lti1 as well as preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0041] Sequence information and homology analysis of the immunomodulatory protein Fip-lti1 gene of Lentinus edodes:

[0042] The cDNA sequence of the immunoregulatory protein Fip-lti1 of shiitake mushroom is 339bp, and the detailed sequence is shown in SEQ ID NO.2. According to the cDNA sequence, the amino acid sequence of Fip-lti1 was deduced, with a total of 112 amino acid residues, a molecular weight of 12.61kDa, a theoretical isoelectric point (pI) of 8.66, an instability coefficient of 11.36, and an aliphatic coefficient of 79.2. For the detailed sequence, see SEQ ID NO .1 sequence shown.

[0043] The amino acid sequence of the immunomodulatory protein Fip-lti1 from Lentinus edodes was searched for protein homology in the Non-redundantGenBank database with the BLAST program, and it was found that it had 61% similarity with the immunomodulatory protein from G. microsporum. %, the similarity with the immunomodulatory protein from Ganoderma lucidum (G. velutipes) immunomod...

Embodiment 2

[0045] Synthesis and expression of the immune regulatory protein Fip-lti1 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-lti1 gene fragment was recovered, connected to the pET32a vector digested with BamHI and XhoI with ligase (T4ligase), and the expression vector pET-Fip-lti1 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-lti1 gene fragment was constructed on the expression ve...

Embodiment 3

[0048] Isolation and purification of the recombinant immunoregulatory protein Fip-lti1 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-lti1 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 The indicated induced proteins were identified by SDS-PAGE and were of correct size.

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Abstract

The invention relates to lentinus-tigrinus immunomodulatory protein Fip-lti1 as well as a preparation method and application thereof. The amino acid sequence of the immunomodulatory protein is as shown by SEQ ID NO. 1. The preparation method comprises the following steps of (1), connecting a cDNA (complementary Deoxyribonucleic Acid) sequence of the lentinus-tigrinus immunomodulatory protein into a carrier, so as to obtain a recombinant carrier; (2), transforming a host cell of the recombinant carrier, so as to obtain a recombinant strain; (3), culturing the recombinant strain, and inducing to recombine the expression of the lentinus-tigrinus immunomodulatory protein Fip-lti1; (4), obtaining the lentinus-tigrinus immunomodulatory protein Fip-lti1 through NI-column purification, dialysis and separation. The immunomodulatory protein is used for preparing health food. The lentinus-tigrinus immunomodulatory protein Fip-lti1 provided by the invention and obtained through in-vitro recombination can be used for obviously promoting the proliferation of a splenic lymphocyte and promoting immunomodulatory effects on the expression of a tumor necrosis factor TNF-alpha, and the like; therefore, the lentinus-tigrinus immunomodulatory protein Fip-lti1 has application potential in the aspect of improving the immunity of an organism.

Description

technical field [0001] The invention belongs to the field of fungal immunoregulatory proteins, in particular to a tiger skin shiitake mushroom immunoregulatory protein Fip-lti1 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 (WuCT, 2011,...

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 高英女王莹汪滢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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