A kind of tiger skin shiitake mushroom immunoregulatory protein fip-lti1 and its preparation method and application

A technology of immunoregulatory protein and tiger skin shiitake mushroom, which is applied in botany equipment and methods, biochemical equipment and methods, applications, etc., can solve the problems of discovering fungal immunomodulatory proteins, so as to promote the proliferation of spleen lymphocytes and improve human immunity. power, expressive effect

Active Publication Date: 2019-07-05
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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  • A kind of tiger skin shiitake mushroom immunoregulatory protein fip-lti1 and its preparation method and application
  • A kind of tiger skin shiitake mushroom immunoregulatory protein fip-lti1 and its preparation method and application
  • A kind of tiger skin shiitake mushroom immunoregulatory protein fip-lti1 and its preparation method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Analysis of the sequence information and homology of the immunomodulatory protein Fip-lti1 gene of Shiitake mushroom:

[0042] The cDNA sequence of the immunomodulatory protein Fip-lti1 of tiger skin mushroom is 339 bp, and the detailed sequence is shown in SEQ ID NO.2. The Fip-lti1 amino acid sequence was deduced from the cDNA sequence. It has 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 aliphatic coefficient of 79.2. The detailed sequence is shown in SEQ ID NO .1 shows the sequence.

[0043] The Fip-lti1 amino acid sequence of the immunomodulatory protein from tiger skin and shiitake mushrooms was searched for protein homology in the Non-redundantGenBank database using the BLAST program, and it was found that the similarity to the immunomodulatory protein derived from G. microsporum reached 61 %, the similarity with the immunomodulatory protein derived from G.appl...

Embodiment 2

[0045] Synthesis and expression of the immunomodulatory protein Fip-lti1 gene of Shiitake mushroom:

[0046] Shanghai Shenggong Bioengineering Co., Ltd. was entrusted to complete the base sequence synthesis of the gene. The synthesized sequence was constructed on the cloning vector pUC57, and the cloned strain DH5a was transformed. The plasmid was extracted and digested with double enzyme digestion (BamHI and XhoI), and the agarose gel After electrophoresis, the Fip-lti1 gene fragment was recovered and ligated to the pET32a vector digested with BamHI and XhoI with 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. The next day, the strain was expanded to 800 mL at 1:50, cultured at 37°C to OD600 = 0.4-0.6, 0.5 mM IPTG was added, and expression was induced at 37°C for 5 hours. The expression was induced by 0.5mM IPTG at 37°C for 5h. Such as figure 2 As shown, the Fip-lti1 ge...

Embodiment 3

[0048] Isolation and purification of recombinant tiger skin mushroom immunomodulatory protein Fip-lti1:

[0049] Centrifuge at 8000 rpm and 4°C for 5 min, collect the induced expression strains, and add 100 mL of crushing liquid for ultrasonic lysis. Cracking conditions: temperature ice bath, power 60%, ultrasound 2s, interval 2s, time 15min. Centrifuge at 12000rpm and 4°C for 15min, and collect the supernatant and precipitate. SDS-PAGE detection to determine the expression form of the target protein. The target protein Fip-lti1 exists in a soluble form. Purify by NI column, collect the flow-through and eluent, and detect the effect of protein purification by SDS-PAGE. Such as image 3 , 4 The indicated induced protein was identified by SDS-PAGE and the size was correct.

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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 immunomodulatory proteins, and particularly relates to a tiger skin mushroom immunomodulatory protein Fip-lti1, and a preparation method and application thereof. Background technique [0002] Fungal immunomodulatory protein (FIP) is a class of small molecule proteins with immunomodulatory activity isolated from higher basidiomycetes. 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 its gene sequence, amino acid sequence and immunophysiological activity. The determination (Kino K, 1989, J BiolChem). In vitro experiments have found that fungal immunomodulatory proteins can promote the proliferation of mouse spleen cells and human peripheral blood lymphocytes (Hsieh KY, 2003, Clin Exp Allergy), and promote T lymphocytes to secrete and express adhesion molecules ICAM (WuCT, 2011, Carcinogenesis) En...

Claims

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Application Information

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Patent Type & Authority Patents(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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