Fungal immunomodulatory protein derived from Morchella conica and application thereof

An immunomodulatory protein and morel apex technology, which is applied in the fields of application, fungi, peptide/protein components, etc., can solve the problems of long cycle, high cost, and low yield, so as to alleviate the inflammatory response, express efficiently, and inhibit Effects of invasion and migration

Inactive Publication Date: 2021-02-05
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The process of extracting FIPs protein from edible fungi is complicated, the cycle is long, the cost is high, and the yield is low, which seriously affects and limits the discovery and mass production of new FIPs, and it is difficult to obtain new properties by homologous cloning. FIPs protein

Method used

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  • Fungal immunomodulatory protein derived from Morchella conica and application thereof
  • Fungal immunomodulatory protein derived from Morchella conica and application thereof
  • Fungal immunomodulatory protein derived from Morchella conica and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Screening, Cloning and Construction of Recombinant Expression Vector of Fungal Immunomodulatory Protein FIP-SH Encoding Gene

[0041] A strain of Morchella conica SH from Yunnan was sequenced, and compared with the existing fungal immunoregulatory protein sequences in the sequenced genome by BLAST, it was found that the fungal immunoregulatory protein FIP-SH was closely related to Versicolor Versicolor, Versicolor versicolor, The sequence homology of the fungal immunoregulatory proteins YZP and ACA of Antrodia camphorata are 41.6% and 50% respectively, the full length of the gene is 399bp, its DNA sequence is shown in SEQ ID NO.2, and the theoretical molecular weight is 13kDa.

[0042] After codon optimization, primers FIP-SH-F containing EcoRI restriction site: 5'-ATGCAGTTTCCACCATCC-3' and primers FIP-SH-R containing NotI restriction site: 5'-GCAAGCAGTCTCCGCATAAG-3' were designed and synthesized , to amplify the synthetic gene with the above primers.

[0043...

Embodiment 2

[0044] Example 2 Preparation of fungal immunoregulatory protein FIP-SH

[0045] 1. Linearization of plasmid SacI

[0046] After the recombinant expression vector pPICZαA-fipsh was restricted by SacI, the linearized carrier fragment was obtained after agarose gel electrophoresis and gel slicing.

[0047] 2. Preparation and transformation of Pichia pastoris X33 competent cells

[0048] Preparation of competent cells: pick Pichia strain X33 on the YPD plate, inoculate into 50ml YPD medium, culture at 30°C, 220rpm to OD=1.3-1.5, then ice-bath for 30min; centrifuge at 4°C, 1500rpm for 5min to precipitate Cells, suspended in 10ml of ice-cold sterile deionized water; 4°C, 1500rpm, centrifuged for 5min to pellet cells, suspended in 5ml of ice-cold sterile deionized water; 4°C, 1500rpm, centrifuged for 5min to pellet cells, pelleted in 2ml of ice-cold sterile Suspend in 1M sorbitol; centrifuge at 1500rpm at 4°C for 5min to pellet the cells, suspend in 200μL of ice-cold sterile 1M sor...

Embodiment 3

[0079] Example 3 Determination of Antitumor Activity of Fungal Immunomodulatory Protein FIP-SH by CCK Method

[0080] Subculture A549 and HepG2 cells in DMEM culture medium, count the tumor cells, and wait for the growth to 80%, divide the cells at 1×10 per well 4 The number of plated in 96-well plate culture, placed in 37 ° C, 5% CO 2 Cultivate in the incubator for 16-24 hours;

[0081] Use the BCA protein concentration determination kit to determine the FIP protein concentration, follow the steps of the BCA kit to configure the standard and then detect the concentration of the FIP sample; after filtering through a 0.22 μm filter, use PBS to prepare a 10 mg / ml stock solution according to the measured concentration and store it for later use ;

[0082] Add FIP factors to the cultured A549 and HepG2 cells according to the concentration of the working solution of 0μg / ml, 5μg / ml, 15μg / ml and 30μg / ml respectively; incubate the culture plate in the incubator for a period of time,...

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Abstract

The invention relates to a fungal immunomodulatory protein derived from Morchella conica and application thereof. The fungal immunomodulatory protein is an immunomodulatory protein FIP-SH derived fromMorchella conica SH, has a total length of 133 amino acids, and has an amino acid sequence shown in SEQ ID NO.1. The fungal immunomodulatory protein has obvious proliferation inhibition, apoptosis induction and migration prevention effects on lung cancer A549 and liver cancer HepG2 cells, and can reduce expression level of inflammatory factors TNF-alpha, IL-1beta and IL-6 in human myeloid leukemia mononuclear THP1 cells. The fungal immunomodulatory protein is a novel fungal immunomodulatory protein with low homology with most reported fungal immunomodulatory proteins, and can be used for preparing immunomodulators and anti-tumor preparations. The invention also provides and synthesizes a gene for encoding the protein and a recombinant engineering yeast strain.

Description

technical field [0001] The invention belongs to the field of fungal immunoregulatory proteins, and in particular relates to a fungal immunoregulatory protein derived from Morchella apexus and application thereof. Background technique [0002] Fungal immunomodulatory proteins (FIPs) were originally extracted from the fruiting bodies of some higher basidiomycetes, such as edible fungi, and are a class of small proteins similar in structure and function to plant lectins and immunoglobulins. A collective term for molecular proteins. [0003] Different FIPs have various important physiological functions such as enhancing immunity, anti-allergy, anti-tumor, anti-virus, anti-fungal, lowering cholesterol, inhibiting immune rejection and promoting bone regeneration. The activity and stability of different FIPs are very different. Therefore, the screening of FIPs proteins with good activity and high stability has become the focus of attention. [0004] At present, there are about 20...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/375C12N15/31C12N15/81C12N1/19A61K38/16A61P29/00A61P37/02A61P35/00C12R1/84
CPCC07K14/375C12N15/815A61P29/00A61P37/02A61P35/00A61K38/00
Inventor 武国干唐雪明邓廷山孙宇曾海娟
Owner SHANGHAI ACAD OF AGRI SCI
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