CN112516288A

CN112516288A - Application of N-glycosylation modified ganoderma lucidum immunomodulatory protein

Info

Publication number: CN112516288A
Application number: CN202011523442.6A
Authority: CN
Inventors: 周选围; 胡大卫; 徐伟民; 李奇璋
Original assignee: Tibet Anada Biomedical Technology Co ltd
Current assignee: Tibet Anada Biomedical Technology Co ltd
Priority date: 2020-12-22
Filing date: 2020-12-22
Publication date: 2021-03-19
Anticipated expiration: 2040-12-22
Also published as: CN112516288B

Abstract

The application of the N-glycosylation modified ganoderma lucidum immunomodulatory protein reduces the expression of proinflammatory mediators by inhibiting a p38 MAPK pathway, improves the expression of the anti-inflammatory mediators at the same time, has higher biological activity and lower cytotoxicity and shows more obvious anti-inflammatory activity.

Description

Application of N-glycosylation modified ganoderma lucidum immunomodulatory protein

Technical Field

The invention relates to a technology in the field of biological pharmacy, in particular to an application of ganoderma lucidum immunomodulatory protein modified by N-glycosylation.

Background

Macrophages, as a cell population with plasticity and pluripotency, exhibit significant functional differences under the influence of different microenvironments in vivo and in vitro. At present, macrophages are classified mainly into M1 type, i.e., classically activated macrophages, and M2 type, i.e., alternatively activated macrophages, according to their activation states and functions. M1-type macrophages are triggered in response to pro-inflammatory cytokines and pathogen-associated molecular patterns (PAMPs) such as interferon gamma (IFN-gamma) and LPS. This type of macrophage produces cytotoxic and inflammatory molecules such as Nitric Oxide (NO) and Reactive Oxygen Species (ROS), proinflammatory cytokines such as tumor necrosis factor (TNF- α), Interleukin (IL) -1 β and IL-6, chemokines such as monocyte chemoattractant protein-1 (MCP-1/CCL-2), and the like. Macrophages of type M2 are responsible for anti-inflammatory cytokines, parasitic infection and injury-associated molecular patterns (DAMPs) such as IL-4 and IL-13, and play an important role in inhibiting acute and chronic inflammatory responses and tissue repair. Macrophages of type M2 are capable of secreting large amounts of anti-inflammatory cytokines such as IL-10 and transforming growth factor-beta (TGF- β). The Ganoderma lucidum immunomodulatory protein is a bioactive protein existing in Ganoderma lucidum (Ganoderma lucidum), and has immunoregulatory function. It is disclosed in the prior art that ganoderma immunomodulatory protein is capable of activating macrophages while promoting the production of pro-inflammatory and anti-inflammatory mediators. However, proinflammatory mediators tend to be the cause of chronic inflammation.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides the application of the N-glycosylation modified ganoderma lucidum immunomodulatory protein, which reduces the expression of proinflammatory mediators by inhibiting the p38 MAPK pathway and improves the expression of anti-inflammatory mediators.

The invention is realized by the following technical scheme:

the invention relates to application of N-glycosylation modified ganoderma lucidum immunomodulatory protein in preparation of an anti-inflammatory drug for inhibiting a p38 MAPK pathway in cells and reducing expression of proinflammatory mediators.

The amino acid sequence of the N-glycosylation modified ganoderma lucidum immunomodulatory protein is shown as Seq ID No.1, Seq ID No.2 and Seq ID No.3, and preferably: seq ID No. 3.

The cells comprise mouse peritoneal macrophage RAW 264.7.

The anti-inflammatory drug is a composition, a nutritional supplement, a food additive, a functional food supplement or a functional beverage composition, and comprises the N-glycosylation modified ganoderma lucidum immunomodulatory protein.

The invention relates to a preparation method of the N-glycosylation modified ganoderma lucidum immunomodulatory protein, which is characterized in that based on the amino acid sequence of the ganoderma lucidum immunomodulatory protein shown in Seq ID No.17, reference is made to pichia pastoris (Pichia pastoris) preference codon shown in Seq ID No.18 for gene synthesis, and primers shown in Seq ID No. 4-7 are used for obtaining the N-glycosylation modified ganoderma lucidum immunomodulatory protein N31S gene shown in Seq ID No. 19; or obtaining the N-glycosylation modified ganoderma lucidum immunomodulatory protein T36N gene shown in Seq ID No.20 through primers shown in Seq ID No.4, Seq ID No.8, Seq ID No.9 and Seq ID No. 7; or the obtained N-glycosylation modified ganoderma lucidum immunomodulatory protein N31S/T36N gene shown in Seq ID No.21 is obtained through primers shown in Seq ID No.4, Seq ID No.5, Seq ID No.10 and Seq ID No. 7. After cloning the N31S gene, the T36N gene and the N31S/T36N gene to the eukaryotic expression vector pPIC 9K: X-His, respectively, the Pichia pastoris GS115 was transformed to obtain yeast transformants, and finally the recombinant protein was obtained by methanol induction.

The primer is specifically as follows: seq ID No. 4: 5'-ccgccgccggaattcatgtctgacaccgctttgatcttc-3', Seq ID No. 5: 5'-gaagttagatgggttacctcttccc-3', Seq ID No. 6: 5'-gggaagaggtaacccatctaacttcatcgacaccgttaccttcccaaag-3', Seq ID No. 7: 5'-atgatgatggggcccgttccattgagcaatgatgaagtcg-3', Seq ID No. 8: 5'-gaagttgtttgggttacctcttccc-3', Seq ID No. 9: 5'-gggaagaggtaacccaaacaacttcatcgacaacgttaccttcccaaag-3', Seq ID No. 10: 5'-gggaagaggtaacccatctaacttcatcgacaacgttaccttcccaaag-3' are provided.

The Ganoderma lucidum immunomodulatory protein is selected from Ganoderma lucidum (Ganoderma lucidum), Ganoderma tsugae (Ganoderma tsugae), Ganoderma sinense (Ganoderma japonica), Ganoderma microsporum (Ganoderma microsporum), Ganoderma sinense (Ganoderma sinensis), Ganoderma atrum (Ganoderma atrum), Ganoderma applanatum (Ganoderma applanatum) and Ganoderma pseudoantler (Ganoderma amboinense), and preferably Ganoderma lucidum (Ganoderma lucidum).

The amino acid sequence of the ganoderma lucidum immunomodulatory protein is preferably Genbank: the sequence shown in LZ8_ GANLU is shown in Seq ID No. 17.

The N-glycosylation modified ganoderma lucidum immunomodulatory protein is derived from a eukaryotic expression system, preferably a pichia pastoris expression system.

Technical effects

The invention obtains the N-glycosylation modified ganoderma lucidum immunomodulatory protein by modifying the ganoderma lucidum immunomodulatory protein; the N-glycosylation modified ganoderma lucidum immunomodulatory protein has an anti-inflammatory effect, and the effect is realized by inhibiting a p38 MAPK pathway. Compared with the prior art, the N-glycosylation modified ganoderma lucidum immunomodulatory protein has higher biological activity, lower cytotoxicity and more obvious anti-inflammatory activity.

Drawings

FIG. 1 is a nucleic acid electrophoresis diagram showing the acquisition of N-glycosylation modified genes of immunomodulating proteins of Ganoderma lucidum (N31S, T36N and N31S/T36N);

in FIG. 1A, lane 1 is the fragment N31S _ F1, lane 2 is the fragment N31S _ F2, lane 3 is the fragment T36N _ F1, lane 4 is the fragment T36N _ F2, lane 5 is the fragment N31S/T36N _ F1, lane 6 is the fragment N31S/T36N _ F2, and lane M is DNA Marker DL 2, 000 (TaKaRa Biotechnology (Beijing) Co., Ltd.). In FIG. 1B, lane 1 is the N31S gene, lane 2 is the T36N gene, lane 3 is the N31S/T36N gene, lane M is the DNA Marker DL 2, 000;

FIG. 2 is a graph showing the expression of ganoderma lucidum immunomodulatory protein and N-glycosylation modified ganoderma lucidum immunomodulatory protein;

in the figure: a is an SDS-PAGE detection image, and B is a Western Blot detection image. Lane M is the protein standard molecular weight (shanghai bi yunnan biotechnology limited), and lane 1 shows the ganoderma lucidum immunomodulatory protein WT; lane 2 shows N-glycosylation modified Ganoderma immunomodulatory protein N31S, lane 3 shows N-glycosylation modified Ganoderma immunomodulatory protein T36N, lane 4 shows N-glycosylation modified Ganoderma immunomodulatory protein N31S/T36N;

FIG. 3 is a graph showing the relative amount of inflammatory mediator gene expression in examples;

the figure shows the effect of an N-glycosylation modified ganoderma immunomodulatory protein on expression of pro-inflammatory and anti-inflammatory mediator genes;

FIG. 4 is a schematic of Western Blot of the p38 MAPK pathway;

the anti-inflammatory mechanism of action of the N-glycosylation modified immunomodulatory proteins of Ganoderma lucidum is shown.

Detailed Description

Specific materials of the following examples include:

1. the strain is as follows: e.coli DH5 α; pichia pastoris GS 115.

2. Animal cells: mouse peritoneal macrophage RAW264.7 purchased from Shanghai Living sciences research institute of Chinese academy of sciences. The culture method comprises the following steps: mouse peritoneal macrophage RAW264.7 was washed with PBS, trypsinized, and the cells were suspended in DMEM cell culture medium. Count cells at 2X 10⁵At a concentration of one/mL, cells were inoculated into a new cell culture flask containing DMEM medium and placed in CO₂The incubator was incubated at 37 ℃.

3. The kit comprises:

AxyPrep^TMplasmid Miniprep Kit and

AxyPrep^TMDNA Gel Extraction Kit, purchased from Corning Life sciencesSchool (wujiang) ltd; t4 DNA Ligase, MiniBEST Universal RNA Extraction Kit and PrimeScript^TMRT Master Mix (Perfect Real Time) was purchased from TaKaRa Biotechnology (Beijing) Ltd; AceQ Universal SYBR qPCR Master Mix, available from nanotechnology ltd, nuozokenza, nj; the BCA method protein concentration determination kit is purchased from Biotechnology engineering (Shanghai) Co., Ltd.

4. Enzymes: 2 × TSINGKE Master Mix (blue), available from seiko biotechnology limited of kyoto; restriction enzymes EcoR I, Apa I and Sac I, available from TaKaRa Biotechnology, Beijing, Inc.

Example 1

The preparation of the N-glycosylation modified ganoderma lucidum immunomodulatory protein specifically comprises the following steps:

1.1 Gene Synthesis

According to the amino acid sequence (Genbank: LZ8_ GANLUU) of Ganoderma lucidum immunomodulatory protein from Ganoderma lucidum (Ganoderma lucidum), sequence synthesis is carried out by referring to Pichia pastoris preferred codon to obtain a synthetic sequence shown as Seq ID No.35, the synthetic sequence is constructed on a cloning vector pUC-57 and transformed into Escherichia coli strain DH5 alpha, and plasmids are extracted.

This example refers to Ganoderma lucidum immunomodulatory protein from Ganoderma lucidum (Ganoderma lucidum) as WT, the original Ganoderma lucidum immunomodulatory protein.

1.2 cloning of mutant genes

Carrying out PCR reaction by taking the plasmid in 1.1 as a template, specifically: carrying out conventional PCR reaction by respectively taking a primer sequence P1 shown in Seq ID No.4, a primer sequence P2 shown in Seq ID No.5, a primer sequence P3 shown in Seq ID No.6 and a primer sequence P4 shown in Seq ID No.7 as primers to obtain two fragments N31S _ F1 and N31S _ F2 of the N-glycosylation modified ganoderma lucidum immunomodulatory protein N31S gene; carrying out conventional PCR reaction by respectively taking a primer sequence P1 shown in Seq ID No.4, a primer sequence P5 shown in Seq ID No.8, a primer sequence P6 shown in Seq ID No.9 and a primer sequence P4 shown in Seq ID No.7 as primers to obtain two fragments T36N _ F1 and T36N _ F2 of the N-glycosylation modified ganoderma lucidum immunomodulatory protein T36N gene; a conventional PCR reaction was carried out using the primer sequences P1 and P2 of Seq ID No.4 and P7 and P4 of Seq ID No.10 and P31S of Seq ID No.7 as primers, respectively, to obtain two fragments N31S/T36N _ F1 and N31S/T36N _ F2 of the N-glycosylation modified ganoderma lucidum immunomodulatory protein N31S/T36N gene (FIG. 1A).

After the gene fragment was purified, N31S _ F1 and N31S _ F2, T36N _ F1 and T36N _ F2, and N31S/T36N _ F1 and N31S/T36N _ F2 were used as primers and templates, respectively, and conventional PCR was performed to obtain N31S, T36N and N31S/T36N genes, respectively (FIG. 1B).

1.3 obtaining of Yeast transformants: the laboratory constructed plasmid pPIC 9K: X-His as well as the WT gene obtained in 1.1 and the N31S, T36N and N31S/T36N genes obtained in 1.2 were digested with the restriction enzymes EcoR I and Apa I at 37 ℃ for 2h according to the following digestion system.

The enzyme cutting system is as follows: 20 μ L of buffer, 3 μ L of EcoR I, 3 μ L of LApa I, 20 μ L of DNA (plasmid pPIC 9K: X-His and gene WT, N31S, T36N and N31S/T36N, respectively), 154 μ L of dH₂O。

1.4 after separation of the digestion products by electrophoresis on a 1% agarose gel in 1 XTAE electrophoresis buffer, staining with ethidium bromide, taking pictures with a gel imaging system, the linearized vector pPIC 9K: His as well as the WT, N31S, T36N and N31S/T36N gene fragments were recovered. The WT, N31S, T36N, and N31S/T36N gene fragments were ligated to the linearization vector pPIC 9K: His, respectively, according to the conventional reaction method to construct the recombinant expression plasmid pPIC 9K: WT-His, pPIC 9K: N31S-His, pPIC 9K: T36N-His, and pPIC 9K: N31S/T36N-His.

1.5 transformation of the ligation product into E.coli DH5 α. The plasmids pPIC 9K: WT-His, pPIC 9K: respectively

N31S-His, pPIC 9K: T36N-His and pPIC 9K: N31S/T36N-His, were digested with the restriction enzyme Sac I at 37 ℃ for 2 h. And recovering the enzyme digestion product, and then carrying out yeast transformation to obtain a yeast transformant.

The yeast transformation comprises the following steps:

1.5.1) selecting Pichia pastoris monoclonals, culturing in YPD liquid medium at 30 ℃ and 220rpmShake culture to OD₆₀₀0.8 to 1.0;

1.5.2) centrifugation at 1, 500 Xg for 10min at room temperature, harvesting the cells, washing with 25mL sterile water, centrifugation under the same conditions, discarding the supernatant, and resuspending the cells with 1mL 100mM LiCl;

1.5.3) transferring the cell suspension into a 1.5mL centrifuge tube, centrifuging for 15s at the maximum rotation speed, and removing LiCl;

1.5.4) resuspend the cells with 400. mu.L of 100mM LiCl and dispense into 1.5mL centrifuge tubes as 50. mu.L of cell suspension;

1.5.5) boiling the single-stranded DNA for 5min and then quickly putting the single-stranded DNA into ice;

1.5.6) centrifuging the cell suspension to remove LiCl;

1.5.7) reagents were added in the order of 240. mu.L of 50% PEG, 36. mu.L of LiCl at a concentration of 1M, 50. mu.g of single-stranded DNA, and 10. mu.g of plasmid DNA.

1.5.8), after cell resuspension, standing and culturing for 30min at room temperature;

1.5.9) carrying out heat shock at 42 ℃ for 20-25 min;

1.5.10) centrifuging at 6,000-8,000 rpm, discarding the transformation solution, and resuspending with 1mL sterile water;

1.5.11) applying appropriate amount of suspension to MD^KanCulturing on a solid culture medium at 30 ℃ for 2-4 days.

1.6 expression and purification of recombinant proteins: selecting yeast transformants, carrying out monoclonal culture on the yeast transformants in a BMGY liquid medium at 30 ℃ and 220rpm under shaking until the OD600 is 2-6; centrifugation at 2, 500 × g for 5min at room temperature to collect cells, supernatant was removed, and cells were resuspended in BMMY liquid medium to an OD600 of 1.0; four days in culture, pure MeOH was added daily to BMMY broth to a final concentration of 1% (V/V).

1.7 at room temperature with 2, 500 Xg centrifugal 5min to collect the supernatant, and the supernatant through 3.5kD dialysis bag dialysis, dialysis bag fluid freeze drying and Lysis Buffer dissolved; his60 Ni Superflow Resin column was equilibrated with 5 volumes of lysine Buffer; loading Lysis Buffer solution; washing with 10 times of volume of Wash Buffer; eluting with 2 times volume of Elution Buffer and collecting the eluate; dialyzing the eluate through a 3.5kD dialysis bag, freeze-drying the dialysate in the dialysis bag, and dissolving the lyophilized dialysate in PBS to obtain PBS solutions of recombinant WT, N31S, T36N, and N31S/T36N proteins. The obtained protein solution was subjected to SDS-PAGE and Western Blot detection. The results showed that the resulting protein was the protein of interest (FIG. 2).

Example 2

The anti-inflammatory application of the N-glycosylation modified ganoderma lucidum immunomodulatory protein specifically comprises the following steps:

at 2X 10⁵Cell/well concentration cells were seeded into 24 well cell culture plates and cultured for 24 h. The medium was discarded, and medium containing 10. mu.g/mL of Ganoderma lucidum immunomodulatory protein (WT) and N-glycosylation modified Ganoderma lucidum immunomodulatory protein (N31S, T36N, and N31S/T36N) was added, respectively, and the culture was continued for 6 hours. Total RNA was extracted using the MiniBEST Universal RNA Extraction Kit. Using PrimeScript^TMRT Master Mix (Perfect read Time), reverse transcribing the extracted RNA into cDNA according to the following system and procedure:

the reverse transcription system is specifically as follows: 4 μ L of 5 XPrimeScript RT Master Mix, 1 μ g RNA, up to 20 μ L dH₂O。

The reaction was carried out at 37 ℃ for 15 minutes, at 85 ℃ for 5 seconds, and then stored at 4 ℃.

After diluting the cDNA by 50 times, real-time fluorescent quantitative PCR reaction was performed using the following primers, reaction system, program and calculation method.

The primer sequence comprises: the IL-6 primer sequence pair shown as Seq ID No.11 and Seq ID No. 12; a pair of TGF-. beta.1 primer sequences as shown in Seq ID No.13 and Seq ID No. 14; the primer sequence pairs of beta-actin as shown in Seq ID No.15 and Seq ID No. 16.

The reaction system is as follows: 5 μ L of 5 × AceQ Universal SYBR qPCR Master Mix, 0.2 μ L Primer F, 0.2 μ L Primer R, 2 μ L cDNA and 2.6 μ L dH₂O。

The amplification procedure was carried out for 5 minutes at 95 ℃ followed by 40 cycles at 10 seconds at 95 ℃ and 30 seconds at 60 ℃ and finally for 5 seconds at 65 ℃ after 10 minutes at 95 ℃.

Data processing: use 2^-ΔΔCtThe method of (4) calculating the expression amount. That is, Δ Ct is Ct_{Target gene}－Ct_{Internal reference gene}；ΔΔCt＝ΔCt_{Experiment ofGroup of}－ΔCt_{Control group}(ii) a Multiple change 2^-ΔΔCt。

As shown in FIG. 1, the N-glycosylation modified Ganoderma lucidum immunomodulatory proteins (N31S, T36N, and N31S/T36N) significantly inhibited the expression of the proinflammatory mediator IL-6 gene and promoted the expression of the anti-inflammatory mediator TGF- β 1, compared to Ganoderma lucidum immunomodulatory protein (WT). Meanwhile, the results also show that the effect of N31S/T36N is more obvious.

Example 3

Analysis of anti-inflammatory mechanism of N-glycosylation modified ganoderma lucidum immunomodulatory protein:

at 2X 10⁵Cell/well concentration cells were seeded into 24 well cell culture plates and cultured for 24 h. Discarding the culture medium, adding culture medium containing Ganoderma immunomodulatory protein with concentration of 10 μ g/mL and N-glycosylation modified Ganoderma immunomodulatory protein, and culturing for 24 hr. After washing the cells twice with PBS, an appropriate amount of RIPA (Shanghai Biyuntian Biotechnology Co., Ltd.) was added, and after pipetting, the cells were centrifuged at 12,000 rpm for 10min at 4 ℃. The supernatant was collected, and the protein concentration was measured using the BCA method protein concentration measurement kit. Western Blot detection was performed using beta-actin, p38 and phosphorylated p38(p-p38) antibody (Abcam) as primary antibodies, and horseradish peroxidase (HRP) -labeled goat anti-mouse and goat anti-rabbit IgG (Biotechnology (Shanghai) Co., Ltd.) as secondary antibodies. The results are shown in fig. 2, compared with ganoderma lucidum immunomodulatory protein (WT), N-glycosylation modified ganoderma lucidum immunomodulatory protein (N31S, T36N and N31S/T36N) can significantly interfere with phosphorylation of p38, thereby inhibiting p38 MAPK signaling pathway. Meanwhile, the effect of N31S/T36N is more obvious.

The invention transforms the ganoderma lucidum immunomodulatory protein through an in vitro mutation technology to obtain the N-glycosylation modified ganoderma lucidum immunomodulatory protein, and compared with the ganoderma lucidum immunomodulatory protein, the N-glycosylation modified ganoderma lucidum immunomodulatory protein has more remarkable biological activity, more excellent anti-inflammatory effect and lower cytotoxicity.

The foregoing embodiments may be modified in many different ways by those skilled in the art without departing from the spirit and scope of the invention, which is defined by the appended claims and all changes that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Sequence listing

<110> Tibet ananda biopharmaceutical science, Limited liability company

Application of <120> N-glycosylation modified ganoderma lucidum immunomodulatory protein

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Thr Tyr Arg Val Ala Val Ser Gly Arg Asn Leu Gly Val Lys Pro Ser

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Tyr Ala Val Glu Ser Asp Gly Ser Gln Lys Val Asn Phe Leu Glu Tyr

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gtcaagccat cctacgctgt tgagtccgac ggttcccaga aggtcaactt cttggagtac 240

aactctggtt acggtatcgc tgacactaac accatccaag ttttcgttgt cgacccagac 300

accaacaacg acttcatcat tgctcaatgg aac 333

<210> 21

<211> 333

<212> DNA

<213> N-glycosylation modified ganoderma lucidum immunomodulatory protein N31S/T36N gene (Artificial Sequence)

<400> 21

atgtctgaca ccgctttgat cttcagactg gcttgggacg tcaagaagtt gtccttcgac 60

tacaccccaa actggggaag aggtaaccca tctaacttca tcgacaacgt taccttccca 120

aaggtcttga ctgacaaggc ctacacctac agagttgccg tttctggtcg taacctgggt 180

gtcaagccat cctacgctgt tgagtccgac ggttcccaga aggtcaactt cttggagtac 240

aactctggtt acggtatcgc tgacactaac accatccaag ttttcgttgt cgacccagac 300

accaacaacg acttcatcat tgctcaatgg aac 333

Claims

1. The application of the N-glycosylation modified ganoderma lucidum immunomodulatory protein is characterized in that the N-glycosylation modified ganoderma lucidum immunomodulatory protein is used for preparing an anti-inflammatory drug to inhibit a p38 MAPK pathway in cells and reduce the expression of proinflammatory mediators;

the amino acid sequence of the N-glycosylation modified ganoderma lucidum immunomodulatory protein is shown as Seq ID No.1, Seq ID No.2 and Seq ID No. 3;

the Ganoderma lucidum immunomodulatory protein is selected from Ganoderma lucidum (Ganoderma lucidum), Ganoderma tsugae (Ganoderma tsugae), Ganoderma sinense (Ganoderma japonicum), Ganoderma japonicum (Ganoderma japonicum), Ganoderma microsporum (Ganoderma microsporum), Ganoderma sinense (Ganoderma sinense), Ganoderma atrum (Ganoderma atrum), Ganoderma applanatum (Ganoderma applanatum) or Ganoderma pseudoantler (Ganoderma amboinense).

2. The use according to claim 1, wherein the amino acid sequence of the N-glycosylation modified ganoderma lucidum immunomodulatory protein is Seq ID No. 3.

3. The use according to claim 1 or 2, wherein the cells comprise mouse peritoneal macrophage RAW 264.7.

4. The use according to claim 1, wherein the anti-inflammatory agent is a composition, a nutritional supplement, a food additive, a functional food supplement, or a functional beverage composition comprising an N-glycosylation modified ganoderma immunomodulatory protein.

5. The use of claim 1, wherein the amino acid sequence of the ganoderma lucidum immunomodulatory protein is Genbank: the sequence shown in LZ8_ GANLU is shown in Seq ID No. 17.

6. A method for preparing an N-glycosylation modified ganoderma lucidum immunomodulatory protein according to any one of claims 1-5, wherein the amino acid sequence of the ganoderma lucidum immunomodulatory protein shown in Seq ID No.17 is subjected to gene synthesis by referring to a codon preferred by Pichia pastoris (Pichia pastoris) shown in Seq ID No.18, by:

firstly, obtaining the N-glycosylation modified ganoderma lucidum immunomodulatory protein N31S gene shown in Seq ID No.19 by using the primers shown in Seq ID No. 4-7; or by

Secondly, primers shown as Seq ID No.4, Seq ID No.8, Seq ID No.9 and Seq ID No.7 are used to obtain the N-glycosylation modified ganoderma lucidum immunomodulatory protein T36N gene shown as Seq ID No. 20; or by

③ obtaining the N-glycosylation modified ganoderma lucidum immunomodulatory protein N31S/T36N gene shown in Seq ID No.21 by the primers shown in Seq ID No.4, Seq ID No.5, Seq ID No.10 and Seq ID No. 7;

then the N31S gene, the T36N gene and the N31S/T36N gene were cloned into the eukaryotic expression vector pPIC 9K: X-His, respectively, and then Pichia pastoris GS115 was transformed to obtain yeast transformants, and finally the recombinant protein was obtained by methanol induction.

7. The method of claim 6, wherein the primers are specifically: seq ID No. 4: 5'-ccgccgccggaattcatgtctgacaccgctttgatcttc-3', Seq ID No. 5: 5'-gaagttagatgggttacctcttccc-3', Seq ID No. 6: 5'-gggaagaggtaacccatctaacttcatcgacaccgttaccttcccaaag-3', Seq ID No. 7: 5'-atgatgatggggcccgttccattgagcaatgatgaagtcg-3', Seq ID No. 8: 5'-gaagttgtttgggttacctcttccc-3', Seq ID No. 9: 5'-gggaagaggtaacccaaacaacttcatcgacaacgttaccttcccaaag-3', Seq ID No. 10: 5'-gggaagaggtaacccatctaacttcatcgacaacgttaccttcccaaag-3' are provided.

8. The method as set forth in claim 6, wherein the N-glycosylation modified immunomodulatory protein of Ganoderma lucidum is derived from the Pichia pastoris expression system.