CN114805518B

CN114805518B - Protein WG-SP01 with immunoregulatory activity

Info

Publication number: CN114805518B
Application number: CN202210609841.7A
Authority: CN
Inventors: 侯银臣; 黄继红; 廖爱美; 潘龙; 魏兆军; 董得平; 杨盛茹; 连伟帅; 李琰; 袁晓晴
Original assignee: Henan University of Animal Husbandry and Economy
Current assignee: Henan Jianjiu Industrial Co.,Ltd.
Priority date: 2022-05-31
Filing date: 2022-05-31
Publication date: 2023-10-13
Anticipated expiration: 2042-05-31
Also published as: CN114805518A

Abstract

The invention discloses an immunoregulatory active protein WG-SP01, wherein the protein WG-SP01 can activate LDH and ACP activities, so that macrophages of an organism are in an activated state; can obviously improve the ATP activity of lymphocytes; the proliferation capacity of spleen T lymphocytes and B lymphocytes is obviously improved; the protein WG-SP01 has remarkable immunoregulatory efficacy. Therefore, the protein WG-SP01 can be used as an active ingredient of an immunomodulator or an immunopotentiator for preparing medicines or functional foods for treating immune diseases such as systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism and the like, and can also be used for other medicines or functional foods with the effect of regulating the immunity.

Description

Protein WG-SP01 with immunoregulatory activity

Technical Field

The invention relates to the field of protein engineering, in particular to a protein WG-SP01 with immunoregulatory activity.

Background

Immune diseases (immune diseases) refer to diseases caused by the influence of the imbalance of immune regulation on the immune response of the body. Autoimmune diseases are mainly caused by the accumulation of large numbers of immune cells and immunoglobulins generated by an excessive immune response, thereby damaging normal tissues and causing lesions and functional impairment. Occurs in skin, such as allergic dermatitis and various skin lesions, in joints, such as arthritis, and in kidneys, such as nephritis … …

Common autoimmune diseases are: systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, juvenile diabetes, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, many kinds of skin diseases, chronic liver disease … …, and the like.

The treatment of such diseases has in common that immunosuppressants are required to suppress immune responses against the body itself. The most common are adrenocortical hormone formulations such as: prednisone, hydrocortisone, dexamethasone, and the like, which are often referred to as "hormones" for short. If the efficacy is poor, it is also possible to use so-called cytotoxic drugs such as cyclophosphamide, methotrexate, etc., which can be used both for suppressing immunity and for treating cancer. All immunosuppressants have a major common adverse effect and they affect the anti-infective, anti-tumour immune function of the body to varying degrees. Thus, many professionals have been studying other therapies and have found that certain biological agents and natural drugs inhibit autoimmune responses, but have no or little adverse effect on immune function against infections, antineoplastic agents. There is a need for an effective food or drug for treating immune disorders.

Disclosure of Invention

The present invention aims to provide a protein WG-SP01 with immunoregulatory activity and application thereof, so as to solve the problems.

According to a first aspect of the present invention there is provided a protein WG-SP01 having immunomodulatory activity, the amino acid sequence of which is shown in SEQ ID NO. 1. Thus, the protein WG-SP01 with the amino acid sequence can activate LDH and ACP activities, so that macrophages of an organism are in an activated state; can obviously improve the ATP activity of lymphocytes; the proliferation capacity of spleen T lymphocytes and B lymphocytes is obviously improved; WG-SP01 has remarkable immunomodulatory efficacy.

In certain embodiments, the secondary structure of the protein WG-SP01 comprises an alpha helix 17%, a beta sheet 27%, a random coil 17%, a beta turn 21%. Thus, the protein WG-SP01 with the secondary structure can activate LDH and ACP activities, so that macrophages of the organism are in an activated state; can obviously improve the ATP activity of lymphocytes; the proliferation capacity of spleen T lymphocytes and B lymphocytes is obviously improved; WG-SP01 has remarkable immunomodulatory efficacy.

According to a second aspect of the present invention, there is provided the use of a protein WG-SP01 having immunomodulatory activity in the preparation of a food or pharmaceutical product having immunomodulatory activity, the amino acid sequence of said protein WG-SP01 being shown in SEQ ID NO. 1; further, the secondary structure of the protein WG-SP01 comprises 17% of alpha helix, 27% of beta sheet, 17% of random coil and 21% of beta turn. Thus, the food or medicine containing the protein WG-SP01 has high-efficiency immunoregulation function, and can be used for treating immune diseases such as systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism and the like.

According to a third aspect of the present invention, there is provided an immunomodulator comprising a protein WG-SP01 having immunomodulatory activity, the amino acid sequence of said protein WG-SP01 being shown in SEQ ID NO. 1; further, the secondary structure of the protein WG-SP01 comprises 17% of alpha helix, 27% of beta sheet, 17% of random coil and 21% of beta turn. Therefore, the immunomodulator has remarkable immunoregulation effect, and can be used for preparing foods or medicines with immunoregulation effect.

In certain embodiments, the immunomodulator comprises the protein WG-SP01 as an active ingredient.

According to a fourth aspect of the present invention there is provided a functional food or pharmaceutical product comprising an immunomodulator comprising a protein WG-SP01 having immunomodulatory activity, the amino acid sequence of said protein WG-SP01 being shown in SEQ ID NO: 1. Further, the secondary structure of the protein WG-SP01 comprises 17% of alpha helix, 27% of beta sheet, 17% of random coil and 21% of beta turn. Therefore, the functional food or medicine has high-efficiency immunoregulation function, and can be used for treating immune diseases such as systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism and the like.

According to a fifth aspect of the present invention, there is provided an immunopotentiator comprising a protein WG-SP01 having immunoregulatory activity, the amino acid sequence of said protein WG-SP01 being shown in SEQ ID NO. 1. Further, the secondary structure of the protein WG-SP01 comprises 17% of alpha helix, 27% of beta sheet, 17% of random coil and 21% of beta turn. Therefore, the immunopotentiator has remarkable immunopotentiating effect, can enhance the immune function of organisms, and can be used for preparing foods or medicines with the immunoregulation effect.

In certain embodiments, the immunopotentiator comprises the protein WG-SP01 as an active ingredient.

According to a sixth aspect of the present invention there is provided a functional food or pharmaceutical product comprising an immunopotentiator comprising a protein WG-SP01 having immunomodulatory activity, the amino acid sequence of said protein WG-SP01 being as shown in SEQ ID NO. 1. Further, the secondary structure of the protein WG-SP01 comprises 17% of alpha helix, 27% of beta sheet, 17% of random coil and 21% of beta turn. Therefore, the functional food or medicine has high-efficiency immunity enhancing function, and can be used for treating immune diseases such as systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism and the like.

In conclusion, the invention has the beneficial effects that:

1. the immunoregulatory active protein WG-SP01 can activate LDH and ACP activities, so that macrophages of an organism are in an activated state; can obviously improve the ATP activity of lymphocytes; the proliferation capacity of spleen T lymphocytes and B lymphocytes is obviously improved, and the WG-SP01 has obvious immunoregulation effect.

2. The immunoregulatory active protein WG-SP01 can be used as an active ingredient of an immunoregulator or an immunoenhancer for preparing medicines or functional foods for treating immune diseases such as systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism and the like, and can also be used for other medicines or functional foods with immunoregulatory effects.

3. The protein WG-SP01 is derived from wheat germ, is used as an effective component of foods or medicines for treating immune diseases, is used for treating the immune diseases, and is safe and free of side effects.

Drawings

FIG. 1 is a three-dimensional map of the secondary and tertiary structures of immunoregulatory active wheat germ protein WG-SP01 with hydrophilic and hydrophobic sites and charge distribution: wherein figure A, B, C is a three-stage block diagram of WG-SP01 (front view a, left view B, top view C). D is a secondary structure diagram of WG-SP01, as above; FIG. E shows the hydrophilicity/hydrophobicity profile of WG-SP01, with the hydrophobicity/non-polarity being light gray areas and the hydrophilicity/polarity being dark gray area portions; FIG. F shows the charge distribution of WG-SP01, with the white short arrow indicating positively charged (primarily arginine and lysine), the white long arrow indicating negatively charged (primarily aspartic acid and glutamic acid), and the white square indicating uncharged;

FIG. 2 is a graph showing the effect of WG-SP01 on mouse spleen acid phosphatase and lactate dehydrogenase activity;

FIG. 3 is a two-dimensional lattice diagram of peripheral blood lymphocyte subpopulations of normal control mice;

FIG. 4 is a two-dimensional lattice diagram of cell subsets of peripheral blood lymphocytes from cyclophosphamide model group mice;

FIG. 5 is a two-dimensional lattice plot of peripheral blood lymphocyte subpopulations of WG-SP01 low dose group mice;

FIG. 6 is a two-dimensional lattice diagram of peripheral blood lymphocyte subpopulations of mice in the WG-SP01 dose group;

FIG. 7 is a two-dimensional lattice plot of peripheral blood lymphocyte subpopulations of WG-SP01 high dose group mice.

Detailed Description

The invention is described in further detail below with reference to the accompanying drawings.

Example 1 has the preparation, screening and identification of the immunomodulatory active protein WG-SP01.

Wheat germ is taken as a raw material, and after enzymolysis by alkaline protease, the protein WG-SP01 with immunoregulatory activity is obtained through centrifugation, ultrafiltration, separation and purification, screening and identification.

The specific method comprises the following steps: wheat germ and 3% NaCl solution are mixed according to the proportion of 1:10, alkaline protein (2000U/g) is added, the addition amount is alkaline protease and the mass ratio of protein in a sample=1:200, enzymolysis is carried out for 0.5h under the condition of pH9.0 at 55 ℃, naOH is used for adjusting pH7.0 to finish the reaction, centrifugation is carried out for 10min at 5000rpm, supernatant fluid is taken, protein is separated by membrane filtration and column chromatography, immunoregulation active protein WG-SP01 is obtained by screening in vitro lymphocyte proliferation experiments, and data information such as amino acid sequence, tertiary structure and the like of the immunoregulation active protein is obtained by means of mass spectrometry and the like.

The amino acid sequence of the immunoregulatory active wheat germ protein WG-SP01 is shown as SEQ ID NO. 1: MASKGGNKGEGPAIGIDLGTTYSCVGVWQHDRVEIVANDQGNRTTPSYVAFTDTERLIGDAAKNQVAMNPTNTVFDAKRLIGRRFSDASVQSDMKMWPFKVVPGAGDKPMIVVTYKGEEKTFSAEEISSMVLTKMREIAEAFLSTTINNAVVTVPAYFNDSQRQATKDAGVIAGLNVMRIINEPTAAAIAYGLDKKATSTGEKNVLIFDLGGGTFDVSILTIEEGIFEVKSTAGDTHLGGEDFDNRMVNHFVQEFKRKNKKDISGNPRALRRLRTACERAKRTLSSTAQTTIEIDSLYEGIDFYATITRARFEELNMDLFRKCMEPVEKCLRDAKMDKTQIHDIVLVGGSTRIPKVQQLLQDFFNGKELCKSINPDEAVAYGAAVQAAILSGEGNQKVQDLLLLDVTPLSLGLETAGGVMTTLIPRNTTIPTKKEQVFSTYSDNQPGVLIQVYEGERTRTKDNNLLGKFELSGIPPAPRGVPQITVTFDIDANGILNVSAEDKTTGQKNKITITNDKGRLSKEEIERMVQEAEKYKSEDEQVRHKVEARNALENYAYNMRNTLRDDKIASKLPADDKKKIEDSIEDAIKWLDGNQLAEADEFEDKMKELESICNPIISKMYQGAGPGGAAGMDEDMPGGGAGTGGGSGAGPKIEEVD. The molecular weight is 71847MW, contains 657 amino acids, the secondary structure comprises alpha helix 17%, beta sheet 27%, random coil 17%, beta corner 21%, and the tertiary structure is shown in figure 1.

Example 2 immunoregulatory Activity assay of protein WG-SP01.

1. Determination of in vitro immunocompetence-mouse spleen lymphocyte proliferation assay

(1) Preparation of spleen cell suspensions

Taking Balb/c mice with 6-8 weeks old, removing necks, killing, soaking in 75% ethanol, placing the mice on an ultra-clean workbench for asepsis, opening the abdomen, taking out the spleen, placing the spleen on a 200-mesh cell sieve above a culture dish containing Hank's liquid, cleaning the spleen with Hank's liquid, squeezing and grinding the mice with a disposable glass syringe core, repeatedly cleaning the mice twice with Hank's liquid, transferring the mice to a centrifuge tube, preparing suspension, centrifuging at 1000rpm for 5-10 min, and discarding supernatant. Taking 6mL of cell lysate which is subjected to high-pressure sterilization or filtration by a 0.22 mu m filter, adding into a centrifuge tube to lyse red blood cells, blowing uniformly, adding Hank's solution after acting for 1-2min, centrifuging at 1000rpm for 5-10 min, and discarding supernatant. Adding Hank's solution, blowing uniformly, centrifuging at 1000rpm for 5min, and discarding the supernatant. Then adding a proper amount of Hank's solution, blowing uniformly, taking a small amount of cell suspension for living cell counting, centrifuging at the rest 1000rpm for 5min, and discarding the supernatant to obtain cell sediment (at the moment, if the sediment is red, the red blood cells are not completely lysed, and then the cells can be lysed again according to the steps).

And adding a proper amount of precooled RPMI-1640 complete culture solution into the obtained cell sediment, blowing to prepare a cell suspension, and counting living cells by using a trypan blue staining method. The method comprises taking a certain volume of cell suspension, adding RPMI-1640 complete culture solution for dilution, and then adding trypan blue solution with a final concentration of 0.04%. The blood cell counting plate counts, and living cells are not stained and dead cells are stained dark blue with trypan blue when viewed under a low power microscope. The cell count formula is shown in (1):

(2) Inoculation culture and MTT activity detection

According to the cell density of the cell suspension obtained by counting, adding precooled RPMI-1640 complete culture solution to prepare the lymphocyte suspension with the cell density of 1.0-2.0X106/mL. mu.L of mouse spleen lymphocyte suspension was added to a 96-well plate. A blank control group and an experimental group are respectively established, and the capacity of the experimental sample to promote the proliferation of spleen lymphocytes is detected. Wherein, the blank control group is added with 100 mu L of complete culture solution, and the experimental groups are added with 100 mu L of sample to be detected. After gentle mixing, the mixture was incubated in an incubator at 37℃with 5% CO2 for 48 hours.

The culture was terminated after 4 hours before the end of the cell culture by adding 10. Mu.l of 5mg/mL MTT solution per well, followed by further culturing for 4 hours. Centrifuge at 3000rpm for 10min, remove liquid in the wells, add 150 μl DMSO per well, shake for 5min, and dissolve the crystals thoroughly. The wavelength of 570nm is selected, the light absorption value of each hole is measured on an ELISA tester, and the result is recorded.

The greater the stimulation index, the greater its immunomodulatory activity. The stimulation index of protein WG-SP01 was measured as described above with different concentrations, and the stimulation index of protein WG-SP01 reached 237% at 5. Mu.g/mL with EC 50=1.2. Mu.g/mL.

Determination of in vivo immunocompetence animal experiments were carried out and evaluated according to the experimental protocols for enhancing immune functions in the technical specification implementation manual for health food inspection and evaluation.

(3) Grouping and administration of laboratory animals

Mice were randomly divided into 6 groups of 12, the specific groupings are shown in table 1. An immunosuppression experimental animal model is established by intraperitoneal injection by adopting Cyclophosphamide (CPA).

Table 1 animal model group (n=12)

Note that: NC, normal control group (Normal control group); CPA, cyclophosphamide group (Cyclophosphamide group).

Table 2 WG-SP01 effect on organ index of mice (n=12)

Note that: * *. is extremely different from the normal group (P < 0.01); * Significant differences compared to normal group (P < 0.05); the difference was extremely significant compared to cyclophosphamide group (P < 0.01); #. is extremely distinct compared to cyclophosphamide group (P < 0.01); the following is the same.

Table 2 shows that CPA has no significant effect on the liver, kidney and heart indices of mice, and that the indices of spleen and thymus are significantly reduced (P < 0.01). The spleen index of mice was affected to some extent by WG-SP01 at each dose compared to CPA, where the difference in spleen index between the high, medium and low doses of WG-SP01 and CPA groups was more pronounced (P < 0.01), but the effect between doses was not pronounced. The spleen index of the mice is increased by WG-SP01 of each dose, and the increasing trend of low dose and high dose is obvious compared with that of CPA group; the results show that the wheat germ globulin has a certain weight increasing effect on immune organ indexes of the immunosuppressed mice, can improve the atrophy state of immune organs of the immunosuppressed mice, and improves the immune function of the mice to a certain extent.

The results of the mouse spleen lactate dehydrogenase and acid phosphatase activity detection show that when the mouse is injected with cyclophosphamide, the spleen acid phosphatase activity and the lactic acid dehydrogenase activity are remarkably reduced (P < 0.01); each dose of WG-SP01 can antagonize immunosuppression caused by cyclophosphamide, has obvious influence on the enzyme activity of mouse spleen acid phosphatase and lactic dehydrogenase in immunosuppression state (P < 0.01), can obviously improve the decline of the enzyme activity of mouse spleen acid phosphatase and lactic dehydrogenase caused by immunosuppression, and tends to return to normal enzyme activity. Experiments show that WG-SP01 can regulate the immune function of the organism in an immunosuppression state, and can improve the activation state of macrophages, thereby achieving the aim of improving the immune function of the organism.

TABLE 3 influence of WG-SP01 on the phagocytosis of neutral Red and iNOS Activity by macrophages in the abdominal cavity of immunosuppressive mice

Table 3 shows that the ability of the macrophages in the abdominal cavity of the cyclophosphamide mice to phagocytose neutral red is significantly lower than that of the normal control group (P < 0.01); WG-SP01 can effectively and significantly improve the ability of macrophages in the abdominal cavity of immunosuppressive mice to phagocytose neutral red (P < 0.01); except for the WG-SP01 low dose group, which was significantly lower than the normal group (P < 0.01), the other treatment groups had no significant difference from the normal group level (P > 0.05). Compared with the normal control group, the activity of the cyclophosphamide group mice abdominal cavity macrophage iNOS is significantly lower than that of the normal control group (P < 0.01); after administration, the vigor of the abdominal cavity macrophage iNOS of each dose group of WG-SP01 is obviously higher than that of cyclophosphamide group (P < 0.01), and compared with the normal group, the vigor of the abdominal cavity macrophage iNOS of each dose group is not significantly different (P > 0.05).

TABLE 4 influence of WG-SP01 on mouse serum IL-2, IL-4, IFN-gamma and TNF-alpha

As can be seen from table 4, the levels of IL-2, IL-4, IFN- γ and TNF- α in the serum of mice were significantly reduced (P < 0.01) compared to the normal control group, indicating that the immunosuppressive model was successful and the immunity of mice was at a low level; compared with the model group, each treatment group can obviously improve the serum cytokine concentration (P < 0.01) of the immunosuppressed mice; experiments show that WG-SP01 has significant recovery effect on immunosuppressive mouse serum cytokines (P < 0.01). The increase in the level of the related immune molecules indicates that the proportion or activity of the lymphocyte subpopulation related to the immune molecules is also obviously increased, thereby promoting the recovery of immune functions.

The lymphocyte CD3+, CD4+, CD8+, and CD25+ cell percentages in the peripheral blood of mice were detected using flow cytometry diabody labeling. After the separated mouse blood mononuclear cells are marked by the specific monoclonal fluorescent antibody, lymphocyte clusters with obvious areas can be obtained on a flow cytometer according to the cell size. Analysis and processing are carried out through CELLQuest software, and the analysis and processing are expressed in a two-dimensional lattice diagram form. The results are shown in FIGS. 3, 4, 5, 6 and 7. The cell numbers of each subpopulation were calculated after analysis of the two-dimensional bitmap of the lymphocyte subpopulation as shown in table 5.

TABLE 5 influence of WG-SP01 on the lymphoid subpopulations of immunosuppressive mice

Lymphocytes are the main cell population constituting the immune system of the body, and can be divided into a plurality of populations with different phenotypes and functions, and when infection occurs or the immune function is changed, the change of each subpopulation of T lymphocytes can often reflect the state of the body very sensitively. As can be seen from table 5, the proportion of cd3+ and cd4+ cells in the peripheral blood of the mice of the CPA immunosuppression model group showed a significant decrease (P < 0.01) compared to the normal group; this is consistent with previous studies and also demonstrates that the CPA immunosuppression model is valid. After WG-SP01 treatment, the proportion of CD3+ and CD4+ T cells in peripheral blood of the immunosuppressed mice is obviously improved, which is obviously higher than that of CPA immunosuppression model group (P < 0.01) and is obviously higher than that of normal group mice (P < 0.01).

The relative state of immune balance in the body is largely maintained by the influence of cd4+ cells and cd8+ cells on each other, and a disorder in the proportion of the two subgroups causes immune dysfunction, so that cd4+/cd8+ represents the balance of overall immune function, and its decrease may reflect a disorder in the immune function of the body. The CD4+/CD8+ ratio of the peripheral blood of the CPA model group mice is found to be significantly lower than the normal group level (p < 0.01) in the experiment; the cd4+/cd8+ ratio of WG-SP01 high, medium, low concentration treatment groups was significantly higher than that of the CPA model group (P < 0.01) and there was no significant difference (P > 0.05) from the normal group.

Cd4+cd25+ regulatory T cells have a very important role in maintaining immune tolerance and immune response homeostasis in the body, and table 5 shows that the proportion of cd4+cd25+ T cells in the peripheral blood of mice in the CPA immunosuppression model group was significantly reduced, with a significant difference (P < 0.01) compared to the normal control group, indicating that the CPA immunosuppression model was successful. After the WG-SP01 is infused, the proportion of CD4+CD25+T cells in peripheral blood of an immunosuppressed mouse is obviously increased, and compared with a CPA immunosuppression model group, the proportion is obviously different (P < 0.01); slightly higher than normal group mice, but without significant differences (P > 0.05). Experiments show that each dose of WG-SP01 has an increasing effect on the proportion of CD4+CD25+T cells in the peripheral blood T cell subset of CPA immunosuppressive mice.

What has been described above is merely some embodiments of the present invention. It will be apparent to those skilled in the art that various modifications and improvements can be made without departing from the spirit of the invention.

Sequence listing

<110> academy of Henan animal husbandry economics

<120> A protein WG-SP01 having immunomodulatory Activity

<130> 20220530

<141> 2022-05-30

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 657

<212> PRT

<213> Artificial

<400> 1

Met Ala Ser Lys Gly Gly Asn Lys Gly Glu Gly Pro Ala Ile Gly Ile

1 5 10 15

Asp Leu Gly Thr Thr Tyr Ser Cys Val Gly Val Trp Gln His Asp Arg

20 25 30

Val Glu Ile Val Ala Asn Asp Gln Gly Asn Arg Thr Thr Pro Ser Tyr

35 40 45

Val Ala Phe Thr Asp Thr Glu Arg Leu Ile Gly Asp Ala Ala Lys Asn

50 55 60

Gln Val Ala Met Asn Pro Thr Asn Thr Val Phe Asp Ala Lys Arg Leu

65 70 75 80

Ile Gly Arg Arg Phe Ser Asp Ala Ser Val Gln Ser Asp Met Lys Met

85 90 95

Trp Pro Phe Lys Val Val Pro Gly Ala Gly Asp Lys Pro Met Ile Val

100 105 110

Val Thr Tyr Lys Gly Glu Glu Lys Thr Phe Ser Ala Glu Glu Ile Ser

115 120 125

Ser Met Val Leu Thr Lys Met Arg Glu Ile Ala Glu Ala Phe Leu Ser

130 135 140

Thr Thr Ile Asn Asn Ala Val Val Thr Val Pro Ala Tyr Phe Asn Asp

145 150 155 160

Ser Gln Arg Gln Ala Thr Lys Asp Ala Gly Val Ile Ala Gly Leu Asn

165 170 175

Val Met Arg Ile Ile Asn Glu Pro Thr Ala Ala Ala Ile Ala Tyr Gly

180 185 190

Leu Asp Lys Lys Ala Thr Ser Thr Gly Glu Lys Asn Val Leu Ile Phe

195 200 205

Asp Leu Gly Gly Gly Thr Phe Asp Val Ser Ile Leu Thr Ile Glu Glu

210 215 220

Gly Ile Phe Glu Val Lys Ser Thr Ala Gly Asp Thr His Leu Gly Gly

225 230 235 240

Glu Asp Phe Asp Asn Arg Met Val Asn His Phe Val Gln Glu Phe Lys

245 250 255

Arg Lys Asn Lys Lys Asp Ile Ser Gly Asn Pro Arg Ala Leu Arg Arg

260 265 270

Leu Arg Thr Ala Cys Glu Arg Ala Lys Arg Thr Leu Ser Ser Thr Ala

275 280 285

Gln Thr Thr Ile Glu Ile Asp Ser Leu Tyr Glu Gly Ile Asp Phe Tyr

290 295 300

Ala Thr Ile Thr Arg Ala Arg Phe Glu Glu Leu Asn Met Asp Leu Phe

305 310 315 320

Arg Lys Cys Met Glu Pro Val Glu Lys Cys Leu Arg Asp Ala Lys Met

325 330 335

Asp Lys Thr Gln Ile His Asp Ile Val Leu Val Gly Gly Ser Thr Arg

340 345 350

Ile Pro Lys Val Gln Gln Leu Leu Gln Asp Phe Phe Asn Gly Lys Glu

355 360 365

Leu Cys Lys Ser Ile Asn Pro Asp Glu Ala Val Ala Tyr Gly Ala Ala

370 375 380

Val Gln Ala Ala Ile Leu Ser Gly Glu Gly Asn Gln Lys Val Gln Asp

385 390 395 400

Leu Leu Leu Leu Asp Val Thr Pro Leu Ser Leu Gly Leu Glu Thr Ala

405 410 415

Gly Gly Val Met Thr Thr Leu Ile Pro Arg Asn Thr Thr Ile Pro Thr

420 425 430

Lys Lys Glu Gln Val Phe Ser Thr Tyr Ser Asp Asn Gln Pro Gly Val

435 440 445

Leu Ile Gln Val Tyr Glu Gly Glu Arg Thr Arg Thr Lys Asp Asn Asn

450 455 460

Leu Leu Gly Lys Phe Glu Leu Ser Gly Ile Pro Pro Ala Pro Arg Gly

465 470 475 480

Val Pro Gln Ile Thr Val Thr Phe Asp Ile Asp Ala Asn Gly Ile Leu

485 490 495

Asn Val Ser Ala Glu Asp Lys Thr Thr Gly Gln Lys Asn Lys Ile Thr

500 505 510

Ile Thr Asn Asp Lys Gly Arg Leu Ser Lys Glu Glu Ile Glu Arg Met

515 520 525

Val Gln Glu Ala Glu Lys Tyr Lys Ser Glu Asp Glu Gln Val Arg His

530 535 540

Lys Val Glu Ala Arg Asn Ala Leu Glu Asn Tyr Ala Tyr Asn Met Arg

545 550 555 560

Asn Thr Leu Arg Asp Asp Lys Ile Ala Ser Lys Leu Pro Ala Asp Asp

565 570 575

Lys Lys Lys Ile Glu Asp Ser Ile Glu Asp Ala Ile Lys Trp Leu Asp

580 585 590

Gly Asn Gln Leu Ala Glu Ala Asp Glu Phe Glu Asp Lys Met Lys Glu

595 600 605

Leu Glu Ser Ile Cys Asn Pro Ile Ile Ser Lys Met Tyr Gln Gly Ala

610 615 620

Gly Pro Gly Gly Ala Ala Gly Met Asp Glu Asp Met Pro Gly Gly Gly

625 630 635 640

Ala Gly Thr Gly Gly Gly Ser Gly Ala Gly Pro Lys Ile Glu Glu Val

645 650 655

Asp

Claims

1. Use of an immunomodulator for improving immunosuppression of mice caused by cyclophosphamide, wherein the immunomodulator uses protein WG-SP01 as an active ingredient in the immunomodulator, and the amino acid sequence of the protein WG-SP01 is shown as SEQ ID NO. 1; the secondary structure of the protein WG-SP01 comprises an alpha helix 17%, a beta sheet 27%, a random coil 17% and a beta corner 21%.