TWI299735B - An immunomodulatory protein cloned from ganoderma microsporum - Google Patents

Description

1299735 IX. Description of the invention: [Technical field of the invention] The present invention relates to an immunomodulatory protein and a preparation method thereof, and particularly to an immunomodulatory protein which is selected from microspores and has a ratio of / cz. Immunomodulatory protein with immunomodulatory efficiency. [Prior Art] The stem belongs to the genus of the fungus (Kindom Fungi), Basidiomycota, Hymenomycetes, Aphyllophorales, Ganodermataceae, and G. oderma. (Alex〇poui〇s et al 1996). At present, there are about 300 species of Ganoderma in the literature, but in the pharmacological and clinical research, only Ganoderma lucidum, G. tsugae, G. sinensis g, and G. sinensis (G. Like,), sessile Ganoderma lucidum (G raz·Listen and other red Ganoderma lucidum and Ganoderma lucidum (GjZ•like, Sakamoto Ganoderma lucidum (10), such as purple Ganoderma lucidum and Ganoderma lucidum (G plus (4)) Species were studied and utilized (Xu, 1993). After 30 years of music theory research, Ganoderma lucidum found that its extract has sedative, analgesic, antitussive, cardiac, liver-protecting (Lin et al 1993), lowering blood pressure (Lee and Rhee) 1990), hypolipidemic (Kabir et al. 1988), cholesterol lowering (K〇m〇da et al 1989), antiallergic (Chen et al 1992), anti-inflammatory (Lin et al. 1993), antiviral (el -Mekkawy et al. 1998), anti-tumor and immunomodulatory functions (Chen et al 2004) and other active ingredients.

In 1971, SaSakl et al. discovered that Ganoderma lucidum (G叩; 7/(10) deuterated polysaccharide has antitumor activity, making the polysaccharide the first active ingredient of Ganoderma lucidum 1299735 (Sasaki et al. 1971). The mechanism of action is not to directly kill or inhibit cancer cells, but to indirectly express its anticancer activity by activating T cells and enhancing the ability of natural killer cells (Lei and Lin 1991) to improve immunity. In addition, for mononuclear Eucalyptus cells can enhance their ability to swallow and promote cytokines such as interleukin (IL-2, IL-4), interferon (IFN-γ) and tumor necrosis factor (TNF-α). Synthesis and release (Lieu et al. 1992); by strengthening natural killer cells and giant stalk cells, directly attacking abnormal tumor cells to achieve anti-cancer and anti-cancer effects. Another active substance of Ganoderma lucidum, immunomodulatory protein (immunomodulatory protein), isolated from G. lucidum mycelium by 曰本 scholar Kino et al. in 1989, named LZ-8 (Ling Zhi-8) (Kinoetal.l989hLZ-8 from 110 amino acids Composition, molecular weight 1 2,420 Da, and a certain degree of similarity to the amino acid sequence and secondary structure of the variable region of the immunoglobulin heavy chain region (31^1^6 〖31·1989). The LZ-8 state is The form of homodimer exists to promote lymphocyte proliferation and to inhibit systemic anaphylaxis reaction and arthusic reaction. Because LZ-8 is not for human red blood cells. Any agglutination reaction (Kino et al. 1989) shows that it has potential for application in human medicine. Therefore, Meiji Group of Japan applied for LZ-8 patents in Japan, Europe and the United States, respectively: LZ- 8 as anti-AIDS drug and nucleic acid sequence patents JP2032026, JP3172184 and JP5068561; LZ-8 protein properties and as an immunosuppressive agent patent EP0288959B1, US5334704. In addition, LZ-8-Leu-Ala-Trp-Asp-Val- Lys- and 1299735-Asn-Leu-Gly-Val-Lys-Pro-Ser_Tyr-Ala-Val- two-part glycoprotein sequences are also protected by patents (US5334704). LZ-8 and lectin (lectin) ) - like It has the ability to agglutinate cells and promote lymphocyte proliferation. Because of the ability of lectins to specifically bind to sugars, they are also called glycoproteins. This combination of specificity of carbohydrates allows it to bind to specific sugar groups on the cell surface, thereby stimulating the ability of cells to elicit a subsequent immune response. Since 1989, Kino found that LZ-8 can stimulate the proliferation of spleen cells in I mice and avoid local and systemic allergic reactions. Later studies have shown that LZ-8 can effectively inhibit the growth of nonobese diabetic (NOD) mice. The development of autoimmune type 1 diabetes (Kino et al. 1990). In addition, LZ-8 can significantly delay rejection time in pancreatic allografts. Compared to other immunomodulatory drugs, CsA (cyclosporin A, a peptide with immunosuppressive effects from fungi) and FK506 (tacrolimus, an antibiotic secreted by soil fungi) are toxic to the pancreas. • The danger of harm, LZ-8 did not find any toxic effects on islets (van der Hem et al. 1995). LZ-8 exhibits immunomodulatory activity regardless of whether it is in vitro or in vivo, but its exact mechanism of action is unclear. Kino et al. found in 1991 that LZ-8 inhibited the production of mouse antibodies, suggesting that LZ-8 inhibits systemic and local allergic reactions by blocking antibody production (Kino et al. 1991). Since then, LZ-8 has been found to regulate intercellular interactions by regulating molecules attached to the cell surface (Miyasaka et al. 1992), and this interaction is lacking in patients with autoimmune diseases. 1299735 to Chiba immunoregulatory protein LZ-8 confirmed that the simple peptide has immunomodulatory and nodular functions, while other studies have also found that the molecular weight of the mycelium from Ganoderma lucidum (G· Μ (10)) is about 13kD. #immunomodulatory protein 四 (4) such as correcting immunomodulatory protein-gts), exactly the same sequence as Lz_8. FIP-gh can promote human pedpherai lymphocytes and mouse spleen cell proliferation, and the highest proliferative effect on human peripheral lymphocytes can be achieved at a concentration of 5 μ§/ιη1. In addition to this, immunomodulatory proteins with a molecular weight of approximately 13 kD can be purified from some non-Ganoderma lucidum mushrooms, such as FIP-/ve (KoetaL 1995) of A. chinensis and FIP-vvo of Grasses ( Hsu et al. 1997). LZ-8, FIP-/ve and FIP-vvo both contain similarities to the variable regions of immunoglobulin heavy chains. FIP-/ve and FIP-vvo are similar in sequence and structure to lz-8, and have similar physiological activities. The health effects of medicinal fungi are well known, and the active ingredients are gradually purified and isolated. However, most of the active ingredients are currently known as polysaccharides or tri-type compounds, but these active substances cannot be purified by other active substances. Such as peptide polysaccharides and glycoproteins and other interference, so there are many doubts about the study of physiological mechanisms. In addition, these active substances are not cell wall components or secondary metabolites, and it is difficult to mass produce them through heterogeneous expression systems. SUMMARY OF THE INVENTION The object of the present invention is to provide an immunomodulatory protein gene selected from microspore ganoderma lucidum, whose gene sequence and the translated protein sequence are different from the patented LZ-8, and have more efficiency than LZ_8. Immunomodulatory ability. 1299735. An immunomodulatory protein gene according to the object of the present invention is selected from the genus Ganoderma lucidum Gimoderma m/croworwm having the amino acid sequence of SEQ ID NO 2 and SEQ ID NO 3 of the Sequence Listing. An immunomodulatory protein of the present invention having the above immunomodulatory protein gene has a molecular weight of 15,863.79 Da. The immunomodulatory protein selected from the above-mentioned microspore ganoderma lucidum has a protein sequence which is different from the patent LZ-8, and thus has a more efficient immunomodulatory ability than LZ-8. Hereinafter, the applicability and functional characteristics of the present invention will be described by way of preferred embodiments only: [Embodiment] Materials For the strains, plastids and primers used in the present invention, please refer to Table 1, Table 2 and Table 2. The microspores Ganoderma lucidum RSH 0821 used in the present invention is stored at 25QC in a slant medium of Potato dextrose agar (PDA, Difco, Detroit, MI) of Difco Corporation of Detroit, Michigan, USA. The heterologous expression system was an expression kit of a sterol yeast yeast from Invitrogen, Inc. (Invitrogen, Carlsbad, CA, US). Escherichia coli five co/z' JM109 is the host cell for the meristematic operation and plastid preservation of the present invention, and the general culture is a solid plate medium (LB, Luria-) using Alpha Bioscience (Baltimore), Baltimore, Maryland, USA. Bertani agar) or liquid medium (LB broth), the culture temperature is 37 ° C; liquid culture needs to be shaken at 250 rpm 1299735. Long-term storage of LB broth table containing 25% glycerol at -80 °C ^ _ strain characteristics source

Ganoderma spp. G. microsporum RSH 0821 G. tsugae RSH 1109 E. coli JM109

Heterokaryote, gmi gene source Heterokaryote, /z-S gene source This laboratory This laboratory Φ

Rosetta origami B (DE3) plastid structure and storage Mdl, sStratagene hsdRll, gyrA96, relAl, ihiA (lac-proAB), (US F1 [traD36, proAB\ laclq, lacZAU\5] Lavule) host expression F·, οττφΓ, m/), ga/, Novagen (beautiful dcm, lacYl, aphC, gor5 22: :Tnl 0, trxB, California sacred site pRARE(CamR} KanR, TetR) 牙哥) P. pastoris KM71 host expression Muts, ZnX,

Invitrogen (American State 17 Carls Bay) Table, plastid characteristics source

E. coli yT&A pGEM-T pGEX-4T-l pGEXL P. pastoris pPICZa A pPLZ pPGMI TA selection of lean vector, ZacZ, Ampr TA selection vector, /acZ, Ampr expression vector, tac promoter (promoter) Amp1" , GST-tag pGEX-4T-l and /z- at the N terminus (5 genes are born in BamHI/EcoRI position 1

Yeastern (Taipei, Taiwan) Promega (Madison, Wisconsin, USA) Amersham Pharmacia (Apse, Sweden) This experimental performance vector, promoter, Zec/, a-factor message peptide, c-terminus with c- Wyc epitope and His-tag pPICZaA and /z-<S genes are produced in EcoRl/Xbai position 1 pPICZaA and g/m·genes are born in jEcoRUJCba'

Invitrogen (Carls Bay, California, USA) This experiment 10 C;. 1299735 Table, primer primer sequence (5'-3') Reference source This experiment This experiment This experiment This experiment This experiment This experiment is Kilstrup and Kristiansen, 2000 Kilstrup and Kristiansen, 2000 Kilstrup and Kristiansen, 2000

LZ8-F LZ8-R LZ8-BF LZ8-ER LZ8-EF LZ8-XR 3,GW-F MKP22 MKP23 MKP24 0821GW-R1 0821GW-R2 GMI-XR

TCCGACACTGCCTTGATCTTCAGG

GTTCCACTGGGCGATGATGAAGTC

GGATCCATGTCCGACACTGCCT

GAATTCCTAGTTCCACTGGGCGA

GAATTCATGTCCGACACTGCC

TCTAGATAGTTCCACTGGGCG

CGTTCGACTACACCCCGAACTGGGGC

GCGCTGCAGGCATGCGAGCTCCCAAGCTTGATCG

AATTCGATCAAGCTTGGGAGCTCGCATGCCTGCAGCGC

GCGCTGCAGGCATGCGAGCTG

GAATTCGATGGCCCGCCGAGC

CCCTTCTAGTTCCACTGGGCAAC

TCTAGATAGTTCCACTGGGCA

The restriction enzyme cleavage point in the primer is indicated in bold, 10,000 amH I : GGATCC, EcoR I : GAATTC ^ Hindm : AAGCTT ^ Xba I : TCTAGA microspores Ganoderma lucidum immunoregulatory protein gene selection microspore ganoderma lucidum chromosome DN A extraction: Refer to the method of AI-Samarrai and Schmid (Al-Samarrai and Schmid 2000). The microspores of Ganoderma lucidum mycelium were placed in PDB liquid medium by inoculation, and cultured at 25 ° C for one to several weeks. The mycelium was collected by suction filtration and washed several times with distilled water to remove water. It is ground to a powdery state with liquid nitrogen and stored in a refrigerator at -20 °C.

11 1299735 Polymerase chain reaction (PCR) analysis: Using the chromosomal DNA of microspore ganoderma lucidum as a template, LZ8-F/LZ8-R is the primer (Table 2) 'PCR amplification' and its adhesion temperature Ta (annealing temperature) is 48 ~6 (TC, extension time te (el〇ngati〇) n time) is 30 sec· (Table 4), the obtained nucleic acid fragment is purified, and the plastid yT&A is transferred by ΤΑ ligation. After the formation of the five. eo" JM109 (Table 1), perform nucleic acid sequencing analysis. _ Genome Walking (Reference to Kilstrup and Kristiansen (Kilstrup and Kristiansen 2000), using restriction enzymes to cut chromosomal DNA into A small fragment, and a adaptor (adaptor) with a known sequence at both ends of the fragment is used as a template DNA', and then a PCR-amplification reaction is performed using a gene-specific primer and a specific primer on the adaptor, and finally a large number of Target gene fragment. Results · In the PCR analysis, the partial sequence of the new gene of G·spring m/cro orwm immunoregulatory protein was approximately 330 bp (Fig. 1) using LZ-8 primer. The sequence was known for this new gene. Another design The 3' genome walking of 3' GW-F can obtain the three-terminal unknown sequence of this new gene (Fig. 1). In order to select the entire gene of the new immune prion protein once, the sequence is known at the three ends. Two specific primers 0821GW-R1 and 0821 GW-R2 (Table 3) were designed for 5' genome walking, and a gene fragment of about 450 bp in length was obtained (Fig. A). The three-terminal sequence was selected before the merger. Gene fragment, G. immunoregulatory new gene total length 666 bp and named gwz· (third B map). 12 < S)- 1299735 Table 4 DNAa template primer b PCR strip; ~ Ta ( °C ) c Te ( min. ) d 3 ▼ partial genomic step 0821 / ^ coR I ? 0821 / i7zndHI 3rGW-R / MKP24 54, 56, 58, 2 2 5 'end complete genome step 0821 / £coR I , 1st PCR 0821GW -R1/MKP24 60 3 0821/^/^(1111 2nd PCR 0821GW-R2/MKP24 56 2 a : Chromosomal DNA cut by restriction enzymes b: Please refer to Table 3. • cTa: annealing temperature 〇d te : extension time (elongation time). Ganoderma lucidum immunoregulatory protein LZ-8 is a heterologous expression of pGEXL expression vector: It is obtained by PCR analysis and stored in yT&A/ζ-δ gene as template, with restriction enzymes mHIII and EmRIV, respectively. c) The cleavage LZ8-BF and LZ8-ER are bow 1 and PCR amplification (Ta 2 60 〇C te 2 _ 30 sec·), the obtained fragment is stored in yT&A and is inserted into PGEX- according to general operation. The relative position of 4T-1 can be obtained as the expression vector pGEXL. It is then converted to five co/z'Rosetta-gamiB (RGB) for performance. jE. Performance of fusion protein:

1% of the overnight cultured inoculum was inoculated to 900 mi of LB liquid medium containing 1 〇〇 gg/ml ampicillin (amibillin) at 37. 〇 〇 培养 培养 培养 培养 〇〇 〇〇 D6〇〇 = 0.6, with 0.5 mM iPTG (isopropyl_D-thiogalactopyranoside isopropyl-d-thiogalactopyranoside) at 30C induced spacer. Centrifuge (3000^^10]^^ 4. 〇) harvest 隹 (g. }. 13 1299735 cells, suspended in 5 ml phosphate buffer PBS (140m M NaCl, 2.7 mM KC1, 10 mM Na2HP04, 1.8 mM After KH2P04, pH 7·3), ultrasonic shock was performed. Before the centrifugation (14000 g, 20 min., 4 °C), Triton X-100 was added to the final concentration of 1% to increase the solubility of the fusion protein. The supernatant was purified by Glutathione Sepharose 4B column (Glutathione Sepharose-4B affinity chromatography column), and the obtained fusion protein was prepared for rabbit polyclonal antibody. Ganoderma lucidum immunoregulatory protein expressed pPLZ and pPGMI Construction of the expression vector: and the primers LZ8-EF/LZ8-XR and LZ8-EF/GMI-XR derived from the restriction enzyme cleavage site of five coR II, respectively. c) After amplification by PCR amplification, the resulting fragment is stored in pGEM-T and inserted into the relative position of pPICZaA according to the general meristem operation. The expression vector pPLZ and pPGMI can be obtained. The expression vector can make the N-terminus of the target protein a. A_factor signal pepetide, C terminated with c-myc epitope (e Pitope) and His-tag. While the recombinant protein is sent extracellularly, the message peptide will be excised into reGMI (sixth image). P· Electroporation transformation method: Refer to Invitrogen's corpse/c/ni expression set The expression kit instructions and the transformation method published by Wu and Letchworth (Wu and Letchworth 2004), the corpse KM71 in 200 mL YPD (Yast Peptone Dextrose), liquid culture 14 1299735, to 30 Incubate at 0 °C to 0D_ (absorbance value) about ΐ·〇-2·0 (1 unit OD600 is approximately 5 X 107 cells/mL), centrifuge after centrifugation, and suspend in 100-200 mL of pretreatment buffer (1 mM CH3COOLi (lithium acetate), 10 mM DTT (dithiothreitol dithiothreitol), 0.6 M sorbitol (sorbitol), 10 mM Tris-HCl (trishydroxymethylaminodecane-salt) pH 7.5), After standing at room temperature for 30 minutes, the cells were centrifuged, and the cells were washed three times with 1 mL of ice 1 M sorbitol and suspended in 1 mL of ice 1 Μ _ sorbitol (~1010 cells/mL), which was electroporation. ) Competent cells used. The plastids were extracted and the plastids were cut into linear lines by SacI. After purification, l//g DNA (10//L) was mixed with 80//L of competent cells and transferred to an electroporation glass tube (cuvette, 0.2 cm, BTX) ice bath for 5 minutes, electroporation with ECM 630 electroporation system (BTX, San Diego, CA), set at 1.5 kV, 25 μF, 200 Ω. Immediately after electroporation, add 1 mL of ice to YPDS liquid challenge medium (1% yeast extract); 2% peptone (gland); 2% dextrose (glucose) and 1M sorbitol (sorbitol) at 30 ° After standing for 1-2 hours, the bacterial solution was applied to a YPDS plate medium containing 1 〇〇pg/ml zeocin (antibiotic), and cultured at 30 ° C; generally, the higher resistance of the transformed strain was inserted. The greater the number of performance vectors, the higher the performance of recombinant proteins (Baneyx 2004). P· Performance of the fusion protein: The transgenic strain is BMGY liquid medium (l% yeast extract) containing 1〇〇gg/mlzeocin; 2% peptone (gland); pH 6

15 < S 1299735 potassium phosphate (potassium citrate) 100 mM; 1.34% ΥΝΒ; 4χ 10-5% biotin (auxin) and l% glycerol (glycerol)) after 30 times of culture and activation at 30 ° C, will reach static The sterile phase of the inoculum was connected to 500 ml BMGY to give a final OD_ of 0.1. After 24 hours of culture, the medium was replaced with 100 ml of BMMY liquid medium (1% yeast extract); 2% peptone (脒); pH 6 of potassium phosphate 100 mM; 1.34% YNB; 4χ 1 (T5% biotin (auxin) 0 and 0.5% MeOH (sterol)) induced fusion protein expression, followed by addition of sterol every 24 hours to a final concentration of 0.5% for a total of two consecutive days of induction, and the extracellular supernatant was collected. As shown in the fourth figure, the recombinant protein reGMI was induced by 0.5% sterol, and the protein concentration increased with the number of induction days. P· Fusion of the fusion protein: wash buffer with ten times colloidal volume pH 7.4 , 50 mM NaH2P04 (sodium dihydrogen phosphate), 300 mM NaCl (sodium chloride), 10 mM imidazole (isopyrazole)) washed Ni-NTA (nickel-nitrilotriacetic acid) tube column, recombinant protein reLZ-8 And reGMI can be completely adsorbed in the column. After passing through the sample, the non-specifically adsorbed heteroprotein is washed away with a ten-fold colloidal volume of wash buffer; finally, the fusion protein is washed down with a phosphate buffer containing different imidazole concentrations. , respectively: reLZ-8 at 40~1 00 mM ; reGMI is between 1 〇〇 and 250 mM (figure 5). The purified fusion protein is dialyzed against PBS phosphate buffer and stored in solution state or in dry powder at 4 C. Although households are similar (10) Except for the target protein, the extracellular supernatant has almost no other heterologous proteins, but the purification of the pigment in the extracellular fluid can be removed only by the purification of the affinity column, and the interference of the recombinant protein to reduce the immunological activity is reduced 16 < S > 1299735 Factor. 特性 Characterization of fusion proteins Analysis of molecular weight and _ group modification: Regardless of the recombinant proteins reLZ-8 and reGMI, the individual theoretical molecular weights can be inferred from known sequences, but the molecular weights of these recombinant proteins on electrophoretic films Both are larger than the theoretical molecular weight. To obtain a more accurate molecular weight, refer to reGMI for MALDI-TOF ( Matrix Assisted Laser

Desorption /Ionization- Time Of Flight, matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of mass spectrometry results are shown in Figure 7. The measured value of reGMI is 15863.79 Da, which is almost the same as the theoretical value of 15847.47 Da, indicating that SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) overestimates the molecular weight of reGMI under the Tris buffer system. In addition, it can be inferred from the MALDI-TOF results that the modification of reGMI is not performed. ♦ Homologous dimer analysis: The original LZ-8 is in the form of a homologous duplex (Tanaka, i989), and the physiological activity of the same Flp-gb with the amino acid sequence and LZ-8 can form homology. The twins have an absolute relationship (Lin, 1997). For the initial judgment, whether the recombinant protein reGMI is present in the original state or in the form of a homologous duplex, the protein solution is chemically cross-linked with different concentrations of glutaraldehyde (cr〇sslinking). ). The principle is that the glutenic acid can make the lySine (ionic acid) on the surface of the protein form a covalent bond with each other through the amine group, while the different proteins with similar spatial positions 17 < S > 1299735 have the opportunity to Combined with fixation. The experimental results are shown in the eighth figure. Under the action of 0.01% glutaraldehyde, the signal of the reGMI homologue begins to appear. As the concentration of glutaraldehyde increases, the proportion of homologous dimers gradually increases. reGMI exists in the form of a homologous duplex in its original state. Protein Conformation Analysis: As indicated by the box in Figure 6, the two-stage amino acid sequence of the LZ-8 patent is: (1) -Leu-Ala-Trp-Asp-Val-Lys- ( LAWDVK ) And (2) -Asn-Leu-Gly-Val-Lys-Pro-Ser-Tyr-Ala-Val-(NLGVKPSYAV); and the GMI from G. microsporum and the amino acid sequence of the same segment of the LZ-8 patent pending (1) -Leu-Ala-Trp-Asn-Val-Lys-(LAWNVK) and (2) -Asp-Leu-Gly-Val-Arg-Pro-Ser-Tyr-Ala-Val-(DLGVRPSYAV). GMI differs significantly from the proprietary amino acid sequence of LZ-8, and differences in these amino acids may result in changes in protein conformation, which in turn cause differences in physiological activity between each other. In order to investigate the difference in configuration between reGMI and reLZ-8, Western hybridization analysis showed that reGMI could be recognized by LZ-8 antibody but the signal was weak (ninth). It is shown that reGMI does have a similar configuration to reLZ-8 'but because of the difference in sequence, reGMI is structurally different from reLZ-8, and this difference may affect the activity of immunomodulation. Therefore, the following immunomodulatory activity test was conducted to understand the effect of the difference in the amine 18 1299735 base acid on the immunomodulatory activity of both. Immunomodulatory activity assay The purified reGMI was assayed for immunomodulatory activity using dendritic cells as follows: Bone marrow cells from 5 to 6 weeks old BALB/c mice were taken to contain 10% FCS (calf serum) The RPMI-1640 medium was cultured; the cells were differentiated by IL-4 and GMCSF (granulocyte/macrophage colony stimulating factor) on the second day; the cells were replaced on the fourth day. The plate was further cultured to remove macrophages (Macrophage); on the sixth day, immature dentritic cells were added with different concentrations of protein samples, and after 20 hours, the supernatant was collected for IL-12 ELISA assay. (ELISA, Enzyme-linked immunosobent assay enzyme binding immunosorbent assay). The purified reGMI is immunomodulated by macrophages and T cells. The activity is as follows: The giant cell line J774A.1 and the human T cell line Jurkat cells (human lymphoblasts) are each contained in RPMI-1640. The medium of %FCS was cultured, and protein samples of different concentrations were added. After six hours, the supernatant was collected for ELISA determination of TNF-α and IL-2, respectively. Results · To exclude the possibility of interference with the P· paWorzi cell wall component (Muller et al. 1997), the P. KM71 transformant of pPICZaA was subjected to the same conditions for expression, purification and dialysis without expression genes.

19 < S 1299735 vector (vector) is the negative control group; LPS is the positive control group (tenth to twelfth), regardless of BALB/c mouse bone marrow dendritic cells, macrophage cell line J447A.1 or Jurkat cells Does not trigger an immune response. The purified reLZ-8 and reGMI were administered to BALB/c mice bone marrow dendritic cells at four recombinant protein concentrations of 0, 0.625, 1.25 and 2.5 pg/ml. cell

Secretion of IL-12 p40, in which reGMI stimulates dendritic cells to secrete IL-12p40 at a dose of 2.5 pg/ml six times the same concentration as reLZ-8 (the tenth figure) IL-12p40 is one of IL-12 Increased expression of IL-12p40 means that IL-12 expression may also increase, while IL-12 is a key cytokine that dendrites initiate and even maintain TH1 immune cell population. The presence of IL-12 also inhibits TH2 cell growth and differentiation. (Park and Scott 2001). Ganoderma lucidum immunoregulatory proteins can also act directly on T cells. For example, in Figure 12, 10 pg/ml of reLZ-8 and reGMI can be administered to human butyl cell line Jurkat cells, all of which can activate cells. Secretion of IL-2 (white matter). Luke promotes the differentiation and differentiation of undifferentiated T cells. In addition, IL-2 is also one of the cytokines secreted by τΗΐ cells. TNF-α (tumor necrosis factor) is triggered by giant sputum cells. One of the cytokines of inflammatory response is also an indicator for judging whether or not megaloblastic cells are activated. Figure 11 shows that reLZ-8 and reGMl can activate mouse macrophage cell line j774a 1 at 5 〇 gg/ml. 1 secretion of TNF-a 〇 is known by the above immunological test, The immunoregulatory protein reGMI of the present invention not only has the ability to promote activation of various cells, but also has higher immunological efficiency than reLz ^. < S &gt; - 20 1299735 The above describes the feasibility of the present invention and the various embodiments. The results of the present invention are only the preferred embodiments of the present invention and are not to be construed as limiting the scope of the invention. The specific scope of the invention is defined by the scope of the invention.

21 &lt; S &gt; 1299735 [Simplified Schematic] The first figure shows the results of PCR amplification of G· and G· with LZ-8 primer (LZ8-F/LZ8-R); The electrophoresis result of the amplified fragment, the first B picture is the sequencing result of the amplified fragment; the arrow in the first A picture shows the used primer, wherein 3'GW-F is used for the 3' genome walking 〇 the second picture G · 3' genome walking results of the immunomodulatory protein gene; wherein the second A picture is the sequencing result of the PCR amplified fragment of the template DNA by the 0'GW-F/MKP24 and the like; and the second B picture is the expansion The electrophoresis results are increased; the arrows in the second A diagram show the primers used, and the 0821 genome-free 〇3 map is the 5' genome walking 〇3 map G· mkro printed orz/m immunomodulatory protein gene 5' The genome walking result; after the first PCR amplification with 5, GW_R1/MKP24, the second #PCR amplification was performed with 5'GW-R2/MKP24; the third A picture is the sequencing of the amplified fragment. The result is also the G. immunoregulatory protein gene g ship·full sequence; the third B picture is the electrophoresis result after amplification; the third A picture shows the LZ-8 order The patent section. The fourth panel is a heterologous expression of G. microsporum immunomodulatory protein GMI with ?lCZaA/Pichia pastoris KM71. The fifth picture shows the results of affinity column purification of recombinant protein reLZ-8 and reGMI with different concentrations of isopyrazole flow washing solution; the fifth A picture is reLZ-8; the fifth B picture is reGMI. 22 1299735 The sixth figure is the patented segment of the LZ-8 and GMI recombinant protein reLZ-8, the amino acid sequence of reGMI where the box is not the LZ-8 sequence. The seventh graph is the MALDI-TOF analysis result of reGMI. The eighth figure is a cross-linking diagram of reGMI with different concentrations of glutaraldehyde. The ninth figure shows the results of Western heterozygous reactions with equal amounts of reLZ-8 and reGMI. The tenth figure is the result of reGMI promoting the secretion of IL-12p40 by BALB/c mouse bone marrow dendritic cell line. P11 shows that reGMI promotes the secretion of TNF-α by mouse macrophage cell line J774A.1. Figure 12 shows the results of reGMI promoting the secretion of IL-2 by human T cell line Jurkat cells. [Main component symbol description] None

23 1299735 Sequence Listing &lt;110&gt; Wobate Technology Co., Ltd. /-World Bio-Tech Alliance Corp. &lt;120&gt; Immunoregulatory protein selected from microspore ganoderma lucidum &lt;160 3

&lt;210&gt; 1 &lt;211&gt;666 &lt;212>DNA &lt;213&gt; Ganoderma microsporum

&Lt; 400> 1 cttccgcggc aatcccatac agcttatccg tactgcgctg agcattatgt ttcgactaca ttcccgacgg ctcggcgtgc gaatacaact cccgacaccg cccagacggc cagcgc 666 aggttgtcgc gcgatgttga gcatggcata caggcatgcg ccgacactgc ctccaaactg tcctgactga gcccctcata ccggctacgg gcaacaattt ctgctcggcg agctcccaag cttgatcttc gggccgcggg caaggcgtac cgcggtcgag catcgcggac cattgttgcc ggccatcgaa gggccatcga cacgtgtcca agctccgcaa cttgatcgaa acgctcgcct cgtcccagca acgtaccgcg agcgacggct acgaacacga cagtggaact ggcgctcggc aggcgccata gggtccttgg ttcgatcaag attcacgaac tctacgccaa cttacaccca ttccctgacc ggaatgtgaa gctttatcga tcgtcgtttc cgcagaaaat tccaagtgta agaagggagg cttgggagct ctgcagaacc tggacgcgtc acgccctcca tctgccccgc gcagctcgcg caccgtcacc cggaaaggac caacttcctc cgtcattgac agtgatcgcc cgcatgcctg 60 120 180 240 300 360 420 480 540 600 660

&lt;210&gt;2 &lt;211&gt;6 &lt;212&gt; PRT &lt;213&gt; Ganoderma microsporum &lt;400>2

Leu Ala Trp Asn Val Lys 1 5 1299735

&lt;210>3 &lt;211&gt;10 &lt;212&gt; PRT &lt;213&gt; Ganoderma microsporum &lt;400&gt;3

Asp Leu Gly Val Arg Pro Ser Tyr Ala Val 1 5 10

Claims (1)

1299735 Announcement 10. The scope of patent application: • 1. An immunomodulatory protein selected from microspores of Ganoderma lucidum, whose amino acid sequence is deduced by the nucleotide sequence of SEQ ID NO 1 of the Sequence Listing. 2. For example, the immunomodulatory protein of the first application of the patent scope is selected from the small scorpion Ganoderma lucidum RSH 0821 〇# 3. The immunomodulatory protein of the first application of the patent scope, the molecular weight of the MALDI-TOF analysis is 15863.79 Da . 4. The immunomodulatory protein of claim 1 is in the form of a homologous duplex. 5. An immunomodulatory protein according to claim 1 of the patent scope, comprising the amino acid sequence of SEQ ID NO 2 and SEQ ID NO 3 of the Sequence Listing, J. Φ 6. A nucleotide sequence as shown in SEQ ID NO 1 of the Sequence Listing. hX3S^ -- 1