CN103073632B - There is infection and potential new cytokine LYG1 of anti-tumor activity and application thereof - Google Patents

Abstract

The present invention provides a kind of and has infection and potential new cytokine LYG1 of anti-tumor activity and application thereof, the sequence of described LYG1 (i.e. ripe potential new cytokine LYG1 with function) is the aminoacid sequence shown in SEQ ID NO:1, described application is LYG1 in preparation for preventing and/or treat the application in the medicine of immune correlated disease and test kit, the medicine of described treatment immune correlated disease is antitumor drug, anti-inflammatory medicaments, anti-infectious disease, treatment anaphylactic disease and/or the medicine for the treatment of autoimmune disease, illustrate that potential new cytokine LYG1 is in tumor, infectious disease, inflammation, anaphylactic disease and autoimmune disease aspect may play a significant role, there is potential clinical value.

Description

There is infection and potential new cytokine LYG1 of anti-tumor activity and application thereof

Technical field

The present invention relates to a kind of new Human cytokine and application thereof, particularly relate to a kind of potential new cytokine LYG1 application at aspects such as anti-inflammatory, anti-infectious disease, antitumor and autoimmune diseases with infection and anti-tumor activity.

Background technology

Human immune system goes through the evolution of 3,000,000,000 years, forms complicated and fine regulator control system, has vital effect at body.It externally can defend the invasion of pathogenic microorganism, the most then can remove the cell of aging, pathological changes and death, and in maintaining, environment stablizes.Immune system completes above-mentioned functions by innate immune response and adaptive immune response.As the exchange language between immunocyte, cytokine is respectively provided with important regulating and controlling effect to innate immune response and adaptive immune response.

Cytokine is having regulating cell Growth and Differentiation, regulation immunologic function and physiological reaction and participating in the small protein of pathological reaction by the various emiocytosis of body, plays a role by being combined with receptor.Cytokine mainly includes interleukin (Interleukin, IL), colony stimulating factor (Colony-StimulatingFactor, CSF), interferon (Interferon, IFN), tumor necrosis factor (Tumor-NecrosisFactor, TNF) etc., it is the exchange language between immunocyte, innate immune response and adaptive immune response are respectively provided with important regulating and controlling effect.

CD4+T cell plays pivotal role in adaptive immune response, and it can produce antibody by Help B Cells, assists CD8+T cell killing target cell.Cytokine according to secretion and the difference of function, CD4+T cell can be divided into Th1, Th2, Th17 and Treg cell, and newfound Th9 and Th22 cell etc..Cytokine has pivotal role in differentiation, activation and the Function of Th cell.First, cytokine can determine the differentiation direction of initial CD4+T cell, if IL-12, IL-4 are to determine cytokine necessary to Th1, Th2 differentiation respectively.Secondly, cytokine has regulating and controlling effect to differentiated Th cell, IL-12 Yu IL-18 can work in coordination with promotion Th1 emiocytosis IFN-γ in the way of TCR non-dependent.Additionally, cytokine or the major way of Th cells play effect.Th1 and Th2 cell is two classes very important CD4+T cell subsets, and both balances are immunoregulatory importances.Th1 cell Major Secretory IFN-γ, TNF-α etc., in antiviral, the infection of anti-intra-cellular pathogens, antitumor, delayed hypersensitivity, play critical function by cellullar immunologic response, and participate in the generation of the autoimmune disease such as rheumatoid arthritis, diabetes, development；Th2 cell Major Secretory IL-4, IL-5, IL-13 etc., participate in anti-parasitic-infectious and anaphylaxis, plays an important role in terms of the tolerance of xenograft and pregnancy period fetus simultaneously.The further investigation differentiation of Th1, Th2 cell, physiology and pathologic function and the regulatory mechanism balanced between the two have important theory significance and potential using value.

At present, utilize recombinant cytokine that technique for gene engineering produces, recombinant soluble receptor, neutrality antibody to receive good efficacy at aspects such as treatment tumor, hematopoietic disorders, autoimmune diseasees, become a new generation's medicine.Recombinant cytokine has a lot of superior part as medicine, as: cytokine is human body self component, and the physiological process of scalable body and raising immunologic function, very low dosage can play a role, thus evident in efficacy, it has also become the indispensable treatment means of some difficult and complicated illness.The Cytokines Drug that approved produces at present includes IFN-α, β, γ, Epo, GM-CSF, G-CSF, IL-2 etc..According to incompletely statistics, the most at least have 26 and enter clinical research by genomic drug, including new recombinant cytokine, recombinant soluble receptor and neutrality antibody etc..Meanwhile, cytokine or cell surface receptor detection are also the indexs judging body's immunity and immune cell differentiation, have important value at the aspect such as the diagnosis of numerous disease, course of disease observation, Outcome measure and cytokine therapy monitoring.Such as, IFN-γ and recombiant protein thereof have been used for treat anaphylactic disease, treat the such as autoimmune disease such as rheumatoid arthritis and systemic sclerosis, treatment bacterial fungus catch, treat viral infectious diseases, anti-parasitic-infectious, anti-fibrosis effect and treatment tumor.

Summary of the invention

Therefore, it is an object of the invention to provide a kind of potential new cytokine LYG1 and the application at aspects such as anti-inflammatory, anti-infectious disease, antitumor and autoimmune diseases thereof.

For above-mentioned purpose, technical scheme is as follows:

On the one hand, the present invention provides a kind of potential new cytokine LYG1 (the i.e. ripe LYG1 with function) for preventing and/or treat immune correlated disease, and the sequence of described LYG1 is the aminoacid sequence shown in SEQIDNO:1.

Preferably, described potential new cytokine LYG1 is Human cytokine LYG1 or mouse cytokine LYG1.

Wherein, the precursor sequence of described LYG1 is the aminoacid sequence that N end does not excises signal peptide, and the sequence of described signal peptide is the aminoacid sequence shown in SEQIDNO:2.

On the other hand, the present invention provides the antibody of a kind of anti-above-mentioned LYG1.

Another further aspect, the present invention provides potential new cytokine LYG1 described above or antibody described above or the nucleotide sequence encoding LYG1 described above or the carrier of the nucleotide sequence containing coding LYG1 described above or by the vector introduction of the nucleotide sequence of the LYG1 described above containing coding or be transfected into recombiant protein that HOST ORGANISMS produces in preparation for preventing and/or treating the application in the medicine of immune correlated disease.

Preferably, the medicine of described treatment immune correlated disease is antitumor drug, anti-inflammatory medicaments, anti-infectious disease, treatment anaphylactic disease and/or the medicine for the treatment of autoimmune disease.

Preferably, described tumor is by promoting Th1 cell to break up, promoting cytokine IFN-γ secretion and/or suppress Complement Regulatory Protein CD46, CD55 and CD59 to express the tumor suppressing and/or treating.

It is further preferable that described tumor is melanoma or the tumor sensitive to immunization therapy.

Preferably, described inflammation, infectious disease, anaphylactic disease and autoimmune disease are inflammation, infectious disease, anaphylactic disease and the autoimmune disease by promoting Th1 cell to break up, to promote cytokine IFN-γ secretion to suppress and/or treat.

It is further preferable that described inflammation is inflammatory bowel.

Another aspect, the present invention provides potential new cytokine LYG1 described above or antibody described above or the nucleotide sequence encoding LYG1 described above or the carrier of the nucleotide sequence containing coding LYG1 described above or by the vector introduction of the nucleotide sequence of the LYG1 described above containing coding or be transfected into recombiant protein that HOST ORGANISMS produces in preparation application in the medicine promoting the differentiation of Th1 cell, promoting cytokine IFN-γ secretion and/or suppression Complement Regulatory Protein CD46, CD55 and CD59 to express.

Another further aspect, the present invention provides a kind of test kit for diagnosing immune correlated disease, described test kit comprises antibody described above and/or for the DNA of the LYG1 nucleotide sequence described above of composite coding in PCR and/or the PCR primer of its cDNA chain, the detection in the detection by quantitative of LYG1 albumen, relevant disease of the described antibody and auxiliary diagnosis；Described primer is for the detection by quantitative of LYG1 gene expression.

Another aspect, the present invention provides a kind of mentioned reagent box preparing the application in the medicine diagnosing immune correlated disease.

Preferably, described immune correlated disease is tumor, inflammation, infectious disease, anaphylactic disease and/or autoimmune disease.

It is further preferable that described immune correlated disease is tumor or inflammation.

On the other hand, a kind of pharmaceutical composition for preventing and/or treat immune correlated disease of the present invention, described pharmaceutical composition includes potential new cytokine LYG1 described above or antibody described above or the nucleotide sequence encoding LYG1 described above or the carrier of the nucleotide sequence containing coding LYG1 described above or by the vector introduction of the nucleotide sequence containing the LYG1 described in coding preceding claim or be transfected into the recombiant protein that HOST ORGANISMS produces, and one or more pharmaceutical excipients or pharmaceutical carrier.

Preferably, the nucleotides sequence of the LYG1 described in described coding claim 1 is classified as the nucleotide sequence shown in SEQIDNO:3, described carrier be pcDNA3.1/myc-His (-) B, described HOST ORGANISMS is escherichia coli, HEK293T or 293-6E cell, it is further preferable that described escherichia coli are DH5 α.

This research uses immunogene group strategy, utilizes secretion checking, expression pattern analysis and systemic function research to find that human gene LYG1 encodes a classical secretory protein.PERIPHERAL BLOOD MONONUCLEAR CELL (the PBMC that it stimulates at lipopolysaccharide (LPS), Peripheralbloodmononuclearcell) in, LPS stimulate mouse macrophage in and the inflammatory bowel tissue of inflammatory bowel patient in down-regulated expression, up-regulated in the A549 cell that IL-1 β stimulates, in the THP1 cell of PMA and LPS combined stimulation, express notable rise, express after mononuclear cell is divided into dendritic cell and substantially raise.Restructuring LYG1 albumen can lower prostate cancer cell line DU145 surface Complement Regulatory Protein CD55 and the CD59 expression in mRNA level in-site, lowers CD46 and the CD59 expression at protein level.Additionally, restructuring LYG1 albumen can also promote that the in vitro and in vivo of mice Th1 cell is broken up, mouse DTH model plays protective effect, promote the secretion of IFN-γ, and the growth of tumor in Murine melanoma B16 cells tumor model can be suppressed.And Th1 cell Major Secretory IFN-γ, in antiviral, the infection of anti-intra-cellular pathogens, antitumor, delayed hypersensitivity, play critical function by cellullar immunologic response, and participate in the generation of the autoimmune disease such as rheumatoid arthritis, diabetes, development.The expression of suppression Complement Regulatory Protein can make complement system play antitumor action.Result above is all pointed out, and LYG1 is a potential new cytokine, may play a significant role, have potential clinical value in terms of inflammation, infectious disease, tumor, anaphylactic disease, autoimmune disease.

Accompanying drawing explanation

Hereinafter, describe embodiment of the present invention in detail in conjunction with accompanying drawing, wherein:

Fig. 1 is BFA inhibition test, the result of the secretory pathway of detection LYG1, in figure pcDB be transfected empty carrier pcDNA3.1/myc-His (-) negative control group of B (hereafter abbreviated with pcDB), LYG1 for transfected pcDNA3.1/LYG1-myc-His (-) experimental group of B (hereafter abbreviated with pcDB-LYG1-myc-his), wherein anti-actin (anti-actin) is internal reference；

Fig. 2 is purification LYG1 albumen and carries out SDS-PAGE qualification and the result of N end order-checking, and figure A is the result of the test of SDS-PAGE detection LYG1 purity of protein, and in figure, 1 is protein labeling, and 2 is the LYG1 albumen of reduction, and 3 is non-reducing LYG1 albumen；Fig. 2 B is the result of the test of N end sequencing analysis, and Fig. 2 B-1 is the result of blank order-checking；Fig. 2 B-2 is the sequencing result of standard substance；Fig. 2 B-3～12 is respectively first amino acid residue to the 10th amino acid residue sequencing result；

nullFig. 3 is the qualification result of LYG1 polyclonal antibody，The identification ability of albumen in the LYG1 polyclonal antibody 293T culture supernatant to overexpression pcDB and LYG1 is detected by the method for Western blotting (Westernblot)，In figure，Anti-LYG1 (anti-LYG1) is the rabbit source serum (containing LYG1 polyclonal antibody) after LYG1 protein immunization，Normal serum (normalserum) is the rabbit source serum before immunity、Anti-myc (anti-myc) is positive control antibodies、Anti-rabbit IgG (anti-rabbitIgG) is for directly hatching two anti-detection cell pyrolysis liquids，As blank，PcDB is the negative control group having transfected empty carrier pcDB，LYG1 is the experimental group having transfected pcDB-LYG1-myc-his；

Fig. 4 is the expression pattern analysis result of the test of LYG1 (mice is Lyg1), and Fig. 4 A represents that LYG1 wide spectrum in people respectively organizes is expressed, and in figure, 1 is brain, 2 is heart, and 3 is kidney, and 4 is liver, 5 is lung, and 6 is pancreas, and 7 is Placenta Hominis, 8 is skeletal muscle, 9 is colon, and 10 is ovary, and 11 is prostate, 12 is small intestinal, and 13 is testis；It is higher that Fig. 4 B represents that LYG1 expresses in immune system, and in figure, 1 is leukocyte, and 2 is bone marrow, and 3 is lymph node, and 4 is spleen, and 5 is thymus, and 6 is tonsil, and 7 is tire liver；Fig. 4 C represents Lyg1 thymus in mice normal structure, and in harmonization of the stomach brain, higher level is expressed, and in figure, 1 is thymus, and 2 is lymph node, and 3 is small intestinal, and 4 is colon, and 5 is brain, and 6 is kidney, and 7 is lung, and 8 is spleen, and 9 is liver, and 10 is heart；Fig. 4 D represents that LYG1 is divided into dendritic cell (Dendriticcells at mononuclear cell, DC) up-regulated during, in figure, 1 is PBMC, 2 is mononuclear cell, 3 is immature dendritic cell (imDC), and 4 is that zymosan (Zymosan) stimulates ripe DC cell (mDC-zymosan) 5 to stimulate ripe DC cell (mDC-LPS) for LPS；Fig. 4 E represents Lyg1 expression in Th1, Th17 and the Treg cell of mice tranquillization and activation；Fig. 4 F represents that real-time quantitative PCR detects LYG1 down-regulated expression in inflammatory bowel patient's intestinal tissue, and Fig. 4 G represents that real-time quantitative PCR detects TNF-α expression, as positive control；

Fig. 5 is the LYG1 (mice the is Lyg1) result of the test expressing change under the following 4 kinds of inflammatory factors built stimulate in inflammatory model, and figure A is the PBMC that LPS (10ng/mL) stimulates；B is the A549 cell that IL-1 β stimulates；C is PMA and the THP1 cell of LPS stimulation；D is the mouse macrophage that LPS stimulates, and extracts mRNA, and prepare cDNA library from above cell, with cDNA library as template, and the expression of the mRNA level in-site of detection LYG1 (mice is Lyg1).Wherein IL-6, IL-8, IL-1 β is respectively corresponding positive control；

Fig. 6 is to use RT-PCR and the mRNA level in-site of flow cytometer detection surface of cell membrane CD46, CD55 and CD59 and the expression situation of change of protein level after addition recombined human LYG1 eukaryotic protein in prostate cancer cell line DU145, Fig. 6 A represents that LYG1 lowers the mrna expression level of CD46 and CD59, wherein IL-1 β is positive control, Fig. 6 B represents that LYG1 lowers the protein expression level of CD46 and CD59, and wherein CD59 expresses situation of change selection IFN-γ as positive control；

Fig. 7 is the effect in mice Th1 cells in vitro breaks up of the recombined human LYG1 eukaryotic protein, and Fig. 7 A is to detect by the method for streaming to add Th1 cell (IFN-γ positive ratio is reacted) percentage result after variable concentrations recombined human LYG1 eukaryotic protein；Fig. 7 B show by the secretion situation of Th1 cell IFN-γ after ELISA method detection addition variable concentrations recombined human LYG1 eukaryotic protein.Wherein IL-18 is positive control；

nullFig. 8 is the effect in mouse DTH model of the recombined human LYG1 eukaryotic protein，Wherein，Fig. 8 A show mBSA and excites latter 24 hours mouse insole swelling situations，Fig. 8 B is by the level of IFN-γ in the method detection mice serum of ELISA，Fig. 8 C and Fig. 8 D be external addition mBSA mice LN cells and supernatant in IFN-γ and secretion situation (Fig. 8 B of IL-17A、8C、In 8D, abscissa represents mouse number，Such as PBS-1 represents first mice of PBS group)，Fig. 8 E is to cultivate the cell proportion situation after 72 with the mouse boosting cell adding mBSA outside the method detection bodies of streaming，Sets forth the result of representational two of PBS and LYG1 group，Figure analyzes the ratio of CD4+ cell shared by Th1 (IFN-γ positive ratio) and Th17 cell (IL-17A positive ratio)；

Fig. 9 is the effect in murine melanoma model of the recombined human LYG1 eukaryotic protein, measures tumor major diameter (a) and minor axis (b) with slide gauge, and Fig. 9 A is that tumor is substantially seen；Fig. 9 B is tumor growth curve (often organizing gross tumor volume mean variation curve every day after inoculated tumour)；Fig. 9 C is tumor weight (often organizing the meansigma methods of tumor weight).

Detailed description of the invention

The term used in the present invention, unless otherwise specified, typically has the implication that those of ordinary skill in the art are generally understood that.Unless otherwise indicated, for the ease of difference, with the LYG1 that all letter is all capitalized, (rather than in claims) represent that the Lyg1 of mankind LYG1, only initial caps represents that mice Lyg1, LYG1 albumen are recombined human LYG1 eukaryotic protein in this manual.

Below in conjunction with concrete preparation embodiment and Application Example, and the present invention is described in further detail with reference to data.These embodiments present invention solely for the purpose of illustration, rather than limit the scope of the present invention by any way.

Human lung adenocarcinoma cell line A549's cell in following example, person monocytic cell's type lymphoma cell line THP1 cell, PC-3 DU145 are purchased from ATCC；Mice is C57BL/6J mice (being purchased from Laboratory Animal Science portion of Department Of Medicine, Peking University).

The clone of the cDNA of embodiment 1LYG1 gene

By bioinformatic analysis, find UniGene:Hs.164589, its corresponding gene be LYG1 (sequence is SEQIDNO:3:

AGCAACTGGGGATGCTATGGAAACATCCAAAGCCTGGACACCCCTGGAGCATCTTGTGGGATTGGAAGACGTCACGGCCTGAACTACTGTGGAGTTCGTGCTTCTGAAAGGCTGGCTGAAATAGACATGCCATACCTCCTGAAATATCAACCCATGATGCAAACCATTGGCCAAAAGTACTGCATGGATCCTGCCGTGATCGCTGGTGTCTTGTCCAGGAAGTCTCCCGGTGACAAAATTCTGGTCAACATGGGCGATAGGACTAGCATGGTGCAGGACCCTGGCTCTCAAGCTCCCACATCCTGGATTAGTGAGTCTCAGGTTTCCCAGACAACTGAAGTTCTGACTACTAGAATCAAAGAAATCCAGAGGAGGTTTCCAACCTGGACCCCTGACCAGTACCTGAGAGGTGGACTCTGTGCCTACAGTGGGGGTGCTGGCTATGTCCGAAGCAGCCAGGACCTGAGCTGTGACTTCTGCAATGATGTCCTTGCACGAGCCAAGTACCTCAAGAGACATGGCTTCTAA Italicized item is LYG1 non-coding sequence, nucleotide sequence added with the signal peptide that part is encoding precursor LYG1 of frame, remaining is LYG1 coded sequence), Ref:NM_1748982.2, GeneID:149999339, it it is a unknown function human gene, Human_est data base is utilized to carry out sequence calibration by BLASTn method errorless, then according to the special primer of this sequential design LYG1 full length gene reading frame, and in forward primer, add NotI restriction enzyme site gcggccgc and kozak sequence cacc, downstream primer addition KpnI restriction enzyme site sequence ggtacc and polishing base gc:

Forward primer: gcggccgccaccatgtctgcattgtggctgctgc (SEQIDNO:4)

Downstream primer: ggtaccgcgaagccatgtctcttgaggtacttg (SEQIDNO:5)

Use above-mentioned primer, carrying out pcr amplification reaction with the hybrid template of people's normal spleen tissue cDNA library (Clontech:Cat.No.636743), people's normal tire hepatic tissue cDNA library (Clontech:Cat.No.636748) and people's normal granulocytes cDNA library, reaction condition is as follows:

Reaction volume 50 μ L, wherein contains:

PCR reaction condition: 94 DEG C, degeneration 5 minutes；Then 94 DEG C of degeneration 30 seconds, anneal 30 seconds for 61-58 DEG C, and 72 DEG C extend 30 seconds, expand 10 circulations；94 DEG C of degeneration 30 seconds, anneal 30 seconds for 58 DEG C, and 72 DEG C extend 30 seconds, expand 20 circulations；Last extension 7 minutes at 72 DEG C.

Amplified production is 3 ' 3 ' the prominent cohesive end fragments having base A, test kit (Qiagen is reclaimed with QIAquick glue, 28706) it is purified by product description, then the linear pGEM-TEASY carrier (Promega of base T is had with 3 ', A1360) connect 8 hours at 16 DEG C, connect product and convert bacillus coli DH 5 alpha, conversion product grows on the LB plating medium containing ampicillin, selected clone, extract plasmid, use AbIPRISM3700DNA analyser (Perkin-Elmer/AppliedBiosystem) order-checking.

The structure of embodiment 2 recombinant mammalian expressing vector pcDB-LYG1-myc-his

In order to detect the function of LYG1, the first structure eukaryon expression plasmid containing LYG1 open reading frame: pcDNA3.1/LYG1-myc-His (-) B.nullWith NotI and KpnI enzyme action pGEM-T-LYG1，Simultaneously with NotI and KpnI enzyme action eukaryotic expression vector pcDNA3.1/myc-His (-) B (hereafter abbreviated with pcDB) (Invitrogen，V85520)，The cDNA genetic fragment of the LYG1 after enzyme action is connected 8 hours with carrier at 16 DEG C，Convert bacillus coli DH 5 alpha，Conversion product grows on the LB plating medium containing ampicillin，Select the bacterium colony of growth，Extract plasmid，PCR identifies，Select positive colony，(ABIPRISM3700DNA analyser is used by order-checking，Ibid)，Select the cDNA gene plasmid of the LYG1 being properly inserted sequence，Named pcDB-LYG1-myc-his (with c-myc and 6XHis label).

Embodiment 3LYG1 is the secretory protein of a classical pathway

1. cell is cultivated and transfection

HEK293T cell (being professor's TadashiMatsuda present of Japan), this room Secondary Culture, with the DMEM culture medium culturing containing 10% hyclone (FBS).HEK293T cell Vigofect (prestige lattice Lars) cation transfection reagent, operates according to description, transfected plasmids pcDB-LYG1-myc-his and pcDB.After transfecting 6 hours with 1 × PBS washed cell of room temperature once, change fresh HEKG serum-free medium, in cell culture supernatant, add 10 μ g/mLBFA (Brefeldin-A) and ethanol (as negative control) respectively.

2. cells and supernatant and total protein of cell extract and Western blotting (Westernblot) is analyzed

The collection of HEK293T cell culture supernatant: after 48 hours, when cell confluency about reaches 90%, collects each experimental group cell and cell culture supernatant, in 4 DEG C, 2000g is centrifuged 10 minutes, abandons precipitation, then at 4 DEG C, 15000g is centrifuged 15 minutes, removes cell debris, leaves and takes cell culture supernatant.

The extraction of HEK293T total protein of cell: the cell after leaving and taking supernatant is placed on ice, and ice-cold 1 × PBS washes twice, and ice-cold 1 × PBS blows down cell, collects in 1.5mL centrifuge tube by cell, and in 4 DEG C, 2000g is centrifuged 5 minutes.Remove supernatant, precipitation adds RIPA cell pyrolysis liquid (20mMTris-HCl, pH7.4,150mMNaCl, 1mMEDTA, 1mMEGTA, 1%TritonX-100, proteinase inhibitor C ocktail), placing 30 minutes on ice, 4 DEG C, 15000g is centrifuged 15 minutes, supernatant proceeds to newly manage, be stored in-80 DEG C standby.

Protein quantification: the method that the albumen of cell extraction provides according to BCATM protein detection kit (BCATMProteinAssayKit) (Pierce, 23227) description carries out protein quantification.

Western blotting: often group cell pyrolysis liquid takes total protein 30ug, and often group cell culture supernatant takes 40 μ L, adds albumen sample-loading buffer (Beijing Bao Sai Bioisystech Co., Ltd), boils 10 minutes in 99 DEG C.12.5%PAGE electrophoresis, 100V electricity turns 1.5 hours, and TBST liquid balances, and closes 1 hour by 5% milk room temperature, adds corresponding one and resists, and 4 DEG C overnight, fully washes film with TBST 3 times, each 10 minutes；Being subsequently adding two anti-(1: 10000) of corresponding IRDyeTM700/800 labelling, room temperature lucifuge is reacted 1 hour；Film is fully washed 3 times again with TBST, each 10 minutes；Finally use infrared imaging system scanning imaging system (OdysseyInfraredImager, LI-CORBioscience company of the U.S.) detection signal.

As it is shown in figure 1, result shows, after adding BFA, LYG1 secretion significantly reduces, it was demonstrated that LYG1 is the secretory protein secreted by classical pathway (endoplasmic reticulum Golgi bodies approach).

The purification of embodiment 4 recombined human LYG1 eukaryotic protein and N terminal Sequence Analysis

In order to carry out the functional study of potential new cytokine LYG1, we are expression and purification LYG1 recombiant protein in 293-6E cell.293-6E cell is purchased from Canadian National Research Council (nationalresearchcouncilofcanada).By 293-6E cell DMEM (Dulbecco ' smodifiedEagle ' smedium) culture medium containing 10% hyclone, at 5%CO₂, the incubator of 37 DEG C is cultivated 24 hours.Use PEI cation infection protocol transfection pcDB-LYG1-myc-his eukaryon expression plasmid.

After transfecting 16 hours with 1 × PBS washed cell of room temperature once, fresh HEKG serum-free medium is changed, at 5%CO₂, the incubator of 37 DEG C is cultivated.Collecting cell culture supernatant after 48 hours, in 4 DEG C, 2000g is centrifuged 10 minutes, remove the cell of culture supernatant, abandon precipitation, then at 4 DEG C, 18000g is centrifuged 20 minutes, removes the finely ground particle substance in supernatant, leaves and takes the cells and supernatant after process for subsequent purification.

nullThe cell culture supernatant after above-mentioned process is purified: first by supernatant after 0.45 μm filter filters with Ni2+ post material，Imidazoles (10mM)/NaCl (200mM) is added in supernatant，Be combined with post material again，Flow speed control is per minute at 10，Post material is rinsed again with level pad [imidazoles (20mM)/NaCl (200mM) in1 × PBS (pH7.4)]，To wash away unconjugated foreign protein，After being back to baseline to light splitting value，With eluent [imidazoles (500mM)/NaCl (200mM) in1 × PBS (PH7.4)] elution of bound albumen until light splitting value no longer declines，Collect eluent in super filter tube，With 1 × PBS (pH7.4) 12mL of pre-cooling，4 DEG C are centrifuged 2 times，It is concentrated into 500 μ L，Sucking-off albumen，In 4 DEG C，18000g be centrifuged 20 minutes degerming，Subpackage be stored in-80 DEG C stand-by.

The 5 μ L albumen BCA methods that take carry out protein quantification to it, take sample segment and add albumen sample-loading buffer, boil 5 minutes in 100 DEG C of water-baths, carry out SDS-PAGE and Western blotting detection, identify the purity of LYG1 protein purification, as shown in Figure 2 A, the purity of albumen is higher, more than 90% for result, endotoxin residual is less than 1EU/mg, there may be glycosylation modified, and ExPASy database analysis prediction LYG1 contains the glycosylation modified site of O, this is consistent therewith.Not adding beta-mercaptoethanol in sample is non-reducing LYG1 albumen, adds the LYG1 albumen that beta-mercaptoethanol is reduction.The molecular weight of SDS-PAGE electrophoresis detection is the apparent molecular weight of protein, and non-real molecular size range.Under the reducing conditions, intrachain disulfide bond fully opens, and presents strip during protein SDS-PAGE electrophoresis；Under non reducing conditions, albumen due to the existence of intrachain disulfide bond, than under reducing condition comparatively speaking closer to spherical.So, under non reducing conditions, albumen apparent molecular weight is the most smaller.

It addition, take sample segment to carry out SDS-PAGE, and by protein delivery to pvdf membrane, after dyeing and decolouring, for protein N terminal sequence analysis.Result as shown in Figure 2 B, have detected N end 10 aminoacid, respectively S/A, N, W, G ,/, Y, G, N, I, Q, illustrate that 19 aminoacid of N end of precursor LYG1 are signal peptide sequence.Having a higher peak in the most each figure, this is the by-product dptu of system secretion, for relative internal reference.So by our experimental verification, LYG1 maturation protein (the i.e. albumen of function, the most potential new cytokine LYG1) aminoacid sequence be the sequence after having excised front 19 aminoacid of precursor LYG1N end, the recombined human LYG1 eukaryotic protein that the most all of embodiment is used all meets this requirement.

The preparation of embodiment 5LYG1 polyclonal antibody and qualification

Antibody can be used for detecting, diagnosing and treat disease, and then we are prepared for the antibody of LYG1.With the LYG1 protein immune animal of purification.Select adult male New Zealand rabbit (buying in Laboratory Animal Science portion of Department Of Medicine, Peking University), initial immunity is sufficiently mixed with isopyknic Freund's complete adjuvant after the antigen PBS of 300 μ g is diluted to 1mL, in two foots each 0.25mL subcutaneous injection, the subcutaneous multiple spot of remaining back part (6 point) is injected.Once, consumption is the same for the most every 3 weeks booster immunizations, and the subcutaneous multiple spot of whole back parts (8 point) is injected.Within after booster immunization 10 days for the third time, take rabbit ear edge venous samples can detection titer, reach 1: 10 to titer⁵After above, rabbit is put to death heart extracting blood, obtain serum.Carry out preliminary Western blotting to identify.As shown in Figure 3, anti-LYG1 group (anti-LYG1 group) (Post-immunisation serum, 1:: 1000 dilutions, experimental group), anti-myc group (anti-myc) (positive control), control serum (normalserum before immunity, 1: 1000 dilution), anti-rabbit IgG (anti-rabbitIgG) (two anti-comparisons), the polyclonal antibody (anti-LYG1) of anti-LYG1 can LYG1 albumen in specific recognition cell culture supernatant and the LYG1 recombiant protein of purification, can be used for detection and the diagnosis of LYG1 relevant disease.Albumen has two bands, is specificity, and prompting LYG1 albumen there may be modification.

Embodiment 6LYG1 expression pattern analysis (RNA extraction, RT-PCR and Real-timePCR) in tissue and cell

In order to analyze LYG1 expression change in the normal tissue, in immunocyte difference differential period and different cellular inflammation model, we use the Normal human tissue cDNA library of the Clontech company of purchase, mice normal structure cDNA library prepared by laboratory and people and the cell library of mice, use nest-type PRC to expand LYG1.nullCell library includes that preparation is as follows: collect the DC cell of different differential period、The Th cell of mice tranquillization and activation、Do not stimulate and inflammatory factor LPS (100ng/mL) stimulate 6h PBMC (White Blood Cells Concentrate is provided by Beijing Red Cross Blood Center，Lymphocytes separating solution (purchased from Hua Jing bio tech ltd, Shanghai) density－gradient centrifuga－tion method is used to obtain)、Do not stimulate and inflammatory factor phorbol exters (PMA，Phorbol-12-myristate-13-acetate)(10ng/mL，24 hours) stimulate 3h with LPS (100ng/mL) afterwards、6h、The THP1 of 12h、Do not stimulate and inflammatory factor IL-1 β (10ng/mL) stimulates 3h、6h、15h、The A549 cell of 24h、Do not stimulate and inflammatory factor LPS stimulates 2h、4h、(every mouse peritoneal injects 3% sodium thioglycolate 2ml to the mouse macrophage of 6h，After 48 hours, disconnected neck is put to death fixing，To intraperitoneal injection 5mlPBS，Open abdominal cavity behind soft abdominal cavity and reclaim PBS，2000rpm，4 DEG C of centrifugal acquisition peritoneal macrophages)，TRIzol is used to extract total serum IgE.Each sample takes 2 μ g, with Reverse Transcription box [ReversetranscriptTMkit (Invitrogen)] the synthesizing single-stranded cDNA library of reverse transcription.GAPDH is as internal reference in amplification.Real-time quantitative PCR uses SYBRGreen method and quantitative real time PCR Instrument (LightCycler instrument, ABI7500) to carry out.

The amplification of people LYG1: forward primer 5 '-tttcaggagccgtagagcc (SEQIDNO:6) outside use, outside downstream primer 5 '-tgatcatggtcttgggttt (SEQIDNO:7), inner side forward primer 5 '-gcggccgccaccatgtctgcattgtggctgctgc (SEQIDNO:8), inner side downstream primer 5 '-ggtaccgcgaagccatgtctcttgaggtacttg (SEQIDNO:9) carries out nested amplification.One expansion condition is: 94 DEG C (5 minutes) → 94 DEG C (30 seconds), 56 DEG C (30 seconds), 72 DEG C (30 seconds), expands → 72 DEG C (7 minutes) of 30 circulations.One expands product dilution 50 expands template, 94 DEG C (5 minutes) → 94 DEG C (30 seconds), 58 DEG C (30 seconds), 72 DEG C (30 seconds) as two again, expands → 72 DEG C (7 minutes) of 30 circulations.

The amplification of mice Lyg1: forward primer outside use: 5 '-agagtctccaggaggcaactatgtg (SEQIDNO:10), outside downstream primer 5 '-ccatggtccttgaagtatttggct (SEQIDNO:11)；Inner side forward primer 5 '-aacattggcagtggacttggga (SEQIDNO:12), inner side downstream primer 5 '-gcgcaaggacgtcattgcag (SEQIDNO:13) carries out nested amplification, and GAPDH is as internal reference in amplification.Amplification condition is an expansion: 94 DEG C (5 minutes) → 94 DEG C (30 seconds), 56 DEG C (30 seconds), 72 DEG C (30 seconds), expands → 72 DEG C (7 minutes) of 30 circulations.One expands product dilution 50 expands template, 94 DEG C (5 minutes) → 94 DEG C (30 seconds), 58 DEG C (30 seconds), 72 DEG C (30 seconds) as two again, expands → 72 DEG C (7 minutes) of 30 circulations.

Result shows, as shown in Figure 4 A, LYG1 has low expression level in Various Tissues, such as kidney, liver, lungs, pancreas, Placenta Hominis, skeletal muscle, colon, ovary, prostate, small intestinal and testis；As shown in Figure 4 B, express slightly higher in immune system, especially bone marrow, thymus and tonsil.Additionally, we are prepared for mice normal structure cDNA library, consistent with Normal human tissue express spectra, Lyg1 expresses slightly higher in thymus, and also has higher level to express in harmonization of the stomach brain, low expression level (as shown in Figure 4 C) in colon, kidney and lung.We also have detected the expression of LYG1: LYG1 in mononuclear cell is divided into DC cell processes and express relatively low in PBMC and mononuclear cell, imDC expresses slightly higher, and in zymosan and LPS stimulate ripe DC LYG1 up-regulated (as shown in Figure 4 D).Zymosan and LPS have mediated DC respectively to be broken up to Th17 and Th1 cell direction T cells (NaiveT cell), and LYG1 is as a secretory protein, it is up-regulated in the DC that zymosan and LPS stimulate, and prompting LYG1 likely participates in the differentiation of Th cell in microenvironment.Additionally, we also have detected Lyg1 expression in mice difference Th cell subsets, find that Lyg1 expresses higher in Th17 and Treg cell, Th1 cell is expressed relatively low (as shown in Figure 4 E).Additionally, we also have detected LYG1 expression in inflammatory bowel (IBD) patient inflammatory bowel tissue and normal person's intestinal tissue.Finding in patient's inflammatory bowel tissue, the down-regulated expression (as shown in Fig. 4 F, 4G) of LYG1, prompting LYG1 has substantial connection with the generation development of inflammatory bowel.Additionally, the expression of mouse macrophage (Fig. 5 D) Lyg1 of the expression of THP1 cell (Fig. 5 C) LYG1 that we also have detected the A549 cell (Fig. 5 B) of PBMC (Fig. 5 A), IL-1 β stimulation of LPS stimulation, PMA and LPS stimulates and LPS stimulation.As it can be seen, LYG1 down-regulated expression in the PBMC that LPS stimulates, and in the A549 cell that IL-1 β stimulates up-regulated, in the THP1 cell of PMA and LPS Co stituation, the expression of LYG1 is substantially raised.The down-regulated expression of Lyg1 in the mouse macrophage that LPS stimulates.These results prompting LYG1 may participate in generation and the development of inflammation.Inflammatory bowel tissue of patient and inflammation model tormulation spectrum all point out the LYG1 effect with antiinflammatory, and its recombiant protein or antibody have the function for the treatment of inflammation.

The impact on Complement Regulatory Protein molecule of embodiment 7LYG1

The high expressed of Complement Regulatory Protein (CRP) can suppress the complement system attack to tumor cell, makes tumor evasion immunity of organism defend.Cytokine can play critical function by regulating Complement Regulatory Protein in the immunization therapy of tumor.As found in the research to renal carcinoma, IL-1 can lower the expression of CD46, CD59, IL-4 energy sustained down-regulation CD46, CD55 and transforming growth factor, increasing C3 deposition on tumor cell, while using antibody for antitumor related antigen, conjunctive use cytokine will become a kind of new effective immunotherapy method.Then, we observe the impact that tumor cell line surface C RP is expressed by LYG1.Adding restructuring LYG1 albumen in DU145 cultivating system, the cell collecting 48h does RT-PCR test, and 72h cell does fluorescence staining.Result shows, compared with not adding LYG1 protein control group, LYG1 can lower CD55 and the CD59 expression (Fig. 6 A) in mRNA level in-site, lower CD46 and the CD59 expression (Fig. 6 B) at protein level, and mRNA level in-site CD46 is unchanged, protein level CD55 is unchanged, thus it is speculated that it has relation with the time point collecting cell.Experiment shows that LYG1 can stop immunologic escape by lowering the expression of CRP so that complement system can play Graft Versus Tumor, thus suppresses tumor to develop, and further shows that LYG1 albumen is active.

Embodiment 8LYG1 can promote the vitro differentiation of mice Th1 cell

1. separating mouse CD4+T cell and the Differentiation Induction in vitro of mice Th cell

1.1 the separation of mice CD4+T cell and cultivation

Taking 6～8 weeks female C57BL/6J mices, cervical dislocation is put to death, 75% soak with ethanol 1 minute, takes out mice and is placed on aseptic ware, cuts off osculum, tear skin, eye scissors separation connective tissue, take both sides inguinal lymph nodes in the middle part of mice abdomen；Enter abdominal cavity afterwards and take mesenteric lymph node and both sides para-aortic lymph node, put into be placed on 200 eye mesh screens in the culture dish filling RPMI-1640 and grind pressure lymph node with pestle gently, obtain cell suspension, test kit (InvitrogenDYNAL) description is sorted, it is thus achieved that mice CD4+T cell according to mice CD4+T cell feminine gender.Particularly as follows: with buffer 1 resuspended LN cell, add hyclone (FBS) and mixtures of antibodies (anti-mouse B220 (anti-mouseB220), CD11b, Ter-119, CD16/32 and CD8), hatch 20 minutes for 4 DEG C, wash one time with buffer 1, add washed magnetic bead, incubated at room 15 minutes, magnetic frame stands 2min, can obtain CD4+T cell.Taking part CD4+T cell, add anti-mouse CD4-PerCP antibody (anti-mouseCD4-PerCP antibody), lucifuge 4 degree is hatched 20min, PBS and is washed once, and 200 μ L are resuspended, by the purity of the CD4+T cell of the method grouping system of streaming.CD4+T cell is resuspended by the RPMI1640 culture medium containing 10% hot inactivated serum, and concentration is 2X10⁶/ mL, spreads to 48 orifice plates, every hole 500 μ L.

1.2 mice Th cell and the Differentiation Induction in vitros of iTreg cell

Mice CD4+T cell is obtained according to the step magnetic bead sorting described in 1.1, it is incubated in 48 orifice plates that anti-mouse CD3 (anti-mouseCD3) (10 μ g/mL) is pre-coated, add anti-mouse CD28 (anti-mouseCD28) (2 μ g/mL) and rhIL-2 (4ng/mL), and add exogenous LYG1 recombiant protein (0,0.1,1,10ng/ml) and antibody, the differentiation of inducing mouse Th1, Th17 and iTreg cell.The inductive condition of Th1 cell is: IL-12,10ng/mL；Anti-mIL-4 (anti-mIL-4), 10 μ g/mL.The inductive condition of Th17 cell is: TGF-β, 5ng/mL；IL-6,10ng/mL；Anti-mIL-4,10 μ g/mL；Anti-mIFN-γ (anti-mIFN-γ), 10 μ g/mL.Within 4th day, change liquid, cultivate under the conditions of only IL-2 (4ng/mL) and mice Th1 and Th17 cell within two days, can be obtained.The inductive condition of iTreg is: IL-2,4ng/mL；TGF-β, 5ng/mL；External evoked three days.

2. immunofluorescence dyeing

2.1 membrane molecule detections

Gather in the crops different types of immunocyte, wash twice with PBS/0.1%BSA, be directly added into 100 μ L confining liquid (PBS/10% normal sheep serum) re-suspended cells, close 30 minutes for 4 DEG C；Being subsequently added the antibody of labelling, 4 DEG C of lucifuges hatch 40min；Use corresponding IgG as Isotype control.After finally washing twice with PBS/0.1%BSA, collect cell by flow cytometer, use Cellquest software (BD company) that result is analyzed.

2.2 intracellular molecules detections

Gather in the crops different types of immunocyte, wash twice with cold PBS/0.1%BSA, first fix 30min on ice with 4% paraformaldehyde；Then by 0.1%TritonX-100 incubated at room 30min, 1500rpm is centrifuged 5min.Adding 100 μ L confining liquid (PBS/10% normal sheep serum) re-suspended cells, room temperature is closed 30 minutes.Being subsequently added traget antibody, 4 DEG C of lucifuges hatch 40min；Use corresponding IgG as Isotype control.After finally washing twice with PBS/0.1%BSA, collect cell by flow cytometer, use Cellquest software that result is analyzed.

3.ELISA

Cell gathers in the crops supernatant after cultivating the corresponding time, and in 4 DEG C, 2000g is centrifuged 10 minutes, abandons precipitation, and then at 4 DEG C, 15000g is centrifuged 15 minutes, removes cell and fragment in supernatant.Use the secretory volume of IFN-γ, IL-4, IL-17A cytokine ELISA kit (eBioscience) sandwich assay detection cytokine.

As shown in figs. 7 a-b, recombined human LYG1 eukaryotic protein is activated in mice system, it can promote vitro differentiation (raising of IFN-γ percent positive) and the secretion of IFN-γ of mice Th1 cell, and there were significant differences (p=0.0002) with being not added with LYG1 group for LYG1-1ng/mL.

Due to Th1 cell Major Secretory IFN-γ, IL-2, LT α etc., being played a significant role in antiviral, the infection of anti-intra-cellular pathogens, antitumor, delayed hypersensitivity by cellullar immunologic response, therefore LYG1 recombiant protein and antibody can have significant application value by promoting the differentiation of Th1 cell and IFN-γ secretion at antiviral, the infection of anti-intra-cellular pathogens, anti-tumor aspect.

The in vivo functionality research of embodiment 9LYG1

1.LYG1 can suppress mice delayed hypersensitivity (DTH)

In order to study LYG1 in vivo on the differentiation of Th cell and the impact of survival, we use mBSA induction of mouse DTH model, choose 8 week old female C57BL/6J mice, within 0th day, give every mouse web portion Intradermal 2 injection 125 μ g (50 μ L) adequately emulsified mBSA of CFA, every day lumbar injection 100ngLYG1 albumen.Within 7th day, every mice left foot pad subcutaneous injection 150 μ gmBSA/PBS solution (30 μ L) excites, and the right isopyknic PBS of foot pad subcutaneous injection is as negative control.Vernier caliper measurement mouse insole thickness is all used before exciting and after exciting 24 hours.The foot pad thickness change calculations formula weighing DTH response strength is: footpad swelling degree (mm)=(the foot pad thickness that mBSA excites-excite forefoot pad thickness)-(the foot pad thickness of PBS injection-excite forefoot pad thickness) (1. TakayukiYoshimoto, etal.RoleofIL-16indelayed-typehypersensitivityreactionBlood.2000；2869-74；2. AkinaIshii1, KeisukeObok, etal.DevelopmentofIL-17-mediatedDelayed-TypeHypersensiti vityisnotaffectedbydown-regulationofIL-25expression.AllergolInt.2010；399-408).Then eyeball takes blood, and 4 DEG C overnight, and 3000rpm is centrifuged, and takes serum and manages to new Ep, and 12000rpm is centrifuged 10min and removes fragment, and gained serum i.e. can be used for the ELISA detection of IFN-γ.Take spleen, inguinal lymph nodes and mesenteric lymph node, grind and obtain splenocyte and lymph-node cell (LN cell), resuspended one-tenth 5 × 10⁶/ mL is incubated at 24 orifice plates, cultivates respectively 5 days and 3 days under the pre-coated CD3 antibody overnight of 10 μ g/mLmBSA or 3 μ g/mL4 DEG C stimulates, and acquisition cell carries out flow cytometer detection and culture supernatant carries out ELISA detection respectively.

It was found that as shown in Figure 8, LYG1 albumen can suppress mouse insole swelling degree (Fig. 8 A), and in serum, the concentration of IFN-γ is apparently higher than PBS control group (Fig. 8 B).In the lymph-node cell culture supernatant stimulated in vitro, the concentration of IFN-γ (Fig. 8 D) and IL-17A (Fig. 8 C) is all remarkably higher than PBS control group, but IFN-γ multiple is higher.The result (Fig. 8 E) of streaming displays that, LYG1 group Th1 (IFN-γ positive ratio) and Th17 (IL-17A positive ratio) cell ratio in CD4+ cell are all higher than PBS group, but Th1 cell obtains ratio and has significantly more difference.Having result of study to report, in the DTH model of mBSA induction, the CD4+ cell being primarily involved in is Th1 and Th17 cell, and the Th1 cell of secretion of gamma-IFN has protective effect in DTH falls ill, and the Th17 cell of secretion IL-17A then can increase the weight of the development of DTH.The induction of DTH model is divided into two stages, and first 7 days is the differential period of antigen-specific Th cells, for the effective stage of antigen-specific Th cells after exciting.In this experiment; all lumbar injection LYG1 albumen every day in immunity stage; and after exciting, do not inject albumen; in serum, IFN-γ concentration LYG1 group is significantly higher than matched group; in the culture supernatant of the LN cell of same stimulated in vitro, the content of LYG1 group IFN-γ is apparently higher than matched group; in the splenocyte that exo-antigen stimulates, LYG1 group Th1 cell proportion significantly improves, and prompting LYG1 has played protective effect possibly through differentiation and the secretion of IFN-γ of promotion antigen specific T h1 cell to delayed hypersensitivity DTH model.This is consistent with external result of study, and proves that LYG1 can treat the anaphylaxis that Th1 cell (IFN-γ) mediates.

2.LYG1 the tumor growth of B16 tumor can be suppressed

Early stage result prompting LYG1 can promote the differentiation of Th1 cell and the secretion of IFN-γ, and can lower the expression of tumor cell surface Complement Regulatory Protein, and prompting LYG1 may participate in anti tumor immune response.Mice B16 tumor model has reduced immunogenicity, sensitive to immunization therapy, and is difficult to the feature of transfer, is to study the classical animal model by immunity related molecular especially cytokine therapy tumor.Then we utilize this scale-model investigation LYG1 in the effect of anti-tumor aspect.

Choose 8-10 week old female C57BL/6J mice, oxter subcutaneous injection B16 cell (2X10⁵Individual cell/100 μ LPBS/ Mus), within the 5th day, lose hair or feathers to mice, observe tumor growth situation.According to tumor growth situation, by mice group, often organize at least 5.Often group mouse peritoneal injection LYG1 albumen (20 μ L/ days/Mus of g/200 μ), matched group lumbar injection PBS (200 μ L/ days/Mus), co-continuous injection 5 days.Measuring tumor major diameter (a) and minor axis (b) with slide gauge, the volume algorithm of tumor is V=1/2ab².Result is as it is shown in figure 9, compared with PBS group, injection protein groups gross tumor volume is obviously reduced (Fig. 9 A), and slowly (Fig. 9 B), tumor weight substantially reduces (Fig. 9 C) to tumor growth.Result above proves that restructuring LYG1 albumen can substantially suppress the growth of B16 tumor.

3. statistical analysis

The standard deviation of data above represents the difference in once experiment between different multiple holes, and above experiment is the most at least repeated twice above, using data the most once as representative；Whether there is significant difference between each group of Student ' sT check analysis, p ＜ 0.05 thinks have significant difference.

Claims (3)

1. potential new cytokine LYG1 or the nucleotide sequence of the LYG1 described in coding or the carrier of the nucleotide sequence containing the LYG1 described in coding or by the vector introduction of the nucleotide sequence containing the LYG1 described in coding or be transfected into recombiant protein that HOST ORGANISMS produces in preparation for preventing and/or treating the application in the medicine of immune correlated disease；

Wherein, the sequence of described LYG1 is the aminoacid sequence shown in SEQIDNO:1；

Wherein, the medicine of described treatment immune correlated disease is antitumor drug；

Wherein, described tumor is by promoting Th1 cell to break up, promoting cytokine IFN-γ secretion and/or suppress Complement Regulatory Protein CD46, CD55 and CD59 to express the tumor suppressing and/or treating；

Wherein, described tumor is the tumor sensitive to immunization therapy, and described is melanoma tumors to the tumor that immunization therapy is sensitive.

Application the most according to claim 1, it is characterized in that, the nucleotides sequence of the LYG1 described in described coding claim 1 is classified as the nucleotide sequence shown in SEQIDNO:3, described carrier be pcDNA3.1/myc-His (-) B, described HOST ORGANISMS is escherichia coli, HEK293T or 293-6E cell.

Application the most according to claim 2, wherein, described escherichia coli are DH5 α.