CN103073632A

CN103073632A - Potential novel cytokine LYG1 with anti-inflammatory and anti-tumor activities, and application thereof

Info

Publication number: CN103073632A
Application number: CN2011103292898A
Authority: CN
Inventors: 韩文玲; 马大龙; 张岩飞; 付伟伟; 王平章; 刘绘绘; 潘文; 李婷; 王文彦; 石太平
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2011-10-26
Filing date: 2011-10-26
Publication date: 2013-05-01
Anticipated expiration: 2031-10-26
Also published as: CN103073632B

Abstract

The invention provides a potential novel cytokine LYG1 with anti-inflammatory and anti-tumor activities, and an application thereof. The LYG1 (mature and functioning potential novel cytokine LYG1) has an amino acid sequence represented by SEQ ID NO: 1. LYG1 is applicable in preparing medicines and kit used for preventing and/or treating immune-related diseases. The medicines used for treating immune-related diseases comprise antitumor medicines, anti-inflammatory medicines, anti-infectious medicines, and allergic disease and/or autoimmune disease treating medicines. As a result, the potential novel cytokine LYG1 may play an important role in the respects such as tumors, infectious diseases, inflammations, allergic diseases, and autoimmune diseases. LYG1 has potential clinical values.

Description

Have anti-infective and potential new cytokine LYG1 and application thereof anti-tumor activity

Technical field

The present invention relates to a kind of new human cell's factor and application thereof, relate in particular to a kind of potential new cytokine LYG1 with anti-infective and anti-tumor activity in the application of the aspects such as anti-inflammatory, anti-infectious disease, antitumor and autoimmune disease.

Background technology

The human immune system goes through the evolution in 3,000,000,000 years, forms complicated and meticulous regulator control system has vital effect at body.It externally can defend the invasion of pathogenic micro-organism, and is internal then can remove aging, pathology and dead cell, keeps the stable of interior environment.Immunity system is finished above-mentioned functions by innate immune response and adaptive immune response.As the interchange language between the immunocyte, cytokine all has important regulating and controlling effect to innate immune response and adaptive immune response.

Cytokine is by the having the regulating cell Growth and Differentiation, regulate immunologic function and physiological response and participate in the small protein of pathologic reaction of the various emiocytosises of body, by playing a role with receptors bind.Cytokine mainly comprises interleukin-(Interleukin, IL), G CFS (Colony-Stimulating Factor, CSF), Interferon, rabbit (Interferon, IFN), tumour necrosis factor (Tumor-Necrosis Factor, TNF) etc., be the interchange language between the immunocyte, innate immune response and adaptive immune response are all had the important regulating and controlling effect.

The CD4+T cell is brought into play keying action in adaptive immune response, it can assist the B cell to produce antibody, auxiliary CD8+T cell killing target cell.According to cytokine and function different of secretion, the CD4+T cell can be divided into Th1, Th2, Th17 and Treg cell, and newfound Th9 and Th22 cell etc.Cytokine has keying action in differentiation, activation and the Function of Th cell.At first, cytokine can determine the differentiation direction of initial CD4+T cell, is respectively to determine that Th1, Th2 break up necessary cytokine such as IL-12, IL-4.Secondly, cytokine has regulating and controlling effect to the Th cell that has broken up, and IL-12 and IL-18 can be with the collaborative Th1 emiocytosis IFN-γ that promotes of the mode of the non-dependence of TCR.In addition, cytokine or the major way of Th cells play effect.Th1 and Th2 cell are the very important CD4+T cell subsets of two classes, and both balances are immunoregulatory importances.The main secretion of gamma-IFN of Th1 cell, TNF-α etc., infect at antiviral, anti-intra-cellular pathogens, bring into play critical function in antitumor, the delayed type hypersensitivity by cellullar immunologic response, and participate in generation, the development of the autoimmune diseases such as rheumatoid arthritis, diabetes; The Th2 cell is mainly secreted IL-4, IL-5, IL-13 etc., participates in anti-parasitic-infectious and anaphylaxis, simultaneously heterograft and the pregnancy period fetus tolerance aspect play an important role.Differentiation, physiology and the pathologic function of further investigation Th1, Th2 cell and between the two the regulatory mechanism of balance have important theory significance and potential using value.

At present, utilize recombinant cytokine, recombinant soluble acceptor, the neutrality antibody of genetic engineering technique production to receive good efficacy at aspects such as treatment tumour, hematopoietic disorders, autoimmune disorders, become medicine of new generation.Recombinant cytokine has a lot of superior parts as medicine, as: cytokine is the human body self component, can regulate the physiological process of body and improve immunologic function, and very low dosage can play a role, thereby evident in efficacy, become the indispensable treatment means of some difficult and complicated illness.The cytokine medicine of at present approved production comprises IFN-α, β, γ, Epo, GM-CSF, G-CSF, IL-2 etc.According to incompletely statistics, have 26 at least in the world at present and enter clinical study by genomic drug, comprise new recombinant cytokine, recombinant soluble acceptor and neutrality antibody etc.Simultaneously, it also is the index of judging body's immunity and immunocyte differentiation that cytokine or cell surface receptor detect, and has important value at the aspects such as diagnosis, course of disease observation, curative effect judgement and cytokine therapy monitoring of numerous disease.For example, IFN-γ and recombinant protein thereof have been used to treat anaphylactic disease, autoimmune disorders such as rheumatoid arthritis and systemic sclerosis for the treatment of, treatment bacterium fungi infestation disease, treatment viral infection disease, anti-parasitic-infectious, anti-fibrosis effect and treatment tumour.

Summary of the invention

Therefore, the purpose of this invention is to provide a kind of potential new cytokine LYG1 and in the application of the aspects such as anti-inflammatory, anti-infectious disease, antitumor and autoimmune disease.

For above-mentioned purpose, technical scheme of the present invention is as follows:

On the one hand, the invention provides a kind of potential new cytokine LYG1 (namely ripe with the LYG1 performance function) be used to preventing and/or treating immune correlated disease, the sequence of described LYG1 is the aminoacid sequence shown in the SEQ ID NO:1.

Preferably, described potential new cytokine LYG1 is people's type cytokines LYG1 or mouse cytokine LYG1.

Wherein, the precursor sequence of described LYG1 is the aminoacid sequence that the N end does not excise signal peptide, and the sequence of described signal peptide is the aminoacid sequence shown in the SEQ ID NO:2.

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

Again on the one hand, the invention provides the nucleotide sequence of potential new cytokine LYG1 described above or antibody described above or the LYG1 described above that encodes or contain coding LYG1 described above nucleotide sequence carrier or import or be transfected into recombinant protein that host's organism produces for the preparation of the application in the medicine that prevents and/or treats immune correlated disease by the carrier of the nucleotide sequence that contains coding LYG1 described above.

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

Preferably, the tumour of described tumour for expressing by promotion Th1 cytodifferentiation, promotion cytokine IFN-γ secretion and/or inhibition Complement Regulatory Protein CD46, CD55 and CD59 to suppress and/or treat.

More preferably, described tumour is melanoma or to the tumour of immunotherapy sensitivity.

Preferably, described inflammation, infectious diseases, anaphylactic disease and autoimmune disease inflammation, infectious diseases, anaphylactic disease and the autoimmune disease for secreting by promotion Th1 cytodifferentiation, promotion cytokine IFN-γ to suppress and/or treat.

More preferably, described inflammation is inflammatory bowel.

Another aspect, the invention provides the nucleotide sequence of potential new cytokine LYG1 described above or antibody described above or the LYG1 described above that encodes or contain coding LYG1 described above nucleotide sequence carrier or by the carrier of the nucleotide sequence that contains coding LYG1 described above import or be transfected into recombinant protein that host's organism produces for the preparation of promote the Th1 cytodifferentiation, promote cytokine IFN-γ secretion and/or suppress Complement Regulatory Protein CD46, CD55 and medicine that CD59 expresses in application.

Again on the one hand, the invention provides a kind of test kit for the diagnosis immune correlated disease, described test kit comprises antibody described above and/or is used at the DNA chain of PCR composite coding LYG1 nucleotide sequence described above and/or the PCR primer of its cDNA chain, and described antibody is used for the detection by quantitative of LYG1 albumen, detection and the auxiliary diagnosis of relative disease; Described primer is used for the detection by quantitative of LYG1 genetic expression.

Another aspect, the invention provides a kind of mentioned reagent box for the preparation of the diagnosis immune correlated disease medicine in application.

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

More preferably, described immune correlated disease is tumour or inflammation.

On the other hand, the present invention is a kind of pharmaceutical composition be used to preventing and/or treating immune correlated disease also, described pharmaceutical composition comprise the nucleotide sequence of potential new cytokine LYG1 described above or antibody described above or the LYG1 described above that encodes or contain coding LYG1 described above nucleotide sequence carrier or import or be transfected into the recombinant protein that host's organism produces by the carrier of the nucleotide sequence that contains coding claim LYG1 described above, and one or more pharmaceutical excipients or pharmaceutical carrier.

Preferably, the nucleotides sequence of described coding LYG1 claimed in claim 1 is classified the nucleotide sequence shown in the SEQ ID NO:3 as, described carrier is pcDNA3.1/myc-His (-) B, described host's organism is intestinal bacteria, HEK293T or 293-6E cell, more preferably, described intestinal bacteria are DH5 α.

Immunogene group strategy is adopted in this research, utilizes secretion checking, expression pattern analysis and systemic function research to find the secretory protein of classics of human gene LYG1 coding.Peripheral blood mononuclear cell (the PBMC that it stimulates at lipopolysaccharides (LPS), Peripheral blood mononuclear cell) down-regulated expression in, in the mouse macrophage that LPS stimulates and in inflammatory bowel patient's the inflammatory bowel tissue, the A549 cells that stimulates at IL-1 β raises, THP1 cells at PMA and LPS combined stimulation significantly raises, and expresses obviously after monocyte is divided into dendritic cell and raises.Restructuring LYG1 albumen can be reduced prostate cancer cell line DU145 surface Complement Regulatory Protein CD55 and CD59 in the expression of mRNA level, and downward modulation CD46 and CD59 are in the expression of protein level.In addition, restructuring LYG1 albumen can also promote the interior differentiation of external and body of mouse Th1 cell, plays protective effect in the mouse DTH model, promotes the secretion of IFN-γ, and can suppress the growth of tumour in the mouse melanoma B16 cell tumour model.And the main secretion of gamma-IFN of Th1 cell infects at antiviral, anti-intra-cellular pathogens, brings into play critical function in antitumor, the delayed type hypersensitivity by cellullar immunologic response, and participates in generation, the development of the autoimmune diseases such as rheumatoid arthritis, diabetes.The expression that suppresses Complement Regulatory Protein can make complement system performance antitumor action.Above result points out, and LYG1 is a potential new cytokine, may play a significant role aspect inflammation, infectious diseases, tumour, anaphylactic disease, the autoimmune disease, has potential clinical value.

Description of drawings

Below, describe by reference to the accompanying drawings embodiment of the present invention in detail, wherein:

Fig. 1 is the BFA inhibition test, detect the result of the Secretory Pathway of LYG1, the pcDB negative control group of empty carrier pcDNA3.1/myc-His (-) B (below be abbreviated as pcDB) that has been transfection among the figure, the experimental group of pcDNA3.1/LYG1-myc-His (-) B (below be abbreviated as pcDB-LYG1-myc-his) that LYG1 has been transfection, wherein anti-Actin muscle (anti--actin) be confidential reference items;

Fig. 2 is purifying LYG1 albumen and carries out that SDS-PAGE identifies and the result of N end order-checking, and figure A is the test-results that SDS-PAGE detects the LYG1 purity of protein, and 1 be protein labeling among the figure, 2 LYG1 albumen for reducing, and 3 is non-reducing LYG1 albumen; Fig. 2 B is the test-results 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 are respectively 10 amino-acid residue sequencing results of first amino-acid residue to the;

Fig. 3 is the qualification result of LYG1 polyclonal antibody, method with western blotting (Western blot) detects the LYG1 polyclonal antibody to the recognition capability of albumen in the 293T culture supernatant of overexpression pcDB and LYG1, among the figure, anti-LYG1 (anti-LYG1) is the rabbit source serum (containing the LYG1 polyclonal antibody) behind the LYG1 protein immunization, normal serum (normal serum) is the rabbit source serum before the immunity, the positive control antibodies of anti-myc (anti-myc), anti-rabbit igg (anti-rabbit IgG) is for directly hatching two anti-detection cell pyrolysis liquids, as blank, the negative control group of empty carrier pcDB that pcDB has been transfection, the experimental group of pcDB-LYG1-myc-his that LYG1 has been transfection;

Fig. 4 is the expression pattern analysis test-results of LYG1 (mouse is Lyg1), and Fig. 4 A represents LYG1 wide spectrum expression in normal each tissue of people, and 1 is brain among the figure, 2 is heart, and 3 is kidney, and 4 is liver, 5 is lung, and 6 is pancreas, and 7 is placenta, 8 is skeletal muscle, 9 is colon, and 10 is ovary, and 11 is prostate gland, 12 is small intestine, and 13 is testis; Fig. 4 B represents that LYG1 expresses in immunity system higher, and 1 is white corpuscle among the figure, and 2 is marrow, and 3 is lymphoglandula, and 4 is spleen, and 5 is thymus gland, and 6 is tonsilla, and 7 is the tire liver; Fig. 4 C represents Lyg1 thymus gland in the mouse healthy tissues, and higher level is expressed in stomach and the brain, and 1 is thymus gland among the figure, and 2 is lymphoglandula, and 3 is small intestine, 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 (Dendritic cells at monocyte, DC) Expression In The Process raises, 1 is PBMC among the figure, 2 is monocyte, 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 at the Th1 of mouse tranquillization and activation, the expression in Th17 and the Treg cell; Fig. 4 F represents that real-time quantitative PCR detects LYG1 down-regulated expression in inflammatory bowel patient intestinal tissue, and Fig. 4 G represents that real-time quantitative PCR detects TNF-alpha expression level, as positive control;

Fig. 5 is LYG1 (mouse the is Lyg1) test-results that the expression in the inflammatory model changes under the following 4 kinds of inflammatory factors that make up stimulate, and figure A is the PBMC that LPS (10ng/mL) stimulates; B is the A549 cell that IL-1 β stimulates; C is the THP1 cell that PMA and LPS stimulate; D is the mouse macrophage that LPS stimulates, and extracts mRNA from above cell, and the preparation cDNA library, take cDNA library as template, detects the expression of the mRNA level of LYG1 (mouse is Lyg1).Wherein IL-6, IL-8, IL-1 β are respectively corresponding positive control;

Fig. 6 uses RT-PCR and the mRNA level of flow cytometer detection surface of cell membrane CD46, CD55 and CD59 and the expression changing conditions of protein level add recombinant human LYG1 eukaryotic protein in prostate cancer cell line DU145 after, Fig. 6 A represents the mrna expression level of LYG1 downward modulation CD46 and CD59, the wherein positive contrast of IL-1 β, Fig. 6 B represents the protein expression level of LYG1 downward modulation CD46 and CD59, and wherein CD59 expression changing conditions selects IFN-γ as positive control;

Fig. 7 is the effect of recombinant human LYG1 eukaryotic protein in the differentiation of mouse Th1 cells in vitro, and Fig. 7 A is with Th1 cell (IFN-γ is positive, and ratio is reacted) percentage result behind the method detection adding different concns recombinant human LYG1 eukaryotic protein of streaming; Fig. 7 B is depicted as the secretion situation with Th1 cell IFN-γ behind the ELISA method detection adding different concns recombinant human LYG1 eukaryotic protein.The wherein positive contrast of IL-18;

Fig. 8 is the effect of recombinant human LYG1 eukaryotic protein in the mouse DTH model, wherein, Fig. 8 A is depicted as mBSA and excites rear 24 hours mouse insole swelling situations, Fig. 8 B is for detecting the level of IFN-γ in the mice serum with the method for ELISA, Fig. 8 C and Fig. 8 D are secretion situation (Fig. 8 B of IFN-γ and IL-17A in the mouse LN cells and supernatant of external adding mBSA, 8C, X-coordinate represents the mouse numbering among the 8D, for example PBS-1 represents first mouse of PBS group), the cell proportion situation of Fig. 8 E after for the mouse boosting cell cultivation 72 that adds mBSA with the method detection bodies of streaming outward, provide respectively PBS and LYG1 and organized representational two result, analyzed the ratio of Th1 (the positive ratio of IFN-γ) and the shared CD4+ cell of Th17 cell (the positive ratio of IL-17A) among the figure;

Fig. 9 is the effect of recombinant human LYG1 eukaryotic protein in the murine melanoma model, measures tumour major diameter (a) and minor axis (b) with vernier callipers, and Fig. 9 A is that tumour is seen substantially; Fig. 9 B is tumor growth curve (behind the inoculated tumour every group every day gross tumor volume mean variation curve); Fig. 9 C is tumor weight (mean value of every group of tumor weight).

Embodiment

Employed term unless other explanation is arranged, generally has the implication that those of ordinary skills understand usually in the present invention.Indicate unless have in addition, for the ease of difference, (but not in claims) represent human LYG1 with whole alphabetical LYG1 that all capitalize in this manual, and only the Lyg1 of initial caps represents mouse Lyg1, and LYG1 albumen is recombinant human LYG1 eukaryotic protein.

Below in conjunction with concrete Preparation Example and Application Example, and comparable data is described the present invention in further detail.These embodiment are in order to demonstrate the invention, but not limit the scope of the invention by any way.

Human lung adenocarcinoma cell line A549's cell in following examples, person monocytic cell's type lymphoma cell line THP1 cell, PC-3 DU145 are all available from ATCC; Mouse is C57BL/6J mouse (being purchased from Department Of Medicine, Peking University Laboratory Animal Science section).

The clone of the cDNA of embodiment 1LYG1 gene

By bioinformatic analysis, find UniGene:Hs.164589, its corresponding gene be LYG1 (sequence is SEQ ID NO:3:

AGCAACTGGGGATGCTATGGAAACATCCAAAGCCTGGACACCCCTGGAGCATCTTGTGGGATTGGAAGACGTCACGGCCTGAACTACTGTGGAGTTCGTGCTTCTGAAAGGCTGGCTGAAATAGACATGCCATACCTCCTGAAATATCAACCCATGATGCAAACCATTGGCCAAAAGTACTGCATGGATCCTGCCGTGATCGCTGGTGTCTTGTCCAGGAAGTCTCCCGGTGACAAAATTCTGGTCAACATGGGCGATAGGACTAGCATGGTGCAGGACCCTGGCTCTCAAGCTCCCACATCCTGGATTAGTGAGTCTCAGGTTTCCCAGACAACTGAAGTTCTGACTACTAGAATCAAAGAAATCCAGAGGAGGTTTCCAACCTGGACCCCTGACCAGTACCTGAGAGGTGGACTCTGTGCCTACAGTGGGGGTGCTGGCTATGTCCGAAGCAGCCAGGACCTGAGCTGTGACTTCTGCAATGATGTCCTTGCACGAGCCAAGTACCTCAAGAGACATGGCTTCTAA

Italicized item is the LYG1 non-coding sequence, be added with the part of frame and be the nucleotide sequence of the signal peptide of coding precursor LYG1, all the other are the LYG1 encoding sequence), Ref:NM_1748982.2, GeneID:149999339, it is a unknown function human gene, utilizing the Human_est database to carry out sequence by the BLASTn method proofreaies and correct errorless, then according to the special primer of this sequences Design LYG1 full length gene reading frame, and in upstream primer, add NotI restriction enzyme site gcggccgc and kozak sequence cacc, downstream primer adds KpnI restriction enzyme site sequence ggtacc and polishing base gc:

Upstream primer: gcggccgc cacc atgtctgcattgtggctgctgc (SEQ ID NO:4)

Downstream primer: ggtacc gc gaagccatgtctcttgaggtacttg (SEQ ID NO:5)

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

Reaction volume 50 μ L, wherein contain:

The PCR reaction conditions: 94 ℃, sex change 5 minutes; Then 94 ℃ of sex change are 30 seconds, 61-58 ℃ of annealing 30 seconds, and 72 ℃ were extended 10 circulations of increasing 30 seconds; 94 ℃ of sex change 30 seconds, 58 ℃ of annealing 30 seconds, 72 ℃ were extended 20 circulations of increasing 30 seconds; At last 72 ℃ of downward-extensions 7 minutes.

Amplified production is 3 ' 3 ' the outstanding cohesive end fragment of base A to be arranged, reclaim test kit (Qiagen with QIAquick glue, 28706) carry out purifying by product description, then with 3 ' the linear pGEM-T EASY carrier (Promega that base T arranged, A1360) 16 ℃ of lower connections 8 hours, connect product and transform bacillus coli DH 5 alpha, conversion product is grown at the LB plate culture medium that contains penbritin, selected clone, extract plasmid, use AbI PRISM 3700 DNA analysis instrument (Perkin-Elmer/Applied Biosystem) order-checking.

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

In order to detect the function of LYG1, at first make up the eukaryon expression plasmid that contains the LYG1 open reading frame: pcDNA3.1/LYG1-myc-His (-) B.Cut pGEM-T-LYG1 with Not I and Kpn I enzyme, cut carrier for expression of eukaryon pcDNA3.1/myc-His (-) B (below be abbreviated as pcDB) (Invitrogen with Not I and Kpn I enzyme simultaneously, V85520), the cDNA gene fragment of LYG1 after enzyme cut is connected 8 hours with carrier under 16 ℃, transform bacillus coli DH 5 alpha, conversion product is grown at the LB plate culture medium that contains penbritin, select the bacterium colony of growth, extract plasmid, PCR identifies, select positive colony, (use ABI PRISM 3700DNA analyser by order-checking, the same), select the cDNA gene plasmid of the LYG1 of correct insertion sequence, called after pcDB-LYG1-myc-his (with c-myc and 6XHis label).

Embodiment 3LYG1 is the secretory protein of a classical pathway

1. cell cultures and transfection

Cultivations of going down to posterity of HEK293T cell (be Japan Tadashi professor Matsuda present), this chamber, usefulness contains the DMEM culture medium culturing of 10% foetal calf serum (FBS).The HEK293T cell operates transfection plasmid pcDB-LYG1-myc-his and pcDB with Vigofect (prestige lattice Lars) positively charged ion transfection reagent according to specification sheets.1 * PBS washed cell of transfection usefulness normal temperature after 6 hours is once changed fresh HEKG serum free medium, adds respectively 10 μ g/mL BFA (Brefeldin-A) and ethanol (as negative control) in cell culture supernatant.

2. cells and supernatant and total protein of cell extract and western blotting (Western blot) analysis

The collection of HEK293T cell culture supernatant: after 48 hours, when cell confluency approximately reaches 90%, collect each experimental group cell and cell culture supernatant, in 4 ℃, centrifugal 10 minutes of 2000g abandons precipitation, again in 4 ℃, centrifugal 15 minutes of 15000g removes cell debris, leaves and takes cell culture supernatant.

The extraction of HEK293T total protein of cell: the cell that will leave and take after the supernatant liquor places on ice, and ice-cold 1 * PBS washes twice, and ice-cold 1 * PBS blows down cell, with cell harvesting in the 1.5mL centrifuge tube, in 4 ℃, centrifugal 5 minutes of 2000g.Remove supernatant, and adding RIPA cell pyrolysis liquid in precipitation (20mM Tris-HCl, pH 7.4,150mM NaCl, 1mM EDTA, 1mM EGTA, 1%Triton X-100, proteinase inhibitor C ocktail), placed on ice 30 minutes, 4 ℃, centrifugal 15 minutes of 15000g, supernatant liquor changes new pipe over to, be stored in-80 ℃ for subsequent use.

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

Western blotting: every group of cell pyrolysis liquid got total protein 30ug, and every group of cell culture supernatant got 40 μ L, adds albumen sample-loading buffer (Beijing Bao Sai Bioisystech Co., Ltd), boils 10 minutes in 99 ℃.The 12.5%PAGE electrophoresis, the 100V electricity turns 1.5 hours, and TBST liquid balance with 5% milk room temperature sealing 1 hour, adds corresponding primary antibodie, and 4 ℃ are spent the night, and fully wash film 3 times with TBST, each 10 minutes; Then two anti-(1: 10000) that add corresponding IRDyeTM 700/800 mark, room temperature lucifuge reaction 1 hour; Fully wash film 3 times with TBST again, each 10 minutes; Use at last infrared imaging system scanning imaging system (Odyssey Infrared Imager, U.S. LI-CORBioscience company) detection signal.

As shown in Figure 1, the result shows, add BFA after, the LYG1 secretion obviously reduces, and prove the secretory protein of LYG1 for secreting by classical pathway (endoplasmic reticulum golgi body approach).

Purifying and the N terminal Sequence Analysis of embodiment 4 recombinant human LYG1 eukaryotic proteins

In order to carry out the functional study of potential new cytokine LYG1, we 293-6E cells and purifying the LYG1 recombinant protein.The 293-6E cell is purchased from Canadian National Research Council (national research council of canada).The 293-6E cell is used DMEM (Dulbecco ' s modified Eagle ' s medium) substratum that contains 10% foetal calf serum, at 5%CO ₂, cultivated 24 hours in 37 ℃ the incubator.Use PEI positively charged ion infection protocol transfection pcDB-LYG1-myc-his eukaryon expression plasmid.

1 * PBS washed cell of transfection usefulness normal temperature after 16 hours is once changed fresh HEKG serum free medium, at 5%CO ₂, cultivate in 37 ℃ the incubator.Collecting cell culture supernatant after 48 hours, in 4 ℃, centrifugal 10 minutes of 2000g, remove the cell of culture supernatant, abandon precipitation, again in 4 ℃, centrifugal 20 minutes of 18000g removes the finely ground particle substance in the supernatant, and the cells and supernatant of leaving and taking after the processing is used for subsequent purification.

With the cell culture supernatant after the above-mentioned processing of Ni2+ post material purifying: after first supernatant liquor being filtered through 0.45 μ m filter, in supernatant liquor, add imidazoles (10mM)/NaCl (200mM), be combined with the post material again, flow rate control is at 10 per minutes, use again level pad [imidazoles (20mM)/NaCl (200mM) in1 * PBS (pH7.4)] flushing post material, with the unconjugated foreign protein of flush away, after dividing a light value to be back to baseline, with elutriant [imidazoles (500mM)/NaCl (200mM) in1 * PBS (PH7.4)] elution of bound albumen until a minute light value no longer descend, collect elutriant in super filter tube, with 1 * PBS (pH7.4) 12mL of precooling, 4 ℃ centrifugal 2 times, be concentrated into 500 μ L, sucking-off albumen, in 4 ℃, 18000g degerming in centrifugal 20 minutes, packing be stored in-80 ℃ stand-by.

Get 5 μ L albumen and with the BCA method it is carried out protein quantification, get sample segment and add the albumen sample-loading buffer, boiled 5 minutes in 100 ℃ of water-baths, carry out SDS-PAGE and western blotting and detect, identify the purity of LYG1 protein purification, the result is shown in Fig. 2 A, and the purity of albumen is higher, greater than 90%, intracellular toxin is residual less than 1EU/mg, may exist glycosylation modifiedly, and ExPASy database analysis prediction LYG1 contains the glycosylation modified site of O, and this conforms to it.Not adding beta-mercaptoethanol in the sample is non-reducing LYG1 albumen, adds beta-mercaptoethanol and is the LYG1 albumen of reduction.The molecular weight of SDS-PAGE electrophoresis detection is the apparent molecular weight of protein, is not real molecular size range.Under reductive condition, intrachain disulfide bond is opened fully, presents strip during the protein SDS-PAGE electrophoresis; Under non-reduced condition, albumen is because the existence of intrachain disulfide bond, than under the reductive condition comparatively speaking closer to sphere.So under the non-reduced condition, the albumen apparent molecular weight is just smaller.

In addition, get sample segment and carry out SDS-PAGE, and with protein delivery to pvdf membrane, after dyeing and the decolouring, be used for the protein N terminal sequence analysis.The result has detected 10 amino acid of N end shown in Fig. 2 B, be respectively S/A, N, and W, G ,/, Y, G, N, I, Q illustrates that 19 amino acid of N end of precursor LYG1 are signal peptide sequence.A higher peak is wherein arranged among each figure, and this is the by product dptu of system secretion, is relative confidential reference items.So by our experimental verification, the LYG1 maturation protein (is namely brought into play the albumen of function, be potential new cytokine LYG1) aminoacid sequence be the sequence of having excised after precursor LYG1N holds front 19 amino acid, below the recombinant human LYG1 eukaryotic protein used of all embodiment all meet this requirement.

Preparation and the evaluation of embodiment 5LYG1 polyclonal antibody

Antibody can be used for detecting, diagnoses and the treatment disease, so we have prepared the antibody of LYG1.LYG1 protein immune animal with purifying.Select bull new zealand rabbit (buying in Department Of Medicine, Peking University's Laboratory Animal Science section), initial immunity fully mixes with isopyknic Freund's complete adjuvant after the antigen of 300 μ g is diluted to 1mL with PBS, in each the 0.25mL subcutaneous injection of two foots, the subcutaneous multiple spot of all the other back parts (6 point) injection.Afterwards per 3 all booster immunizations once, consumption is the same, all subcutaneous multiple spots of back part (8 point) injections.Get for the third time rabbit ear edge venous samples can behind the booster immunization in 10 days and detect and tire, reach 1: 10 to tiring ⁵After above, rabbit is put to death heart extracting blood, obtain serum.Carrying out preliminary western blotting identifies.As shown in Figure 3, anti-LYG1 group (anti-LYG1 group) (serum after the immunity, 1:: 1000 dilutions, experimental group), anti-myc group (anti-myc) (positive control), control serum (normal serum before the immunity, dilution in 1: 1000), anti-rabbit igg (anti-rabbit IgG) (two anti-contrasts), the polyclonal antibody of anti-LYG1 (anti-LYG1) in can the specific recognition cell culture supernatant LYG1 albumen and the LYG1 recombinant protein of purifying, can be used for the diagnosis and detection of LYG1 relative disease.Albumen has two bands, is specificity, and may there be modification in prompting LYG1 albumen.

(RNA extracts the expression pattern analysis of embodiment 6LYG1 in tissue and cell, RT-PCR With Real-time PCR)

Change in order to analyze the expression of LYG1 in healthy tissues, in different differential periods of immunocyte and the different cellular inflammation model, we use the Normal human tissue cDNA library of the Clontech company of purchase, the mouse healthy tissues cDNA library of laboratory preparation and the cell library of people and mouse adopt nest-type PRC that LYG1 is increased.The cell library comprises and being prepared as follows: the DC cell of collecting different differential periods; the Th cell of mouse tranquillization and activation; (White Blood Cells Concentrate is not provided by the Beijing Red Cross Blood Center PBMC of stimulation and inflammatory factor LPS (100ng/mL) stimulation 6h; adopt lymphocytes separating solution (available from the smart bio tech ltd of Shanghai China) density gradient centrifugation to obtain); do not stimulate and inflammatory factor Buddhist ripple ester (PMA; Phorbol-12-myristate-13-acetate) (10ng/mL; 24 hours) rear with LPS (100ng/mL) stimulation 3h; 6h; the THP1 of 12h; do not stimulate and inflammatory factor IL-1 β (10ng/mL) stimulation 3h; 6h; 15h; the A549 cell of 24h; do not stimulate and inflammatory factor LPS stimulation 2h; 4h; (every mouse peritoneal is injected 3% sodium thioglycolate 2ml to the mouse macrophage of 6h; disconnected neck is put to death fixing after 48 hours; to intraperitoneal injection 5mlPBS; open the abdominal cavity behind the soft abdominal cavity and reclaim PBS; 2000rpm; 4 ℃ of centrifugal peritoneal macrophages that obtain), use TRIzol to extract total RNA.Each sample is got 2 μ g, with reverse transcription test kit [Reverse transcriptTM kit (Invitrogen)] the synthesizing single-stranded cDNA library of reverse transcription.GAPDH is as confidential reference items in amplification.Real-time quantitative PCR adopts SYBR Green method and quantitative real time PCR Instrument, and (the LightCycler instrument ABI7500) carries out.

The amplification of people LYG1: use outside upstream primer 5 '-tttcaggagccgtagagcc (SEQ ID NO:6), outside downstream primer 5 '-tgatcatggtcttgggttt (SEQ ID NO:7), inboard upstream primer 5 '-gcggccgc cacc atgtctgcattgtggctgctgc (SEQ ID NO:8), inboard downstream primer 5 '-ggtacc gc gaagccatgtctcttgaggtacttg (SEQ ID NO:9) carry out the nido amplification.One expansion condition is: 94 ℃ (5 minutes) → 94 ℃ (30 seconds), 56 ℃ (30 seconds), 72 ℃ (30 seconds), → 72 ℃ (7 minutes) of 30 circulations of increasing.One expand production 50 times of thing dilutions are expanded templates, 94 ℃ (5 minutes) → 94 ℃ (30 seconds), 58 ℃ (30 seconds), 72 ℃ (30 seconds), → 72 ℃ (7 minutes) of circulating of increase 30 as two.

The amplification of mouse Lyg1: use outside upstream primer: 5 '-agagtctccaggaggcaactatgtg (SEQ ID NO:10), outside downstream primer 5 '-ccatggtccttgaagtatttggct (SEQ ID NO:11); Inboard upstream primer 5 '-aacattggcagtggacttggga (SEQ ID NO:12), inboard downstream primer 5 '-gcgcaaggacgtcattgcag (SEQ ID NO:13) carry out the nido amplification, and GAPDH is as confidential reference items in amplification.Amplification condition is an expansion: 94 ℃ (5 minutes) → 94 ℃ (30 seconds), 56 ℃ (30 seconds), 72 ℃ (30 seconds), → 72 ℃ (7 minutes) of 30 circulations of increasing.One expand production 50 times of thing dilutions are expanded templates, 94 ℃ (5 minutes) → 94 ℃ (30 seconds), 58 ℃ (30 seconds), 72 ℃ (30 seconds), → 72 ℃ (7 minutes) of circulating of increase 30 as two.

The result shows, shown in Fig. 4 A, LYG1 has low expression level in Various Tissues, such as kidney, and liver, lungs, pancreas, placenta, skeletal muscle, colon, ovary, prostate gland, small intestine and testis; Shown in Fig. 4 B, in immunity system, express slightly high, especially marrow, thymus gland and tonsilla.In addition, we have prepared mouse healthy tissues cDNA library, and are consistent with the Normal human tissue express spectra, and Lyg1 expresses slightly high in thymus gland, and also have higher level to express in stomach and brain, at colon, and low expression level in kidney and the lung (shown in Fig. 4 C).We have also detected the expression that is divided into LYG1 in the DC cell processes at monocyte: LYG1 and have expressed lower in PBMC and monocyte, in imDC, express slightly highly, and stimulate LYG1 up-regulated among the ripe DC (shown in Fig. 4 D) at zymosan and LPS.Zymosan and LPS have mediated respectively DC to be broken up to Th17 and Th1 cell direction T cells (Naive T cell), and LYG1 is as a secretory protein, it is up-regulated in the DC of zymosan and LPS stimulation, and prompting LYG1 might participate in the differentiation of Th cell in the microenvironment.In addition, we have also detected the expression of Lyg1 in the different Th cell subsets of mouse, find that Lyg1 is higher at Th17 and Treg cells, at Th1 cells lower (shown in Fig. 4 E).In addition, we have also detected the expression of LYG1 in inflammatory bowel (IBD) patient's inflammatory bowel tissue and normal people's intestinal tissue.Discovery is in patient's inflammatory bowel tissue, and the down-regulated expression of LYG1 (shown in Fig. 4 F, 4G) points out the genesis of LYG1 and inflammatory bowel to have substantial connection.In addition, the expression of THP1 cell (Fig. 5 C) LYG1 that stimulates of the A549 cell (Fig. 5 B), PMA and the LPS that stimulate of we PBMC (Fig. 5 A), the IL-1 β that have also detected LPS and stimulate and the expression of mouse macrophage (Fig. 5 D) Lyg1 that LPS stimulates.As shown in the figure, LYG1 is down-regulated expression in the PBMC that LPS stimulates, and raises at the A549 cells that IL-1 β stimulates, and in the THP1 cell of PMA and LPS Co stituation, the expression of LYG1 is obviously raised.The down-regulated expression of Lyg1 in the mouse macrophage that LPS stimulates.These results suggest LYG1 may participate in generation and the development of inflammation.The effect that inflammatory bowel tissue of patient and inflammation model tormulation spectrum all point out LYG1 to have anti-inflammatory, its recombinant protein or antibody have the function for the treatment of inflammation.

Embodiment 7LYG1 is on the impact of Complement Regulatory Protein molecule

The high expression level of Complement Regulatory Protein (CRP) can suppress complement system to the attack of tumour cell, makes tumour escape the immunity of organism defence.Thereby cytokine can be regulated albumen by regulate complement and bring into play critical function in tumor immunotherapy.As in to the research of kidney, finding the expression that IL-1 can reduce CD46, CD59, IL-4 can continue downward modulation CD46, CD55 and transforming growth factor, increase the deposition of C3 on tumour cell, when using antibody for antitumor related antigen, unite and use cytokine will become a kind of new effective immunotherapy method.So we have observed the impact that LYG1 expresses tumor cell line surface C RP.Add restructuring LYG1 albumen in the DU145 culture system, collect the cell of 48h and do the RT-PCR test, the 72h cell is done fluorescent dye.The result shows, with do not add LYG1 albumen control group and compare, LYG1 can reduce CD55 and CD59 in the expression (Fig. 6 A) of mRNA level, downward modulation CD46 and CD59 are in the expression (Fig. 6 B) of protein level, and the horizontal CD46 of mRNA is unchanged, protein level CD55 is unchanged, infers that itself and the time point of collecting cell have relation.Experiment shows that LYG1 can stop immunologic escape by the expression of downward modulation CRP, so that complement system can be brought into play anti-tumour effect, thereby the genesis of inhibition tumour shows further that also LYG1 albumen has activity.

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

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

1.1 separation and the cultivation of mouse CD4+T cell

Get 6～8 all female C57BL/6J mouse, the cervical vertebra dislocation is put to death, and 75% alcohol immersion 1 minute is taken out mouse and placed on the aseptic ware, cuts off osculum at mouse abdomen middle part, tears skin, and eye scissors separates reticular tissue, gets the both sides inguinal lymph nodes; Enter afterwards the abdominal cavity and get mesenteric lymph nodes and the other lymphoglandula of both sides aorta abdominalis, put into and place the culture dish that fills RPMI-1640 to grind gently the pressure lymphoglandula with pestle on 200 eye mesh screens, obtain cell suspension, according to negative sorting test kit (Invitrogen DYNAL) specification sheets of mouse CD4+T cell, obtain mouse CD4+T cell.Be specially: with damping fluid 1 resuspended LN cell, add foetal calf serum (FBS) and mixtures of antibodies (anti-mouse B220 (anti-mouse B220), CD11b, Ter-119, CD16/32 and CD8), hatched 20 minutes for 4 ℃, wash one time with damping fluid 1, add the magnetic bead of washing, incubated at room 15 minutes, magnetic frame leaves standstill 2min, can obtain the CD4+T cell.Get part CD4+T cell, add anti-mouse CD4-PerCP antibody (anti-mouse CD4-PerCP antibody), lucifuge 4 degree are hatched 20min, and PBS washes once, and 200 μ L are resuspended, the purity of the CD4+T cell of the method grouping system of usefulness streaming.The CD4+T cell is resuspended with RPMI 1640 substratum that contain 10% hot inactivated serum, and concentration is 2X10 ⁶/ mL spreads to 48 orifice plates every hole 500 μ L.

1.2 the Differentiation Induction in vitro of mouse Th cell and iTreg cell

Obtain mouse CD4+T cell according to 1.1 described step magnetic bead sortings, be incubated in the 48 pre-coated orifice plates of anti-mouse CD3 (anti-mouse CD3) (10 μ g/mL), add anti-mouse CD28 (anti-mouse CD28) (2 μ g/mL) and rhIL-2 (4ng/mL), and add exogenous LYG1 recombinant 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.Change liquid on the 4th day, can obtain mouse Th1 and Th17 cell in two days only having under IL-2 (4ng/mL) condition to cultivate.The inductive condition of iTreg is: IL-2,4ng/mL; TGF-β, 5ng/mL; Got final product in external evoked three days.

2. immunofluorescence dyeing

2.1 membrane molecule detects

Gather in the crops dissimilar immunocyte, use the PBS/0.1%BSA washed twice, directly add 100 μ L confining liquid (PBS/10% normal sheep serum) re-suspended cells, 4 ℃ were sealed 30 minutes; The antibody that adds subsequently mark, 4 ℃ of lucifuges are hatched 40min; Use corresponding IgG to contrast as homotype.After using at last the PBS/0.1%BSA washed twice, by the flow cytometer collecting cell, use Cellquest software (BD company) that the result is analyzed.

2.2 Molecular Detection in the cell

Gather in the crops dissimilar immunocyte, with cold PBS/0.1%BSA washed twice, at first with 4% Paraformaldehyde 96 fixing 30min on ice; Then use 0.1%Triton X-100 incubated at room 30min, the centrifugal 5min of 1500rpm.Add again 100 μ L confining liquid (PBS/10% normal sheep serum) re-suspended cells, room temperature sealing 30 minutes.Add subsequently traget antibody, 4 ℃ of lucifuges are hatched 40min; Use corresponding IgG to contrast as homotype.After using at last the PBS/0.1%BSA washed twice, by the flow cytometer collecting cell, use Cellquest software that the result is analyzed.

3.ELISA

Cell cultures is gathered in the crops supernatant after the corresponding time, and in 4 ℃, centrifugal 10 minutes of 2000g abandons precipitation, and again in 4 ℃, centrifugal 15 minutes of 15000g removes cell and fragment in the supernatant.Use IFN-γ, IL-4, IL-17A cytokine ELISA test kit (eBioscience) sandwich assay to detect the secretory volume of cytokine.

Shown in Fig. 7 A-B, recombinant human LYG1 eukaryotic protein is activated in the mouse system, it can promote the vitro differentiation (IFN-γ is positive, and per-cent improves) of mouse Th1 cell and the secretion of IFN-γ, and there were significant differences (p=0.0002) with not adding the LYG1 group for LYG1-1ng/mL.

Because the main secretion of gamma-IFN of Th1 cell, IL-2, LT α etc., infect at antiviral, anti-intra-cellular pathogens, play a significant role in antitumor, the delayed type hypersensitivity by cellullar immunologic response, thereby so LYG1 recombinant protein and antibody can be by promoting that Th1 cytodifferentiation and IFN-γ secretion is infected at antiviral, anti-intra-cellular pathogens, anti-tumor aspect have significant application value.

Functional study in the body of embodiment 9LYG1

1.LYG1 can suppress mouse delayed type hypersensitivity (DTH)

In order to study LYG1 in vivo on the impact of Th cytodifferentiation and survival, we have induced the mouse DTH model with mBSA, choose female C57BL/6J mouse in 8 ages in week, gave 2 injections of every mouse web portion intracutaneous, 125 μ g (50 μ L) the adequately emulsified mBSA of CFA on the 0th day, every day abdominal injection 100ng LYG1 albumen.The left sufficient lift hemostasis 150 μ g mBSA/PBS solution (30 μ L) of every mouse excited in the 7th day, and the isopyknic PBS of right sufficient lift hemostasis is as negative control.All use vernier caliper measurement mouse insole thickness before exciting and after exciting 24 hours.The sufficient mat thickness change calculations formula of weighing the DTH response intensity is: footpad swelling degree (mm)=(the sufficient mat thickness that mBSA excites-excite forefoot pad thickness)-(the sufficient mat thickness of PBS injection-excite forefoot pad thickness) (1. Takayuki Yoshimoto, et al.Role of IL-16in delayed-type hypersensitivity reaction Blood.2000; 2869-74; 2. Akina Ishii1, Keisuke Obok, et al.Development of IL-17-mediated Delayed-Type Hypersensitivity is not affected by down-regulation of IL-25 expression. Allergol Int.2010; 399-408).Then eyeball is got blood, and 4 ℃ are spent the night, and 3000rpm is centrifugal, gets serum to new Ep pipe, and the centrifugal 10min of 12000rpm removes fragment, and the ELISA that gained serum namely can be used for IFN-γ detects.Get spleen, inguinal lymph nodes and mesenteric lymph nodes, grind and obtain splenocyte and lymph-node cell (LN cell), resuspended one-tenth 5 * 10 ⁶/ mL is incubated at 24 orifice plates, cultivates respectively 5 days and 3 days under 4 ℃ of pre-coated CD3 antibody that spend the night of 10 μ g/mL mBSA or 3 μ g/mL stimulate, and obtains respectively that cell carries out flow cytometer detection and culture supernatant is carried out the ELISA detection.

Found that, as shown in Figure 8, LYG1 albumen can suppress mouse insole swelling degree (Fig. 8 A), and the concentration of IFN-γ is apparently higher than PBS control group (Fig. 8 B) in the serum.In the lymph-node cell culture supernatant of stimulated in vitro, the concentration of IFN-γ (Fig. 8 D) and IL-17A (Fig. 8 C) all is significantly higher than the PBS control group, but IFN-γ multiple is higher.The result of streaming (Fig. 8 E) also shows, LYG1 group Th1 (the positive ratio of IFN-γ) and Th17 (the positive ratio of the IL-17A) ratio of cell in the CD4+ cell all are higher than PBS to be organized, but the Th1 cell gets ratio more significant difference is arranged.The result of study report is arranged, and in the DTH model that mBSA induces, the main CD4+ cell that participates in is Th1 and Th17 cell, and the Th1 cell of secretion of gamma-IFN has provide protection in the DTH morbidity, and the Th17 cells of secretion IL-17A can increase the weight of the development of DTH.Inducing of DTH model is divided into two stages, and front 7 days is the differential period of antigen specific T h cell, is the effective stage of antigen specific T h cell after exciting.In this experiment; the every day in immunity stage is abdominal injection LYG1 albumen all; and do not inject albumen after exciting; IFN-γ concentration LYG1 group is significantly higher than control group in the serum; the content of LYG1 group IFN-γ is apparently higher than control group in the culture supernatant of the LN cell of same stimulated in vitro; LYG1 group Th1 cell proportion obviously improves in the splenocyte that exo-antigen stimulates, the differentiation that prompting LYG1 might be by promoting antigen specific T h1 cell and the secretion of IFN-γ and delayed type hypersensitivity DTH model has been brought into play provide protection.This is consistent with external result of study, and proof LYG1 can treat the anaphylaxis of Th1 cell (IFN-γ) mediation.

2.LYG1 can suppress the tumor growth of B16 tumour

Early stage, results suggest LYG1 can promote the differentiation of Th1 cell and the secretion of IFN-γ, and the expression that can reduce the tumor cell surface Complement Regulatory Protein, and prompting LYG1 may participate in anti tumor immune response.Mouse B16 tumor model has reduced immunogenicity, and is responsive to immunotherapy, and is difficult for the characteristics of transfer, is that research is by the immunity related molecular especially classical animal model of cytokine therapy tumour.So the effect of LYG1 at anti-tumor aspect that we have utilized this model research.

Choose female C57BL/6J mouse in 8-10 age in week, oxter subcutaneous injection B16 cell (2X10 ⁵Individual cell/100 μ L PBS/ mouse), observed the tumor growth situation to the mouse depilation on the 5th day.According to the tumor growth situation, with mice group, at least 5 every group.Every group of mouse peritoneal injection LYG1 albumen (L/ days/mouse of 20 μ g/200 μ), control group abdominal injection PBS (200 μ L/ days/mouse) injected 5 days altogether continuously.Measure tumour major diameter (a) and minor axis (b) with vernier callipers, the volume algorithm of tumour is V=1/2ab ²The result compares with the PBS group as shown in Figure 9, and injection protein groups gross tumor volume obviously reduces (Fig. 9 A), and tumor growth is (Fig. 9 B) slowly, and tumor weight obviously reduces (Fig. 9 C).Above result proves that restructuring LYG1 albumen can obviously suppress the growth of B 16 tumours.

3. statistical analysis

The representative of the standard deviation of above data is the differences between different multiple holes in the experiment once, and above experiment all repeats more than twice at least, with once data wherein as representative; Whether have significant difference between each group of Student ' s T check analysis, p＜0.05 thinks to have significant difference.

Claims

1. potential new cytokine LYG1 who is used for preventing and/or treating immune correlated disease, the sequence of described LYG1 is the aminoacid sequence shown in the SEQ ID NO:1, preferably, described potential new cytokine LYG1 is people's type cytokines LYG1 or mouse cytokine LYG1.

2. the antibody of the LYG1 in the anti-claim 1.

The nucleotide sequence of potential new cytokine LYG1 according to claim 1 or antibody claimed in claim 2 or the LYG1 claimed in claim 1 that encodes or contain the LYG1 claimed in claim 1 that encodes nucleotide sequence carrier or import or be transfected into recombinant protein that host's organism produces for the preparation of the application in the medicine that prevents and/or treats immune correlated disease by the carrier of the nucleotide sequence that contains the LYG1 claimed in claim 1 that encodes.

4. application according to claim 3 is characterized in that, the medicine of described treatment immune correlated disease is the medicine of antitumor drug, anti-inflammatory medicaments, anti-infectious disease, treatment anaphylactic disease and/or treatment autoimmune disease.

5. application according to claim 4, it is characterized in that, the tumour of described tumour for expressing by promotion Th1 cytodifferentiation, promotion cytokine IFN-γ secretion and/or inhibition Complement Regulatory Protein CD46, CD55 and CD59 to suppress and/or treat, more preferably, described tumour is melanoma or to the tumour of immunotherapy sensitivity;

Or described inflammation, infectious diseases, anaphylactic disease and autoimmune disease inflammation, infectious diseases, anaphylactic disease and the autoimmune disease for secreting by promotion Th1 cytodifferentiation, promotion cytokine IFN-γ to suppress and/or treat, more preferably, described inflammation is inflammatory bowel.

6. potential new cytokine LYG1 according to claim 1, or antibody claimed in claim 2, or the nucleotide sequence of the LYG1 claimed in claim 1 that encodes, or contain the carrier of the nucleotide sequence of the LYG1 claimed in claim 1 that encodes, or import or be transfected into recombinant protein that host's organism produces for the preparation of promoting the Th1 cytodifferentiation by the carrier of the nucleotide sequence that contains the LYG1 claimed in claim 1 that encodes, promote cytokine IFN-γ secretion and/or suppress Complement Regulatory Protein CD46, application in the medicine that CD55 and CD59 express.

7. test kit that is used for the diagnosis immune correlated disease, described test kit comprises antibody claimed in claim 2 and/or is used at the DNA chain of PCR composite coding LYG1 nucleotide sequence claimed in claim 1 and/or the PCR primer of its cDNA chain, and described antibody is used for the detection by quantitative of LYG1 albumen, detection and the auxiliary diagnosis of relative disease; Described primer is used for the detection by quantitative of LYG1 genetic expression.

Test kit according to claim 7 for the preparation of the diagnosis immune correlated disease medicine in application, preferably, described immune correlated disease is tumour, inflammation, infectious diseases, anaphylactic disease and/or autoimmune disease, more preferably, described immune correlated disease is inflammation.

9. pharmaceutical composition that is used for preventing and/or treating immune correlated disease, described pharmaceutical composition comprise the nucleotide sequence of potential new cytokine LYG1 claimed in claim 1 or antibody claimed in claim 2 or the LYG1 claimed in claim 1 that encodes or contain the LYG1 claimed in claim 1 that encodes nucleotide sequence carrier or import or be transfected into the recombinant protein that host's organism produces by the carrier of the nucleotide sequence that contains the LYG1 claimed in claim 1 that encodes, and one or more pharmaceutical excipients or pharmaceutical carrier.

10. according to claim 3 each described application in 6 or 9, it is characterized in that, the nucleotides sequence of described coding LYG1 claimed in claim 1 is classified the nucleotide sequence shown in the SEQ ID NO:3 as, described carrier is pcDNA3.1/myc-His (-) B, described host's organism is intestinal bacteria, HEK293T or 293-6E cell, preferably, described intestinal bacteria are DH5 α.