CN103382220A

CN103382220A - Cell factor FAM19A4 with anti-infection and antineoplastic activity and application thereof

Info

Publication number: CN103382220A
Application number: CN2012101355420A
Authority: CN
Inventors: 韩文玲; 马大龙; 王文彦; 张荷钰; 王平章; 张颖妹; 宋泉声; 李婷; 付伟伟; 潘文
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2012-05-03
Filing date: 2012-05-03
Publication date: 2013-11-06

Abstract

The invention relates to a cell factor FAM19A4 with anti-infection and antineoplastic activity and application thereof. Specifically speaking, the invention provides a gene or protein of FAM19A4 or immunological fragments thereof, genetic engineering vectors and host cells including the gene or protein of FAM19A4 or the immunological fragments thereof and antibodies to the protein of FAM19A4 or the immunological fragment thereof. The invention further provides application of the gene or protein of FAM19A4 or the immunological fragments thereof in preparation of medicinal preparations having antiviral, anti-endocellular bacterial, antimycotic, anti-extracellular bacterial and antineoplastic effects. Furthermore, the invention also provides application of a detection reagent in preparation of compositions used for auxiliary diagnosis and prognosis of diseases related to immunization.

Description

There is cytokine FAM19A4 and application thereof anti-infective, anti-tumor activity

Technical field

The present invention relates to a kind of new human cell's factor and application thereof, particularly relate to gene or albumen or their the immunity fragment of FAM19A4, the gene of described FAM19A4 or albumen or their immunity fragment have anti-infective, anti-tumor activity.

Background technology

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 body various kinds of cell secretion, 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), chemokine (Chemokine) etc.Cytokine is the interchange language between immunocyte, and innate immunity and adaptive immune response are all played an important role.

Innate immunity is the first line of defence that body is resisted the pathogenic agent invasion, barrier structure, innate immune molecule and innate immune cells etc., consists of.Innate immune cells comprises phagocytic cell (neutrophil leucocyte, monocytes/macrophages) and dendritic cell etc.Wherein phagocytic cell by sticking, ooze out, chemotaxis is raised and move to the inflammation kitchen range; By Receptor recognitions such as mannose receptor, complement receptor, Toll sample acceptor and IgGFcR with engulf pathogenic agent; Kill and wound with aerobic (H202, oxyradical, the NO) mechanism of killing and wounding by anaerobic and kill and wound pathogenic agent; Secrete multiple inflammatory cytokine and promote inflammatory reaction.Dendritic cell is known as the bridge cell of mediation innate immunity and adaptive immune response.

Adaptive immune response comprises cellullar immunologic response and humoral immunoresponse(HI), CD4 ⁺the T cell is the hinge of adaptive immune response.Cytokine not only can determine CD4 ⁺the differentiation direction of T cell, the also CD4 to having broken up ⁺the T cell has regulating and controlling effect; In addition, cytokine or CD4 ⁺the major way of T cells play effect, characteristic cytokine IFN-γ, IL-4, IL-17 and TGF-β that Th1, Th2, Th17 and iTreg cell produce play a significant role in the processes such as bacterium and fungi, adjusting inflammation and autoimmune disease respectively outside anti-intracellular infection, parasite, born of the same parents.

Cytokine is brought into play important regulative in immunne response, and as the Th1 cell can produce characteristic cytokine IFN-γ, IL-2 and TNF-α etc., IFN-γ is macrophage activating factor (MAF), can strengthen its engulfing and kill capability microorganism in born of the same parents; IFN-γ can also induce the B cell to produce IgG2a and the IgG3 with strong immune opsonization, further promotes the phagocytic function of scavenger cell.IL-2 can promote the Peptide-specific CTL cell activation, for the activation of part CTL provides second signal.CTL is most important antigen-specific killer cell in body, and virus infected cell and tumour cell are had to very strong lethal effect.TNF-α can activate neutrophil leucocyte, promotes it to kill and wound pathogenic agent.In a word, after the Th1 cell activation, produce above-mentioned effector cell's factor, the mediated cell immunity, play a significant role in infected by microbes, antineoplastic immune in anti-born of the same parents, and participate in generation and the development of delayed type hypersensitivity (delayed type hypersensitivity, DTH).Produce cytokine IL-17, IL-22 etc. after the Th17 cell activation, IL-17 further raises, activates neutrophil leucocyte; IL-22 can induce epithelial cell to produce alexin and kill and wound thalline, plays a significant role in antimycotic and anti-extracellular bacteria infects, and participates in generation, the development of autoimmune disease.

Utilize at present the recombinant cytokine of genetic engineering technique production or recombinant soluble acceptor and neutrality antibody to receive good efficacy at aspects such as treatment tumour, hematopoietic disorders, infection, 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 approved production at present comprises IFN-α, β, γ, Epo, GM-CSF, G-CSF, IL-2 etc.According to incompletely statistics, at least have 26 in the world at present and enter clinical study by genomic drug, comprise new recombinant cytokine, recombinant soluble acceptor and neutrality antibody etc.As IL-2 is that first is proved to be the cytokine with oncotherapy effect, successfully listing in 1992, it is mainly used in treating renal cell carcinoma and melanoma.IL-12 can promote the Th1 cytodifferentiation, promotes NK cell and CTL cell activation, strengthens its killing ability, and it is clinical that its research aspect ovarian cancer resistance has entered the II phase.

On the other hand, along with the widespread use of microbiotic, corticosteroid hormone, immunosuppressor, Resistant strain gradually becomes rising tendency clinically, and in various born of the same parents, the outer infected by microbes of born of the same parents is obvious ascendant trend, and immunocompromised host crowd's infection probability is increased substantially.For the anti-infectious ability of enhancing body, developing new cytokine is medicine, and the ability of the Proliferation and invasion of raising immunocyte inhibition pathogenic agent is just most important.

Summary of the invention

An object of the present invention is to provide a kind of new cytokine.

This case contriver adopts immunogene group strategy, utilize that information biology is selected, functional screening and systemic function research found a kind of new cytokine: human cell's factor FAM19A4, the correlative study results suggest, FAM19A4 may play a significant role at the aspect such as anti-infective, antitumor, has potential clinical value.

Thereby, particularly, an object of the present invention is to provide albumen or its derived protein or their the immunity fragment of a kind of FAM19A4.

Another object of the present invention is to provide the albumen of coding FAM19A4 or the polynucleotide sequence of its derived protein or their immunity fragment.

Another object of the present invention is to provide the carrier of a kind of FAM19A4 of comprising.

Another object of the present invention is to provide the host cell of a kind of FAM19A4 of comprising.

Another object of the present invention is to provide the polyclonal antibody of a kind of FAM19A4.

Another object of the present invention is to provide gene or albumen or their the immunity fragment of FAM19A4, or the application of the polyclonal antibody of FAM19A4 in some pharmaceutical preparations of preparation.

Another object of the present invention is to provide the reagent for detection of the gene of FAM19A4 or albumen or their immunity fragment.

Another object of the present invention is to provide the application of the reagent of the gene that detects FAM19A4 and albumen or their immunity fragment.

According to an aspect of the present invention, the invention provides following (a) or (b) shown in albumen or its immunity fragment:

(a) albumen formed by the aminoacid sequence shown in SEQ ID No.2 or its immunity fragment; Or

(b) in the aminoacid sequence that (a) limits through replacing, lack or adding one or several amino acid and there is derivative albumen or its immunity fragment by (a) of identical function with (a).

As the described aminoacid sequence of the SEQ ID No.2 protein sequence that is FAM19A4 of the present invention, totally 140 amino acid, molecular weight 15.7kD, iso-electric point 9.02.This albumen has signal peptide sequence (1st～45 of SEQ ID No.2).

The immunity fragment of the albumen be comprised of the aminoacid sequence shown in SEQ ID No.2 can have immunogenic fragment for FAM19A4 albumen any, the albumen that for example aminoacid sequence shown in 46th～140 of SEQ ID No.2 forms.According to the present invention, can be replaced, be lacked or be added one or several amino acid FAM19A4 albumen or its immunity fragment, and obtain with FAM19A4 albumen or its immunity fragment have identical function by described albumen or the derivative sequence of its immunity fragment.Described replacement, disappearance or add one or several amino acid and the technology that obtains the derivative with identical function is known to the person skilled in the art.For example, those skilled in the art can utilize the classification of common amino acid sequence to carry out between nonpolar amino acid or the replacement between polare Aminosaeren.

The present invention also provides coding above-mentioned (a) or (b) polynucleotide sequence of described albumen or its immunity fragment.

The FAM19A4 gene is the polynucleotide sequence of coding SEQ ID No.2 of the present invention, and it can be amino acid whose encoding sequence shown in SEQ ID No.2, except the encoding sequence of above-mentioned aminoacid sequence, can also comprise non-coding sequence.Described polynucleotide sequence can be DNA or RNA, and wherein DNA comprises the DNA of cDNA, genomic dna and synthetic, can be strand or double-stranded, can be coding strand or noncoding strand.Wherein preferred gene is the polynucleotide sequence shown in SEQ ID No.1,2248 Nucleotide of this sequence total length (SEQ ID No.1), its sequence that comprises coding FAM19A4 albumen (for example encoding sequence (CDS: 444th～866, Nucleotide) and 5 ' non-coding region (1st～443, Nucleotide) and 3 ' non-coding region (867th～2248, Nucleotide)).Perhaps, more preferably a kind of nucleotide sequence of separation, the encoding sequence that it comprises FAM19A4 albumen.

Those of ordinary skills are known, and the nucleotide sequence of FAM19A4 of the present invention can be entirely identical to the encoding sequence as shown in SEQ ID No.1, also can, due to the degeneracy of genetic code, not exclusively be equal to the encoding sequence of above-mentioned Nucleotide.For example, the frequency difference of the codon that concrete prokaryotic hosts or eucaryon host are used according to each, can select corresponding codon, thereby improve described polynucleotide expression efficiency in corresponding host.Also can there are the polynucleotide (as the longer transformation period) of better performance and additive cipher in order to obtain than natural nucleotide sequence.

The immunity fragment of FAM19A4 gene of the present invention or albumen comprises the immunity fragment of FAM19A4 albumen of the present invention or the immunity fragment of FAM19A4 gene.The immunity fragment of FAM19A4 gene of the present invention can be the nucleotide sequence of the immunity fragment of the described FAM19A4 albumen of encoding, for example: the polynucleotide sequence of aminoacid sequence shown in 46th～140 of coding SEQ ID No.4.

Albumen provided by the present invention or its immunity fragment, and polynucleotide sequence, be albumen or its immunity fragment of separating, and polynucleotide sequence.Described " separation " refers to that material separates (if natural substance, primal environment is natural surroundings) from its primal environment.As the polynucleotide under the native state in active somatic cell or albumen or polypeptid acid sequence do not have separation and purification, if but same polynucleotide or albumen or polypeptide from native state, with common other material existed, separate, be separation and purification.Such polynucleotide may be the parts of a certain carrier, polynucleotide that also may be such or albumen or polypeptide are the parts of a certain composition, since carrier or composition are not the compositions of their natural surroundings, these polynucleotide or protein polypeptide remain separation.

Polynucleotide sequence of the present invention can obtain by the existing method in this area.These technology including, but not limited to: (1) is by hybridization technique DNA isolation sequence; (2) artificial chemistry synthetic DNA sequence; (3) by the required polynucleotide of the extensive acquisition in construction cDNA library; (4) pcr amplification technology.For example, the pcr amplification technology that the polynucleotide sequence of FAM19A4 of the present invention or its immunity fragment can establishing criterias is using cDNA, mRNA or genomic dna as template, and chooses suitable Oligonucleolide primers amplification and obtain.Obtain like this Nucleotide and can clone in suitable carrier, then utilize copying in described carrier to obtain.Also can obtain by the standard DNA synthetic technology, for example, use can be synthetic on DNA synthesizer by solid phase phosphorous acid acid amides triester method well known in the art.Gene of the present invention, or the available ordinary method of mensuration of the nucleotide sequence such as various DNA fragmentations, as dideoxy chain termination; Also available business sequencing kit etc.

The albumen of FAM19A4 of the present invention or its immunity fragment can be recombinant protein or polypeptide, native protein or polypeptide, synthetic proteins or polypeptide, semi-synthetic albumen or polypeptide, preferably recombinant protein or polypeptide.The product that albumen of the present invention or its immunity fragment can be natural purifying, or the product of chemosynthesis, or use recombinant technology to produce from protokaryon or eucaryon host.

The albumen of FAM19A4 of the present invention or its immunity fragment can obtain by ordinary method, and it is synthetic that for example available Applied Biosystem synthesizer or PioneerTM synthesizer are pressed the solid state chemistry technology.Also can produce albumen or polypeptide product by the recombinant DNA sequence coding of biological engineering method in host cell routinely, for example comprise the following steps: (1) with polynucleotide of the present invention (or its varient), or with the expression vector conversion that contains these polynucleotide, transfection or transduction appropriate host cell; (2) host cell that culturing step (1) obtains in suitable medium; (3) separation, required albumen or the polypeptide of purifying from substratum or cell.

Polynucleotide of the present invention and albumen or its immunity fragment preferably provide with unpack format, more preferably are purified to homogeneous.

According to a further aspect in the invention, the invention provides a kind of carrier that contains polynucleotide sequence of the present invention.Polynucleotide sequence in the present invention can be inserted in recombinant expression vector.Described engineering carrier can be general carrier, expression vector etc.

The invention still further relates to the host cell that is suitable for expressing albumen of the present invention or its immunity fragment produced through genetically engineered with above-mentioned carrier or polynucleotide of the present invention.Carrier of the present invention and polynucleotide can, for transforming suitable host cell, be secreted the protein of the sexual cell factor can express the mankind.

Those skilled in the art knows the carrier that How to choose is suitable, promotor, enhanser and host cell and the construct that will contain polynucleotide of the present invention import the method for above-mentioned host cell, include but not limited to: the conversion of calcium chloride mediation, calcium phosphate transfection, the transfection of DEAE-15 dextran mediation, electroporation, microinjection, Particle bombardment or particle gun method, cultivate host strain or the cell through transforming in suitable culture condition and substratum, make after it grows into appropriate cell density, for example, induce selected promotor by appropriate means (temperature transition or pharmaceutical chemicals are induced), and cell is cultivated for some time again.For different host strains or cell and expressed target protein or the character of polypeptide, select corresponding culture condition and substratum within those skilled in the art's ken.

The present invention also provides a kind of method of producing described albumen or its immunity fragment: be suitable for, under the condition of expressing, cultivating the host cell that contains coding polynucleotide of the present invention or its fragment; Obtain albumen or the polypeptide of polynucleotide encoding from described cell culture.Recombinant protein in aforesaid method or polypeptide can be coated in cell, extracellular or express or be secreted into extracellular on cytolemma.If necessary, can utilize its physics, chemical separating and purification of recombinant proteins or polypeptide by various separation methods with other characteristic.Specifically, after transformed host cell the host cell that is being converted grow into suitable cell density, with appropriate means (as temperature variation or chemical speciality are induced) evoked promoter, then continue to cultivate.After cultivation completes, available centrifuging collecting cell, and by any known method, these methods are well-known to those skilled in the art, as freeze-thaw method, Ultrasonic treatment, infiltration broken bacterium, N,O-Diacetylmuramidase dissolution method or mechanical crushing method smudge cells.Can from the host cell culture, reclaim by various known methods and purifying albumen of the present invention or polypeptide or its fragment or fusion rotein or polypeptide, these methods comprise the combination of ferric sulfate or ethanol precipitation, acid extraction method, ultracentrifugation, ultrafiltration process, ion exchange chromatography, phosphoric acid fiber chromatography, hydrophobic interaction chromatography method, gel filtration method, affinity chromatography, high performance liquid chromatography and other various liquid chromatography (LC) technology or these methods.

The present invention also provides the antibody for described FAM19A4.Described antibody comprises monoclonal antibody or polyclonal antibody, preferably polyclonal antibody.Described antibody is preferably polyclonal antibody or the monoclonal antibody that the sequence-specific shown in the amino-acid residue 46～140 with SEQ ID No.2 is combined.Described " specific binding " refer to polyclonal antibody or monoclonal antibody specificity identification target antigen and from the different epitopes of target antigen or the characteristic of antigenic determinant combination.Antibody described in the present invention comprise those can in conjunction with and suppress the antibody of FAM19A4 gene product of the present invention, also comprise that those do not affect the antibody of FAM19A4 polypeptide function of the present invention.The antibody of polypeptide protein of the present invention or its immunity fragment can be produced with preparation method for antibody well known in the art.Example has: monoclonal antibody can be produced by hybridoma technology.The available polypeptide protein of the present invention of the production of polyclonal antibody or its immunity fragment immune animal, as rabbit, mouse, rat etc., prepare polyclonal antibody.The cell that each antibody-like of the present invention can utilize albumen of the present invention or its immunity fragment, derivative, analogue or express them is as antigen, by the routine immunization technology, obtain, these fragments or functional zone can utilize the recombination method preparation or utilize Peptide synthesizer synthetic.

According to an aspect of the present invention, the invention provides gene or albumen or their the immunity fragment of FAM19A4, or the antibody of FAM19A4 comprises the application in pharmaceutical preparation, detection preparation at some preparations of preparation.Particularly, the RAW264.7 cell that to be its (gene of FAM19A4 or albumen or their immunity fragment, or the antibody of FAM19A4) stimulate TNF-α in preparation has the application in the preparation of chemotaxis; Application in the preparation of the generation that it has in preparation the phagocytic activity that strengthens scavenger cell, promote ROS; Application in the preparation of the function that it possesses the expression of lowering the macrophage inflammatory factor in preparation, suppress NF-κ B transcriptional activity; Application in the preparation of its differentiation at preparation promotion Th1 and Th17 cell and activation; Application in the preparation of the function of the protein expression that it possesses in preparation the mRNA level of lowering CD55 or CD59, suppress CD46, CD55 or CD59.

According to a further aspect in the invention, the invention provides gene or albumen or their the immunity fragment of FAM19A4, or the antibody of FAM19A4, the application in the pharmaceutical composition for the preparation of preventing and/or treating immune correlated disease.Wherein, described albumen is preferably the albumen that the aminoacid sequence shown in 46th～140 of SEQ ID No.2 forms, and described polynucleotide sequence is preferably the polynucleotide sequence shown in SEQ ID No.1; Described antibody is preferably polyclonal antibody; Described immune correlated disease is such as being infectious diseases, autoimmune disorder, tumour etc.The gene of FAM19A4 of the present invention and albumen can directly be included in the form of gene and albumen in the pharmaceutical composition that is used for the treatment of immune correlated disease and utilize its transient expression product to be treated, and also can be included in the pharmaceutical composition that is used for the treatment of immune correlated disease and utilize instantaneous and stable expression product to be treated to be included in form in expression vector.

The method whether the present invention also provides the expression level of the gene of a kind of vitro detection from FAM19A4 of the present invention in person's to be measured sample or albumen to change, it comprises: the expression level that detects polynucleotide described in testing sample or albumen or polypeptide; The expression level of the polynucleotide of the expression level of polynucleotide described in testing sample or albumen or polypeptide and normal specimens or albumen or polypeptide is compared; Whether the expression level of determining polynucleotide in testing sample or albumen or polypeptide changes.Described normal specimens can obtain from known not ill normal people's cell, and what this cell should be with the testing sample cell is tissue-derived consistent; The expression level of the polynucleotide of normal specimens can be the expression level from the polynucleotide with statistical significance of described normal people's cell acquisition.In wherein said detection testing sample, the method for polynucleotide level can be above-mentioned any detection method, preferably utilizes RT-PCR to detect the expression level of described polynucleotide in nucleic acid level; Or utilize specific monoclonal or polyclonal antibody to detect the expression level of described polynucleotide at protein level, for example immunohistochemistry detects.Described testing sample can obtain from the cell from the experimenter, Tathagata autoblood, urine, saliva, gastric juice, the cell of examination of living tissue and necrotomy material.

The present invention is provided for detecting the gene of FAM19A4 of the present invention or the reagent of albumen or their immunity fragment simultaneously.For example, the reagent in the expression of nucleic acid level for detection of described polynucleotide; Or for detection of described polynucleotide the reagent in the expression of protein level.The present invention also provides the application of reagent in the composition for the preparation of immune correlated disease auxiliary diagnosis, prognosis judgement of the expression of the gene that detects FAM19A4 or albumen or their immunity fragment.Described reagent can be albumen, nucleic acid, carbohydrate etc., is preferably antibody, sense-rna or siRNA (siRNA) etc.The gene of FAM19A4 of the present invention or albumen or their immunity fragment can be used as sign and the treatment target spot of autoimmune disease.Therefore, gene that can be by detecting FAM19A4 of the present invention or the expression level of albumen or their immunity fragment and detection bodies internal cause FAM19A4 of the present invention express the pathological state due to not enough or excessive, and concrete detection method can be to utilize method or their combinations such as restriction fragment length polymorphism analysis (RFLP), reverse transcription-polymerase chain reaction (RT-PCR), fluorescence in situ hybridization (FISH).Equally, also can use the antibody of FAM19A4 albumen of the present invention, by radioimmunoassay, competitive binding method, Western engram analysis method or enzyme-linked immunosorbent assay (ELISA), reach identical purpose.

In specific embodiments of the invention, the present invention finds mouse macrophage RAW264.7 that FAM19A4 stimulates at LPS and induces up-regulated in people's precursor monocytic series THP-1 of differentiation through PMA, points out it to have regulatory function to scavenger cell.RAW264.7 cell after FAM19A4 albumen activates TNF-α has chemotactic activity; Experiment in vivo and vitro proof FAM19A4 can promote RAW264.7 cell and the Turnover of Mouse Peritoneal Macrophages phagocytic function to zymosan, the expression of lowering inflammatory factor TNF-α and IL-6.Restructuring FAM19A4 albumen can be lowered Colon cancer cell line HT-29 surface Complement Regulatory Protein CD46, CD55 and the CD59 expression in the mRNA level, the expression of lowering the CD46 protein level; The CD55 on prostate cancer cell line DU145 surface and the expression of CD59 are played to restraining effect in the mRNA level, lower the protein expression of CD46, CD55 and CD59.GEO database prompting FAM19A4 up-regulated in the DC of chlamydia pneumonia infected patient cells of monocytic origin, it expresses the results show obviously and raises in the DC of lipopolysaccharides LPS and zymosan stimulation, and prompting FAM19A4 may have regulating effect to the differentiation of Th1 and Th17 cell.In the mouse DTH model of inducing at mBSA, the FAM19A4 footpad swelling degree of mouse that can increase the weight of to fall ill, and the expression level of IL-17 in the raising mice serum, in the mouse lymph nodes cells and supernatant, IFN-γ and IL-17 secretion obviously increase, and prove its differentiation that can promote Th1 and Th17 cell, activation.

In sum, the invention provides a kind of new cytokine: FAM19A4, its bacterium and the aspect such as antitumor outside anti-infective, antiviral, anti-intracellular bacteria, antimycotic, anti-born of the same parents may play a significant role, and have potential clinical value.

The accompanying drawing explanation

Fig. 1 shows the partial results of the bioinformatic analysis of FAM19A4 gene.Wherein, picture A, the nucleotide sequence of FAM19A4 and the aminoacid sequence of prediction; Picture B, there is a typical signal peptide zone in the N end of SignalP prediction FAM19A4; Picture C, D, between each kind of FAM19A4, homology is higher, wherein people FAM19A4 and mouse Fam19a4 in the homology of protein level up to 93.3%.

Fig. 2 A shows that FAM19A4 does not detect expression in Normal human tissue.

Fig. 2 B shows that FAM19A4 extensively hangs down and expresses or expression deletion in the various human tumor cell line.

Fig. 2 C shows that the expression of FAM19A4 is relevant to inflammatory reaction.Wherein, picture A, GEO database displaying FAM19A4 up-regulated in the DC of chlamydia pneumonia infected patient cells of monocytic origin; Picture B, FAM19A4 is up-regulated in the ripe DC of LPS and zymosan stimulation; Picture C, what FAM19A4 stimulated at LPS induces up-regulated in the THP-1 of differentiation through PMA; Fam19a4 is up-regulated in the post-stimulatory RAW264.7 of LPS.

Fig. 3 A shows that FAM19A4 is the secretory protein of secreting by classical pathway, and its secretion can be suppressed by BFA.

Fig. 3 B demonstration has obtained FAM19A4 eucaryon recombinant protein.Wherein, picture A, through identifying, obtain high purity FAM19A4 eucaryon recombinant protein; Picture B, N end sequencing result shows that FAM19A4 maturation protein total length is 95 amino acid.

But Fig. 3 C shows the anti-human FAM19A4 polyclonal antibody of rabbit specific recognition exogenous expression's FAM19A4 albumen.

Fig. 4 shows that the RAW264.7 cell that FAM19A4 stimulates TNF-α has chemotaxis, and its chemotactic activity has dose-dependently.

Fig. 5 shows that FAM19A4 can strengthen the phagocytic activity of scavenger cell, promotes the generation of ROS.Wherein, picture A/C, FAM19A4 can significantly improve the average Phagocytic granules number of RAW264.7 cell, increases cytophagous ratio, strengthens on the whole the phagocytic activity of scavenger cell.Picture B, FAM19A4 can strengthen the external phagocytic activity of Turnover of Mouse Peritoneal Macrophages.Picture D, FAM19A4 can strengthen the interior phagocytic activity of body of Turnover of Mouse Peritoneal Macrophages.Picture E, promote the generation of RAW264.7 cell ROS on the basis that FAM19A4 stimulates at zymosan.

Fig. 6 shows that FAM19A4 can lower the expression of the macrophage inflammatory factor, suppresses NF-κ B transcriptional activity.Wherein, picture A, in the RAW264.7 cell, FAM19A4 can weaken the Tnf-alpha expression rise caused by zymosan, and not remarkable on the impact of Il-6.Picture C, in Turnover of Mouse Peritoneal Macrophages, FAM19A4 all has the downward effect to the post-stimulatory Tnf-α of zymosan and Il-6 expression.Picture B, in the RAW264.7 cell, FAM19A4 can weaken the transcriptional activity of NF-κ B.

Fig. 7 show FAM19A4 in the DTH model with the differentiation of Th1 and Th17 cell and activate relevant.Wherein, picture A, FAM19A4 increases the weight of mouse insole swelling degree; Picture B, FAM19A4 improves the expression level of IL-17 in mice serum; Picture C, FAM19A4 is the secretion of IFN-γ in raising mouse lymphocyte culture supernatant under stimulating without mBSA, has or not the stimulation of mBSA all can improve the secretion of IL-17 in the mouse lymphocyte culture supernatant.

Fig. 8 A shows that in the HT-29 cell, FAM19A4 can lower the mRNA level of CD55 and CD59, and heating can make the part inactivation; Significantly suppress the protein expression of CD55.

Fig. 8 B shows that in the DU145 cell, FAM19A4 lowers the mRNA level of CD55 and CD59, suppresses the protein expression of CD46, CD55 and CD59.

Embodiment

In order more clearly to understand the present invention, referring now to the following example and accompanying drawing, further describe the present invention.Embodiment does not only limit the present invention in any way for explanation.In embodiment, the experiment material that some are main and operate as described below, the experimental technique of other unreceipted actual conditionses is ordinary method and the normal condition that affiliated field is known, such as the people such as Sambrook " molecular cloning experiment guide " (third edition) (Cold Spring Harbor Laboratory (CSHL) Press, 2001) condition described in, or the condition of advising according to manufacturers.

1, RNA extraction, RT-PCR, cDNA library with Real-time PCR

Collect 9 kinds of clones, use TRIzol (Invitrogen) to extract total RNA (by specification operation).Collect the A549 cell that not stimulation and inflammatory factor IL-1 β (10ng/ml) stimulate 3h, 6h, 15h, 24h; After inducing differentiation 24h, PMA (10ng/ml) do not stimulate and THP-1 cell that inflammatory factor LPS (100ng/ml) stimulates 3h, 6h, 12h; Stimulation and inflammatory factor LPS (100ng/ml) do not stimulate the RAW264.7 cell of 2h, 4h, 6h; The Turnover of Mouse Peritoneal Macrophages that stimulation and inflammatory factor LPS (100ng/ml) do not stimulate; The mouse Th17 cell that stimulation and PI do not stimulate; Collect respectively the peripheral blood mononuclear cell of the normal donors of 10 example, the monocyte obtained through sorting, the ripe DC cell through inducing the immature DC that obtains and polyI:C, zymosan, LPS etc. to stimulate; Collect DU145 cell and HT-29 cell that not stimulation and FAM19A4 eucaryon recombinant protein (10ng/ml) stimulate 24h; The mouse lung tissue of normal Balb/c mouse and the injection Klebsiella Pneumoniae 3h of lung, 6h, 24h, used TRIzol to extract total RNA.Each sample is got 2 μ g, uses Reverse transcript ^tMthe synthesizing single-stranded cDNA library of kit (Invitrogen) reverse transcription.Real-time PCR adopts SYBR Green method and LightCycler instrument to carry out.

2, cell cultures and transfection

The HEK293T cell is the cultivation of going down to posterity, and with the DMEM (4.5g/L glucose, 4mML-glutamine, 100U/ml penicillin, 100 μ g/ml Streptomycin sulphates) containing 10% foetal calf serum (FBS), cultivates.The A549 cell is purchased from ATCC, by RPMI 1640 culture medium culturing containing 10%FBS, the THP-1 cell is purchased from ATCC, by the RPMI1640 culture medium culturing that contains 10%FBS and 0.9%2M glucose, RAW264.7, DU145 and HT-29 cell are purchased from ATCC, by the DMEM culture medium culturing containing 10%FBS.

HEK 293T cell transfecting method is the positively charged ion infection protocol, and concrete operation step is as follows: (1) cell cultures: by HEK 293T cell (3.0 * 10 ⁵individual) with the DMEM substratum containing 10% foetal calf serum, be layered on six porocyte culture plates, at 5%CO ₂, cultivate 24 hours in the incubator of 37 ℃.(2) prepare the DNA-Vigofect mixture: dilute 5 μ g purpose plasmids with 100 μ l PBS, slowly mix; Dilute 2 μ l VigoFect with 100 μ l PBS equally, slowly mix, after at room temperature placing 5 minutes, slowly mix with the DNA of dilution, room temperature is placed 15 minutes, to form the DNA-VigoFect mixture.(3) transfection: the DNA-VigoFect mixture is slowly splashed into to Tissue Culture Plate (200 μ l/ hole), slightly shake up.5%CO2, cultivate in the incubator of 37 ℃.(4) transfection, after 6 hours, discards substratum, adds the fresh DMEM substratum containing 10%FBS or 293T serum free medium (but the culturing cell factor that contains low albumen) to continue to cultivate 48 hours.

3, cells and supernatant and total protein of cell extract and Western blot analysis

The collection of 293T cells and supernatant: cell transfecting, after 48 hours, is collected respectively each experimental group cells and supernatant, in 4 ℃, centrifugal 10 minutes of 2000g, abandon precipitation, then in 4 ℃, centrifugal 15 minutes of 15000g, then in 37 ℃ of vacuum-dryings concentrated 1 hour, leave and take the cells and supernatant after processing.

The extraction of 293T total protein of cell: the cell that will leave and take after supernatant is placed on ice, and ice-cold 1 * PBS washes twice, and ice-cold 1 * PBS blows down cell, by cell harvesting in the 1.5mL centrifuge tube, in 4 ℃, centrifugal 5 minutes of 2000g.Remove supernatant, in precipitation, add the RIPA cell pyrolysis liquid (20mM Tris-HCl, pH 7.4,150mM NaCl, 1mM EDTA, 1mM EGTA, 1%Triton X-100, proteinase inhibitor phosSTOP (Roche)), place on ice 30 minutes, 4 ℃, centrifugal 15 minutes of 15000g, supernatant proceeds to new pipe, be stored in-80 ℃ standby.

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

Western blot: every group of cell protein got 30 μ g, and every group of cells and supernatant got 40 μ l, adds albumen sample-loading buffer (Beijing Bao Sai Bioisystech Co., Ltd), in 100 ℃ of water-baths, boils 5 minutes.The 12.5%PAGE electrophoresis, the 100mV electricity turns 1 hour, and TBST liquid balance, with 5% milk room temperature sealing 2 hours, adds corresponding primary antibodie, and 4 ℃ are spent the night, and with TBST, fully wash film 3 times, 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; Finally use Odyssey Infrared Imager detection signal.

4, the separation of Turnover of Mouse Peritoneal Macrophages

Choose female C57BL/6J mouse in 6-8 age in week, the thioglycolic acid of every mouse peritoneal injection 3% is received solution (PBS makes solvent) 2ml, 48 as a child disconnected neck put to death mouse, the PBS of abdominal injection 5ml precooling gently rubs stomach wall 1 minute, open peritonaeum and reclaim Intraabdominal PBS and enter aseptic centrifuge tube, centrifugal 5 minutes of 4 ℃ of lower 2000rpm, obtain Turnover of Mouse Peritoneal Macrophages, the RPMI1640 substratum that use contains 10%FBS, 5%CO ₂, cultivate in the incubator of 37 ℃, initial cell density is 1 * 10 ⁶/ ml.Cultivate and renew substratum after 4 hours, add the stimulations such as inflammatory factor LPS or FAM19A4 eucaryon recombinant protein after overnight incubation in culture supernatant.Whole process all completes under sterile closed condition.

5、ELISA

Cell cultures is gathered in the crops supernatant after the corresponding time, and in 4 ℃, centrifugal 10 minutes of 2000g, abandon precipitation, then, in 4 ℃, centrifugal 15 minutes of 15000g, remove cell and fragment in supernatant.Use mouse TNF-α, IL-6 cytokine ELISA test kit (eBIOSCIENCE) sandwich assay to detect the secretory volume of cytokine.

6, dual-luciferase reporter system

6.1 cell transfecting, results and cracking

With pRL-TK (the renilla luciferase carrier, as the fluorescence internal reference), NF-κ B-luc (Photinus pyralis LUC carrier) cotransfection RAW264.7 cell.Transfection renews bright substratum after 12 hours, add FAM19A4 albumen or LPS as the positive control pretreatment cell, after 2 hours, to add zymosan (100ug/ml) to hatch 24 hours, the abundant washed cell of PBS, add Reporter Lysis Buffer (Promega, E4030), place 30 minutes under-70 ℃, naturally melt in room temperature after taking-up, make lysis.

6.2 luciferase assays

20 μ l cell pyrolysis liquids are moved into to fluorescent plate (Gronier, 655075), with Dual-Luciferase Reporter AssaySystem 10-Pack (Promaga, E1960), by Veritas microplate fluorescence analyser, detect luciferase activity.First add Lampyridea (Firefly) luciferase substrate 25 μ l/ holes, detect the Photinus pyralis LUC activity, then add stop buffer and sea pansy (Renilla) luciferase substrate 25 μ l/ holes, detect internal reference renilla luciferase activity.

6.3 data statistic analysis

Use Excel software to carry out data processing.At first, the numerical value that records in each hole is calculated through " Photinus pyralis LUC fluorescence intensity/renilla luciferase fluorescence intensity ", institute's value represents the NF-kB activity in this hole.Second step, be decided to be 1 (100%) by the NF-kB activity of negative control group, calculates the relative NF-kB activity of other each group.The 3rd step, averaging and standard deviation in three multiple holes of every group, represents the NF-kB activity of this group.

7, mouse delayed type hypersensitivity (delayed type hypersensitivity, DTH) model is set up

Choose female C57BL/6J mouse in 8 week age, within the 0th day, give every mouse web portion intracutaneous 2 injection 125 μ g (50 μ l) adequately emulsified mBSA of CFA, every day abdominal injection 100ng FAM19A4 albumen.Within the 7th day, the left sufficient lift hemostasis 150 μ g mBSA/PBS solution (30 μ l) of every mouse are excited, 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).Then pluck eyeball and get blood, 4 ℃ are spent the night, and then 3000rpm is centrifugal, get serum to new Ep pipe, and the centrifugal 10min of 12000rpm removes fragment, and the ELISA that gained serum can be used for IFN-γ and IL-17 detects.Get spleen and grind acquisition splenocyte, resuspended one-tenth 5 * 10 ⁶/ ml is incubated at 24 orifice plates, at 10 μ g/ml mBSA and recombinant proteins, analyzes the propagation of antigenspecific T lymphocyte, detects the secretion of the cytokines such as IFN-γ, IL-17 in culture supernatant.

8, flow cytometer detects cell surface molecule

Harvested cell also is prepared into single cell suspension, after the PBS of precooling washes 2 times, 4%FBS sealing 15 minutes, add anti-CD46-FITC, anti-CD55-PE or 4 ℃ of lucifuges of anti-CD59-FITC antibody (BD) to hatch 30 minutes, add 100 μ lPBS re-suspended cells after PBS after backwashing cell 1 time, the upflowing cell instrument detects FL-1 and FL-2 passage.

9, statistical analysis

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

CDNA clone and the bioinformatic analysis of embodiment mono-, FAM19A4 gene

1, bioinformatic analysis

By Human est database compare of analysis cDNA sequence and the alternative splicing body with NCBI.By the tBLASTn program looks homologous protein of NCBI, simultaneously from genome database ( http:// www.ncbi.nlm.nih.gov/genome/) obtain chromosomal localization.Protein function domain analyses use NCBI CDD database ( http:// www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml).Other albumen correlation analysis use ExPASy database ( http:// us.expasy.org/), comprising: TMHMM ( http:// www.cbs.dtu.dk/services/TMHMM/) for cross-film, distinguish and analyse; Signal P ( http:// www.cbs.dtu.dk/services/SignalP/) for the signal peptide analysis; Psort II ( http:// psort.nibb.ac.jp) for Subcellular Localization, predict; GNF SymAtlas (http://symatlas.gnf.org/SymAtlas/) is for expression pattern analysis; Prosite ( http:// www.expasy.org/prosite) for domain analyses.And use DNAstar software analysis proteantigen, hydrophilic and hydrophobic, surface to expose property etc.

By bioinformatic analysis, find UniGene:Hs.187873, its corresponding gene is FAM19A4, belongs to FAM19/TAFA family.This family gene has been in the news relevant to central nervous system, and FAM19A4 is high expression level in thalamus, but biological function there is no report, is a unknown function human gene.

FAM19A4 is under the jurisdiction of TAFA family, and the mutual homology of this family member FAM19A1-5 is higher, all comprises abundant halfcystine in aminoacid sequence; The family member is extensively low expression in normal cell and tissue, only is expressed in the specific region of brain.Information biology is found, the FAM19A4 assignment of genes gene mapping is in 3p14.1, Ref:NC 000003.11, GeneID:151647, sequence total length 2248bp (referring to SEQ ID No.1), ORF total length 423bp, 140 amino acid of encoding (referring to SEQ ID No.2), the about 15.7kD of molecular weight, iso-electric point 9.02.In Fig. 1, picture A has shown the nucleotide sequence of FAM19A4 and the protein sequence of prediction.SignalP predicts that there is a typical signal peptide in its N end, is 36 amino acid (B in Fig. 1) of N end.10 halfcystines are arranged in the FAM19A4 aminoacid sequence, there is CC and CXC motif simultaneously, be predicted as chemokine-like albumen, but different from classical chemokine structure.

The FAM19A4 evolution conservative, between each kind, homology is higher, and wherein people FAM19A4 and mouse Fam19a4 up to 93.3% (C, D in Fig. 1), point out it to have critical function in the homology of protein level.Electronics express spectra prompter FAM19A4 normally and in the cancerous tissue organ is extensively and hangs down and express at each, and mouse Fam19a4 is high expression level in the ridge neuroganglion only.To sum up prompting, FAM19A4 and mouse homologous gene be function inactive under the normal physiological state.

2, the clone of the cDNA of FAM19A4 gene

Utilizing the Human_est database, by the BLASTn method, the FAM19A4 gene is carried out to sequence proofreaies and correct errorless.Special primer according to this sequences Design FAM19A4 full length gene reading frame:

Forward primer 5 '- gcggccgccaccatgaggtccccaaggatgagagtct-3 ' (SEQ ID No.3)

Reverse primer 5 '- ggtaccgcccgcgttaccttcgtagttttgact-3 ' (SEQ ID No.4)

In above-mentioned primer, underscore is respectively restriction enzyme Not I and KpnI recognition sequence

Use above-mentioned primer, the coding region that recombinant plasmid corresponding to the MGC (Mammalian Gene Collection) of take clone numbering BC031566 is template amplification FAM19A4, amplification reaction condition is as follows:

Reaction volume 50 μ l, wherein contain:

Temperature of reaction, time: 94 ℃, sex change 5 minutes; Then 94 ℃ of sex change are 30 seconds, 60 ℃ of annealing 30 seconds, and 72 ℃ are extended 1 minute, 30 circulations of increasing; Finally 72 ℃ of downward-extensions 10 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) connect and spend the night under 16 ℃, connect product and transform bacillus coli DH 5 alpha, conversion product is being grown containing on the LB plate culture medium of penbritin, selected clone, extract plasmid, use AbI PRISM 3700 DNA analysis instrument (Perkin-Elmer/Applied Biosystem) order-checking, order-checking is correct, acquisition is connected with the pGEM-T EASY carrier of the cDNA of FAM19A4, it is the pGEM-T-FAM19A4 recombinant plasmid.

Embodiment bis-, FAM19A4 expression pattern analysis

The clontech Normal human tissue cDNA library of use buying and according to the human cDNA library of aforementioned preparation, analyze the mrna expression level of FAM19A4 in healthy tissues and Leukemia Cell Lines.Concrete operations are as follows:

With embodiment mono-2 in the record the pcr amplification condition FAM19A4 is increased.Mouse cDNA library for preparation in above-mentioned 1, use 5 ' primer (5 '-TTCACGGTCACTCTCCAGGAC-3 ', SEQ ID No.5) with 3 ' primer (5 '-AGCCACGTAAATGCCACCTTC-3 ', SEQ ID No.6) amplification mouse homologous gene Fam19a4, amplification condition is 94 ℃ (5 minutes) → 94 ℃ (40 seconds), 54 ℃ (40 seconds), 72 ℃ (40 seconds), increase 30 and circulate → 72 ℃ (7 minutes).

Use 5 ' primer (5 '-TGAAGGTCGGAGTCAACGGATTTGGT-3 ', SEQ ID No.7) with 3 ' primer (5 '-CATGTGGGCCATGAGGTCCACCAC-3 ', SEQ ID No.8) amplification GAPDH is as internal reference, amplification condition is 94 ℃ (5 minutes) → 94 ℃ (40 seconds), 58 ℃ (40 seconds), 72 ℃ (40 seconds), increase 22 and circulate → 72 ℃ (7 minutes).

Pcr amplification product is through agarose gel electrophoresis, and after EB dyeing, GENE Snap gel imaging system detects.

Experimental result refers to Fig. 2 A, Fig. 2 B and Fig. 2 C.Result shows:

Use the clontech Normal human tissue cDNA library amplification FAM19A4 bought, its expression (Fig. 2 A) do not detected in the various human healthy tissues.

FAM19A4 is equal expression deletion in 9 kinds of tumor cell lines, only in chronic myelocytic leukemia cancer cells K562 cell, is the expression (Fig. 2 B) of higher level.

GEO database displaying FAM19A4 is up-regulated (picture A in Fig. 2 C) in the DC of chlamydia pneumonia infected patient cells of monocytic origin.Research of the present invention is found, in three different healthy individual that detect, FAM19A4 does not all detect it and expresses in PBMC and monocyte, faint expression in the immature DC of two individualities, up-regulated (picture B in Fig. 2 C) in LPS and zymosan stimulate the ripe DC obtained., at PMA, induce in the THP-1 clone of differentiation, FAM19A4 is up-regulated under LPS stimulates simultaneously; Mouse homologous gene Fam19a4 expresses and is faint rise (picture C in Fig. 2 C) in the post-stimulatory mouse macrophage RAW264.7 of LPS.To sum up prompting, expression and the inflammatory reaction of FAM19A4 have close ties.

The structure of embodiment tri-, FAM19A4 eukaryon expression plasmid and the purifying of eukaryotic protein

1, the structure of FAM19A4 eukaryon expression plasmid

In order to carry out the functional study of FAM19A4, the present embodiment is used Not I and Kpn I to cut processing to pGEM-T-FAM19A4 recombinant plasmid enzyme, reclaim and goal gene segment (coding region of FAM19A4) further is connected to carrier for expression of eukaryon pcDNA3.1-myc/his (-) B (Invitrogen through Not I and Kpn I processing, V85520), thus obtain pcDB-FAM19A4-myc-his (merging with c-myc and his label) eukaryotic expression recombinant plasmid.

2, the expression and purification of restructuring FAM19A4 albumen

The 293T cell is layered in the 10cm culture dish, at 5%CO with the DMEM substratum containing 10%FBS ₂, cultivate 24 hours in the incubator of 37 ℃.Use Vigofect positively charged ion infection protocol transfection goal gene pcDB-FAM19A4-myc-his eukaryon expression plasmid.The PBS washed cell of transfection use normal temperature after 24 hours once, is changed fresh serum free medium (but the culturing cell factor that contains low albumen), at 5%CO ₂, in the incubator of 37 ℃, cultivate.After 48 hours, the collecting cell culture supernatant is in aseptic new centrifuge tube, and in 4 ℃, centrifugal 10 minutes of 2000g, leave and take the cells and supernatant after processing.

Use Ni ²⁺cells and supernatant after the above-mentioned processing of post material purifying: first by supernatant after 0.45 μ m filter filters, add imidazoles (10mM)/NaCl (200mM) in supernatant, with the post material, be combined again, rinse the post material with 20 times of volumetric balance damping fluids again, to wash away unconjugated foreign protein, hatch elution of bound albumen after 15 minutes with 1 times of volume elutriant and post material, collect elutriant in dialysis tubing, with twice of 4 ℃ of dialysis of the PBS (PH7.4) of precooling, each 3 hours, then sucking-off albumen, and get the negative control of the part PBS that dialysis finishes for the second time as functional experiment, respectively at 4 ℃, 18000g degerming in centrifugal 20 minutes, get the supernatant packing be stored in-80 ℃ stand-by, whole process all completes under sterile closed condition.

Get 5 μ l albumen, by the BCA method, it is carried out to protein quantification, detect the residual condition of lipidated protein and LPS with SDS-PAGE and king crab experiment, usually guarantee that the purity of protein is more than 95%, the residual quantity of LPS is lower than 1EU/mg.

Information biology prompting FAM19A4 is secretory protein, and the present embodiment has also been verified this point by the BFA blocking experiment.BFA blocking experiment concrete operations: at first with plasmid pcDB-FAM19A4-myc-his or empty carrier contrast transfection 293T cell, after transfection 24 hours, add respectively 10 μ g/ml Brefeldin A (BFA) or DMSO (as negative control) in cells and supernatant, process cell after 24 hours, collect each experimental group cell and cells and supernatant and analyze for Western blot.

Result as shown in Figure 3A, in the culture supernatant of the 293T of overexpression FAM19A4 cell, can detect FAM19A4, and its molecular weight of albumen is about 18kDa.After adding the inhibitor B FA of classical Secretory Pathway (endoplasmic reticulum golgi body approach), the FAM19A4 secretion obviously reduces, and proves that FAM19A4 is the secretory protein of secreting by classical pathway.

In order to carry out the functional study of FAM19A4, in the present embodiment, the coding region fragment of FAM19A4 is cloned in pcDNA3.1-myc/his (-) B carrier for expression of eukaryon, and insert myc and his sequence label before terminator, successfully built the pcDB-FAM19A4-myc-his eukaryon expression plasmid, be that FAM19A4 PROTEIN C end is with myc and his label, and, by this eukaryon expression plasmid transfection 293T cell, use Ni ²⁺post material purifying cells culture supernatant obtains the FAM19A4 recombinant protein.Through identifying, the expressing quantity of FAM19A4 is up to 2.62mg/L, and after FAM19A4 albumen is carried out to purifying, its purity is more than 95%, LPS<<0.11EU/mg, reach the requirement (picture A in Fig. 3 B) of carrying out functional experiment.

In addition; the present embodiment turns pvdf membrane by the FAM19A4 eukaryotic protein; carry out N end order-checking (Jikang Biotechnology Co Ltd, Shanghai); result is as shown in picture B in Fig. 3 B; the amino acid number of surveying and sequence (from N, holding the C end) totally 5 (HQIKQ), this site originates in the 46th of prediction FAM19A4 aminoacid sequence, prompting FAM19A4 maturation protein total length is 95 amino acid; 45 amino acid that the signal peptide zone is the N end, rather than 36 amino acid of N end of SignalP prediction.

The preparation of embodiment tetra-, FAM19A4 antibody, purifying and evaluation

1, the preparation of FAM19A4 polyclonal antibody

FAM19A4-myc-his eucaryon recombinant protein immune animal with purifying.Select the bull new zealand rabbit, initial immunity fully mixes with isopyknic Freund's complete adjuvant after the antigen of 300ug is diluted to 1ml with PBS, in each 0.25ml subcutaneous injection of two foots, and the subcutaneous multiple spot of all the other back parts (6 point) injection.Once, consumption is the same for every 3 weeks afterwards booster immunizations, all subcutaneous multiple spot of back part (8 point) injections.Within after booster immunization 10 days for the third time, get the detection of rabbit ear edge venous samples can and tire, to tiring, reach 1: 10 ⁵after above, rabbit is put to death to heart extracting blood, obtain serum.

2, the purifying of FAM19A4 polyclonal antibody and evaluation

Serum adopts Protein G post to carry out the polyclonal antibody purifying: first by serum after 0.2 μ m filter filters, with the post material 4 ℃ of overnight incubation.Serum, after the post material is combined, rinses the post material with 20 times of volume PBS, to wash away unconjugated foreign protein.Use again 1 times of volume 0.1M glycine buffer wash-out (pH2.4) and post material to hatch wash-out after 15 minutes.Collect elutriant in dialysis tubing, with 4 ℃ of dialysis twice of PBS (PH7.4) of precooling, each 3 hours, then sucking-off albumen, in 4 ℃, 12000rpm degerming in centrifugal 20 minutes, got supernatant, obtains antibody purification, packing be stored in-80 ℃ stand-by.Overexpression FAM19A4 in the 293T cell, by avidity and the specificity of the methods such as Western blot, FACS, ELISA evaluation antibody.

Result is referring to Fig. 3 C, but shows the anti-human FAM19A4 polyclonal antibody of this rabbit specific recognition exogenous expression's FAM19A4 albumen.

Embodiment five, the experiment of FAM19A4 chemotactic activity

Being rich in halfcystine in information biology prompting FAM19A4 aminoacid sequence, containing the motifs such as CC, CXC, is chemokine-like albumen, and therefore, the present embodiment is detected the chemotactic activity of FAM19A4.

Chemotactic experiment concrete operations: the RAW264.7 cell count is adjusted into to 5 * 10 ⁵/ ml, add TNF-α to stimulate, and concentration is 20ng/ml.Lower hole at the chemotactic cell adds the FAM19A4 eukaryotic protein, and concentration is 10ng/ml, 3 multiple hole ，Mei hole 27 μ l; The positive control hole adds respectively the CCL5 of same concentration, 27 μ l/ holes, 3 multiple holes; Negative control hole adds PBS, 27 μ l/ holes, 3 multiple holes.Cover the poly-carbon membrane (8 μ m) of the coated PVP free spent the night of mouse tail collagen and silicagel pad, add in Kong， hole, upper strata and add cell suspension 55 μ l/ holes.37 ℃, 5%CO ₂take out film after hatching 6h, scrape off the cell of non-specific adhesion, methyl alcohol is fixed, the dyeing of Ji's nurse Sa, micro-Microscopic observation, the cell of counting chemotactic via hole, calculate chemotactic index (mean of the cell count of the mean ÷ control group chemotactic of the cell count of chemotactic index=experimental group chemotactic).

Experimental result finds, FAM19A4 to the monocyte/macrophage of tranquillization without obvious chemotactic activity (A in Fig. 4).

According to FAM19A4 in the results suggest aspect express spectra, infer FAM19A4 play a role may with the expression of cell surface associated receptor under inflammatory conditions, activate relevant, so chemotactic activity of the RAW264.7 cell detection FAM19A4 that the present embodiment further utilizes TNF-α to stimulate, use CCL5/RANTES as positive control simultaneously, result shows that the RAW264.7 cell that FAM19A4 stimulates TNF-α has chemotaxis, and its chemotactic activity has dose-dependently (Fig. 4).

Embodiment six, FAM19A4 can strengthen the phagocytic activity of scavenger cell, promote the generation of ROS

1, phagocytic function detects

Scavenger cell (RAW264.7 cell or Turnover of Mouse Peritoneal Macrophages) is layered on 24 well culture plates with the RPMI1640 substratum that contains 10%FBS, place in advance cover glass in culture hole, 1.5 * 104/ml, cultivate and within 30 minutes, treat cell attachment in 5%CO2, the incubator of 37 ℃, add LPS or restructuring eukaryotic protein FAM19A4 to stimulate after 2 hours and add zymosan particle zymosan or FITC-zymosan, harvested cell after 30 minutes.As add unlabelled zymosan, carry out Wright's staining Microscopic observation counting, calculate respectively phagocytic cell percentage ratio (percentage of phagocytic cells, PP), average Phagocytic granules number (mean number of particles per cell, MNP) and phagocytic index (phagocytic index, PI=PP * MNP).As add the zymosan of FITC mark, and utilize flow cytometer to be detected, calculate RRI (RRI=phagocytic cell percentage ratio * experimental group is engulfed average fluorescent strength MFI/ control group and engulfed average fluorescent strength MFI) data and do to evaluate and test.This system is usingd IFN-γ as positive control.

Result refers to Fig. 5.Result shows, is exposed to RAW264.7 cell and the Phagocytosis By The Peritoneal Macrophages In Mice enhancing (A, C in Fig. 5) of FAM19A4.FAM19A4 can significantly improve cytophagous average Phagocytic granules number, increases cytophagous ratio, strengthens on the whole the phagocytic activity of scavenger cell.

Prompting based on above RAW264.7 clone result, the present embodiment also utilizes thioglycolic acid to receive to induce and obtains Turnover of Mouse Peritoneal Macrophages and does as above processing after external adherent 4 hours, result shows that FAM19A4 can strengthen the phagocytic function of Turnover of Mouse Peritoneal Macrophages equally, consistent with the result in clone (B in Fig. 5).

The result of testing in vitro according to clone and primary scavenger cell, (100ng/ only to the FAM19A4 albumen of 6-8 week C57BL/6J mouse peritoneal injection various dose for the present embodiment, 1 μ g/ is only), system is used IFN-γ (200ng/ is only) as positive control.Inject 5 * 10 after 2 hours ⁶fITC-zymosan, engulf disconnected neck after 30 minutes and put to death mouse, separates abdominal cavity cell, after mark mouse macrophage specific mark thing F4/80, carries out flow cytometry, calculates the ratio of the engulfing FITC of scavenger cell ⁺/ F4/80 ⁺, result is referring to D in Fig. 5, and FAM19A4 can strengthen the phagolysis to zymosan of scavenger cell in Peritoneal Cells of Mice.

2, ROS reaction detection

The ROS reaction detection is used the active oxygen detection kit that is purchased from the green skies, according to its description of product, preloaded reduced form DCFH-DA probe 10 μ M in adherent RAW264.7 cell (process the RAW264.7 cell as identical as the phagocytic function test section, loaded in advance the DCFH-DA probe before adding the stimulation of FAM19A4 albumen), hatch 20 minutes for 37 ℃.Serum free medium washed cell three times to be fully to remove the probe do not entered in born of the same parents, changes full substratum and adds or do not add FAM19A4 albumen to stimulate after 2 hours to add zymosan, and 30 minutes harvested cells carry out flow cytometer and detect its luminous intensity.

Result shows the generation of ROS reaction in RAW264.7 cell that FAM19A4 can strengthen being caused by zymosan, and this albumen itself does not show as above effect (as E in Fig. 5).

The result of the present embodiment, can infer one of function target cell that scavenger cell is FAM19A4, and FAM19A4 plays a significant role in initial infection especially fungi infestation.

Embodiment seven, FAM19A4 can lower the expression of the macrophage inflammatory factor, suppress NF-κ B transcriptional activity

The present embodiment results are through FAM19A4 and the zymsan co-processing cells and supernatant of 24 hours, the secretion situation of Tnf-α and Il-6 in the detection supernatant, found that the secretion not impact of the independent stimulating expression of macrophage of FAM19A4 on its inflammatory factor, but can weaken the up-regulated of the inflammatory factor caused by zymosan when jointly existing with zymosan, show as and can make Tnf-α secretion in the RAW264.7 cell reduce, and Il-6 changes not obvious (A in Fig. 6); Tnf-α and Il-6 to the Turnover of Mouse Peritoneal Macrophages secretion all have downward effect (C in Fig. 6).

According to as above result, since FAM19A4 can weaken the expression of the inflammatory factor caused by zymosan, infer this effect whether this albumen reaches by the activity that has affected its UPSTREAM BINDING FACTOR.The present embodiment is used the index of NF-κ B dual-luciferase reporter system as its detection, adds the collaborative stimulation of FAM19A4 albumen and zymosan in experimental system, arranges simultaneously and adds separately target protein or zymosan in contrast.Result is as shown in B in Fig. 6, and the FAM19A4 independent role can not exert an influence to the transcriptional activity of NF-κ B in the RAW264.7 cell, and collaborative the stimulation than the independent action of zymosan of albumen and zymosan has and significantly weaken effect.

Embodiment eight, FAM19A4 and Th cytodifferentiation and activation

After Th1 and Th17 cell activation, participate in generation and the development of DTH; In the mouse DTH model of inducing at mBSA, the Th1 cell suppresses the process of DTH, and Th17 cells play promoter action.The present embodiment utilizes mBSA inducing mouse DTH model, after sensitization 0-6 days every days abdominal injection FAM19A4 eukaryotic protein, within the 6th day, put to death mouse, measure mouse insole swelling thickness, detect Cytokine of Serum secretion situation, and get its lymphoglandula and detect each index.Found that FAM19A4 can increase the weight of mouse insole swelling degree (A in Fig. 7).In addition, detect cytokine in mice serum and find that in injection FAM19A4 protein groups mice serum, the IL-17 level is compared obvious increase (B in Fig. 7) with control group; Simultaneously, the separating mouse lymph-node cell is external hatches altogether with mBSA, detectable antigens T lymphocyte specific response situation, found that in injection FAM19A4 protein groups mouse lymphocyte culture supernatant, the IL-17 secretion obviously increases, find that isolated lymph-node cell is not having under the mBSA stimulation, the IFN-γ of its secretion and IL-17 also increase (C in Fig. 7) to some extent simultaneously.

The impact that embodiment nine, FAM19A4 express the tumor cell surface Complement Regulatory Protein

An important physiological function of complement system is to dissolve external cell, certainly also comprises tumour cell, and the activation process of this dissolving is mainly passed through following two kinds of approach: the one, and the classical pathway excited by immune complex; The 2nd, by external cell, comprise the alternative pathway that tumour cell excites.Each approach all will form bimolecular mixture and C3 convertase, final C5b, and C6, C7, C8, C9 is combined into complement attack complex thing and is attached to external cell and the tumor cell surface that will attack, causes the dissolving of cell.Think traditionally in body Complement Regulatory Protein (the complement regulatory protein that exists multiple solubility and membrane-binding, CRP), they interact from different complement components in a particular manner, make the Activation and inhibition of complement in meticulous equilibrium state, thereby prevent from autologous tissue is caused damage, can effectively kill external microorganism again.What in tumour immunity, study morely at present is that film is in conjunction with the CD46 in CRP, CD55, CD59.In order further to analyze the relation of the genesis of FAM19A4 and tumour, the present embodiment has been chosen different clone, utilizes the method for RT-PCR and FACS to detect the impact that FAM19A4 albumen is expressed Complement Regulatory Protein.

In CCL188 HT-29, FAM19A4 can lower the mRNA level of CD55 and CD59, and heating can make the part inactivation; Significantly suppress the protein expression of CD55, on the expression impact of CD59 protein level not significantly (Fig. 8 A).

In PC-3 DU145, FAM19A4 can lower the mRNA level of CD55 and CD59, suppresses the protein expression of CD46, CD55 and CD59, and the mRNA level of CD46 be there is no to remarkable effect (Fig. 8 B).Because IFN-γ can significantly suppress the expression of DU145 cell surface CD59, so the present embodiment is used IFN-γ to stimulate the positive control as system.

Claims

Following (a) or (b) shown in albumen or its immunity fragment:

(a) albumen formed by the aminoacid sequence shown in SEQ ID No.2 or its immunity fragment; Or

(b) in the aminoacid sequence that (a) limits through replacing, lack or adding one or several amino acid and there is derivative albumen or its immunity fragment by (a) of identical function with (a);

Described immunity fragment is preferably the albumen that the aminoacid sequence shown in 46th～140 of SEQ ID No.4 forms.
2. the polynucleotide sequence of albumen claimed in claim 1 or its immunity fragment of encoding; Described polynucleotide sequence is preferably the polynucleotide sequence shown in SEQ ID No.1.
3. an engineering carrier, it comprises polynucleotide sequence as claimed in claim 2.
4. engineering carrier according to claim 3, wherein said carrier is plasmid.
5. a host cell, this host cell obtains through the described engineering carrier conversion of claim 3 or 4, transfection or transduction.
6. for the antibody of albumen claimed in claim 1 or its immunity fragment, be preferably polyclonal antibody.
7. albumen claimed in claim 1 or its immunity fragment or polynucleotide sequence claimed in claim 2, the application outside preparation has antiviral, anti-intracellular bacteria, antimycotic, anti-born of the same parents in the pharmaceutical preparation of bacterium and antitumor action; RAW264.7 cell TNF-α stimulated in preparation has the application in the preparation of chemotaxis; Application in the preparation of the generation that there is in preparation the phagocytic activity that strengthens scavenger cell, promotes ROS; Application in the preparation of the function that possess the expression of lowering the macrophage inflammatory factor in preparation, suppresses NF-κ B transcriptional activity; Application in the preparation of the differentiation for preparing promotion Th1 and Th17 cell and activation; Application in the preparation of the function of the protein expression that perhaps in preparation, possess the mRNA level of lowering CD55 or CD59, suppresses CD46, CD55 or CD59.
8. albumen claimed in claim 1 or its immunity fragment, or polynucleotide sequence claimed in claim 2, or antibody claimed in claim 6, the application in the pharmaceutical composition for the preparation of preventing and/or treating immune correlated disease.
9. application according to claim 8, wherein, described albumen be shown in SEQ ID No.2 or 46th～140 of SEQ ID No.2 shown in the albumen that forms of aminoacid sequence; Described polynucleotide sequence is the polynucleotide sequence shown in SEQ ID No.1; Described antibody is polyclonal antibody; Described immune correlated disease is infectious diseases, autoimmune disorder or tumour.
10. test right requires the application of reagent in the composition for the preparation of immune correlated disease auxiliary diagnosis, prognosis judgement of 1 described albumen or its immunity fragment or polynucleotide sequence claimed in claim 2; Described detection reagent is preferably antibody claimed in claim 6.