CN101096386A - Dectin-1 and heat shock protein Hsp60 polymer and coding nucleic acid and application thereof - Google Patents
Dectin-1 and heat shock protein Hsp60 polymer and coding nucleic acid and application thereof Download PDFInfo
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- CN101096386A CN101096386A CNA2007100416534A CN200710041653A CN101096386A CN 101096386 A CN101096386 A CN 101096386A CN A2007100416534 A CNA2007100416534 A CN A2007100416534A CN 200710041653 A CN200710041653 A CN 200710041653A CN 101096386 A CN101096386 A CN 101096386A
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Abstract
The invention discloses an agglutinin acceptor Dectin-1 and heat shock protein Hsp60 polymer and its coded nucleic acid molecule of peptide and application in the molecular biological domain, which is characterized by the following: the Hsp60 activates tyrosine kinase Syk with Dectin-1 connecting zone interacted with Dectin-1 in the immune sediment and cell stream, which guides the maturity of antigen submit cell to secretory cell factor to adjust immune system; The invention provides the application with Hsp60 and its Dectin-1 connecting zone as vaccine adjuvant, adjusting immune function and self-immunne disease to treat chronic infectious arthritis at least.
Description
Technical field
The present invention relates to biology field.Be specifically related to the nucleic acid molecule and the application of agglutinin receptor Dectin-1 and heat shock protein Hsp 60 polymer and these polypeptide of encoding thereof.
Background technology
The Dectin-1 assignment of genes gene mapping belongs to II type agglutinin receptor at karyomit(e) 12p13, is divided in the born of the same parents, strides outer three parts of film and born of the same parents.Wherein extracellular fragment contains single C type agglutinin sequence (C-type lectin-like domains, CTLD) sequence and do not contain the Ca2+ binding site wherein contains QPD block (can discern semi-lactosi and N-acetylamino galactosamine) in C type agglutinin sequence; Born of the same parents' inner segment contain immunity receptor tyrosine activate motif (immunoreceptor tyrosine-based activation-like motif, ITAM).Dectin-1 is because the shearing site difference causes forming two kinds of acceptor form and some non-functional little polypeptide forms existence that mainly contain function.Two kinds of functional receptors are named α respectively, β, and the α acceptor is the glycoprotein of about 33kd, and beta receptor born of the same parents outside part only contains single C type agglutinin sequence, lacks neck area, and whether neck area exists may be relevant with the effect of internalization beta-glucan with identification.Dectin-1 mainly is expressed in and Ia organ, as spleen, and thymus gland, lymphoglandula etc., and at brain, eye is not expressed in the muscle etc.Dectin-1 mainly is expressed in scavenger cell in cell, and dendritic cell on the antigen presenting cells such as neutrophil leucocyte, also have expression in part T cell.
Prior art shows that Dectin-1 is as a kind of pattern recognition acceptor, and it can be familiar with various 1,3 and 1,6 beta-glucan that connects, but nonrecognition monose.Can in conjunction with and the internalization zymosan, also can be in conjunction with complete yeast.Sea-tangle glycan, phosphorylation dextran can compete suppress beta-glucan and Dectin-1 combine the biological function of inhibition beta-glucan.Dectin-1 is as a kind of acceptor of engulfing, the mediation beta-glucan engulf activation with scavenger cell, in the fungi infestation of body, bringing into play important function.Dectin-1 can engulf pathogenic fungus, promotes the generation of ROS, thereby kills pathogenic agent, and Dectin-1 can activate downstream signal simultaneously, promotes TNF-α, IL-12, and IL-10, production of cytokines such as IL-2 are reconciled body's immunological function.Nearest studies show that, Dectin-1 mediate its signal conduction can with the mutual synergy of TLR, improve the ability of cytokine-expressing level and pathogenic microbe killing.
Present studies show that Dectin-1 also can discern the molecule of T cell surface, promotes the activation of T cell.Up to the present, the ligand molecular of the Dectin-1 of this T of existence cell surface is not also identified.In addition, a lot of C type agglutinin receptors not only can be discerned carbohydrate, can also discern intravital endogenic ligand molecule, comprise protein, and lipid molecule etc. play an important role in immunity system.The endogenic ligand molecule of screening Dectin-1, for research Dectin-1 in immunity system function and the signal transduction mechanism of further illustrating them lay the foundation, and for being that the drug development of drug target is provided fundamental basis with Dectin-1.
Hsp60 is one of protein of high conservative on evolving, and wide expression participates in the folding of intracellular protein in vivo, stops proteinic gathering, is very essential for the existence of cell.In Mammals, Hsp60 is positioned in the plastosome, under the pressure of external environment, comprise stress with ill-condition etc., Hsp60 can translocate to endochylema, cytolemma can also be secreted into the extracellular; Under the situation of external world's damage, necrocytosis also can discharge a large amount of Hsp60.In the blood plasma of healthy human body, exist low-level Hsp60, along with the rising at age, the Hsp60 level descends gradually.But in hypertension, atherosclerosis, in the diseases such as diabetes and rheumatoid disease sacroiliitis, the Hsp60 of blood plasma level obviously raises.Recent study shows that Hsp60 all brings into play important regulatory role in the morbidity of autoimmune disorder and chronic infection etc.Under stress situation, comprise infection etc., Hsp60 crosses expression or ectopic expression can be used as a kind of autoantigen by immune system recognition, brings out the protective immune response of body, promotes the immunologic function of body.In addition, being subjected to external stimulus at cell causes necrocytosis also can discharge a large amount of Hsp60 in blood plasma, and Hsp60 can be secreted in the blood plasma by some unknown Secretory Pathways, the Hsp60 of these solubilities can regulate inherent immunity and acquired immunity, thereby regulates body's immunological function.Hsp60 in this blood plasma is bringing into play a kind of effect of regulating immunity in body, therefore usually the Hsp60 in the blood plasma be can be used as a kind of danger signal.In the inherent immunity system, Hsp60 can activate pro-inflammatory cytokine and produce, and comprises TNF-α, pro-inflammatory cytokines such as IL-12, and the expression of activating cells surface costimulatory molecules simultaneously, as CD80, CD86 promotes the maturation of antigen presenting cell etc.In acquired immune system, Hsp60 can reduce the chemotactic function of T cell, strengthens sticking of T cell.Regulate in the cell at T especially, Hsp60 can promote T to regulate the propagation of cell, improves IL-10, the secretion of TGF-1 β, thus regulate developing of inflammation.Therefore the expert infers, Hsp60 may can regulate body's immunological function as a kind of cytokine.
Hsp60 activates the new function of autoimmunization system as it, has functional importance very widely, yet the mechanism of its performance function be it be not immediately clear.The cytokine effect of Hsp60 may be brought into play its function by the TLR-2/4 acceptor, and then activates the transcriptional activity of NFkappaB, promotes the maturation and the production of cytokines of antigen-presenting cell.Yet more and more evidences shows, on the antigen-presenting cell surface, has the acceptor of engulfing of Hsp60, and this receptor brought into play important biological function, but is not also identified at present.To the research of the immunoloregulation function of Hsp60, identify that the Hsp60 acceptor on the immunology cell seems very necessary for further.The evaluation of Hsp60 acceptor will be for illustrating the biological function of Hsp60 in autoimmune disorder and the theoretical basis that necessity is provided as the research of immunological adjuvant or Parmaceutical for treating disease of autoimmunization system thing with Hsp60.
Summary of the invention
The purpose of this invention is to provide the nucleic acid molecule and the application thereof of agglutinin receptor Dectin-1 and heat shock protein Hsp 60 polymer and these polypeptide of encoding thereof.
The invention discloses the interaction of agglutinin receptor Dectin-1 and its endogenic ligand heat shock protein Hsp 60, more particularly, heat shock protein Hsp 60 is as the endogenic ligand molecule of agglutinin receptor Dectin-1, and Hsp60 is by tyrosine kinase mediated its biologic activity of Syk.The amino acid polypeptide segment P540 (aa540-573) among the Hsp60 has wherein mediated combining of Hsp60 and Dectin-1.
Plain acceptor Dectin-1 of collection of the present invention and heat shock protein Hsp 60 polymer, wherein,
A) comprise the polypeptide of isolating Mammals Dectin-1 binding domains, wherein said isolating Mammals Dectin-1 binding domains is by being made up of the aminoacid sequence of at least 70% homogeny with the Hsp60 Dectin-1 binding domains shown in the SEQ ID NO:2; Or,
B) comprise the polypeptide of Dectin-1 binding domains, wherein said Dectin-1 binding domains is by be made up of the aminoacid sequence of 70% homogeny with the Hsp60 Dectin-1 binding domains shown in the SEQID NO:2 at least, and condition is that described polypeptide is not a total length Hsp60 polypeptide; Or,
C) comprise the polypeptide of the Dectin-1 binding domains shown in the SEQ ID NO:2.
Done the conservative amino acid displacement in the described polypeptide.
Described isolating Dectin-1 binding domains is made up of the aminoacid sequence shown in the about 540-573 amino acids shown in the SEQ ID NO:2.
The present invention also provides the method for authenticating compound, and this compound is regulated the maturation and the cytokine secretion of the mediation antigen presenting cell of Dectin-1 binding domains, regulates the ability of immunologic function, and this method comprises:
The acellular miscellany that contains the Dectin-1 binding domains is contacted with test compounds;
Measure test compounds and regulate the ability of Dectin-1 binding domains, thereby identify the maturation and the cytokine secretion of the mediation antigen presenting cell that can regulate the Dectin-1 binding domains, regulate the compound of immunologic function; Or,
The cell of expressing the Dectin-1 binding domains is contacted with test compounds;
Measure test compounds and regulate the ability of Dectin-1 binding domains, thereby identify the maturation and the cytokine secretion of the mediation antigen presenting cell that can regulate the Dectin-1 binding domains, regulate the compound of immunologic function.
Aforesaid method comprises the activity of regulating Hsp60 polypeptide or its Dectin-1 binding domains, or, comprise the expression of regulating Hsp60 or its Dectin-1 binding domains.
The invention provides the application of agglutinin receptor Dectin-1 and heat shock protein Hsp 60 polymer, described application includes but not limited to that immunological adjuvant, adjusting immunologic function, treatment comprise the application of the autoimmune disease of rheumatoid disease sacroiliitis.
The present invention is by yeast two-hybrid, and immunoprecipitation and cell streaming etc. experimental results show that the endogenic ligand molecule of Hsp60 as Dectin-1, and P540 mediates the combination between them.Hsp60 and its receptors bind activate propylhomoserin kinases Syk later on, thereby promote the maturation of antigen presenting cell.
The present invention by following technical proposals proof Hsp60 as the endogenic ligand molecule of Dectin-1 and activate the tyrosine kinase mediated biological function of syk:
By yeast two-hybrid screening, find that Hsp60 can interact with Dectin-1 extracellular fragment (aa112-247), the expression of activation yeast reporter gene that can be very strong shows the very strong interaction of existence between them;
By the immunoprecipitation technology, the cell streaming technology, experiments such as immunofluorescence and energy resonance have proved the endogenic ligand of Hsp60 as Dectin-1, can be engulfed by Dectin-1;
By immunological experiment, confirm that Hsp60 can regulate function of immune system, in antigen presenting cell, Hsp60 can activate the Syk Tyrosylprotein kinase, raises CD80, the expression level of CD86, the maturation of promotion antigen-presenting cell;
Research and analyse Hsp60 and Dectin-1 bonded zone by deletion mutantion Hsp60, find that the amino acid segment P540 (aa540-573) of Hsp60 can combine with Dectin-1 and engulf.
The present invention also provides a kind of fusion rotein, and this albumen comprises first polypeptide be made up of isolating Dectin-1 binding domains and second polypeptide of non-Hsp60.
The present invention also provides a kind of carrier, and it comprises wherein a kind of of following nucleotide sequence:
A) nucleotide sequence of the isolating Mammals Dectin-1 binding domains of coding, the Dectin-1 binding domains shown in wherein said isolating Mammals Dectin-1 binding domains and the SEQ ID NO:2 has 70% aminoacid sequence homogeny;
B) nucleotide sequence of the isolating Mammals Dectin-1 binding domains of coding, wherein said nucleotides sequence is listed among 45 ℃ of 6 * SSC, then 50-65 ℃ down with 0.2SSC and 0.1%SDS washing one time or multipass after with the complementary sequence hybridization of the nucleotide sequence shown in the SEQ ID NO:1 of encoding D ectin-1 binding domains;
C) nucleotide sequence of the isolating Mammals Dectin-1 of the coding binding domains shown in SEQ ID NO:1.
Description of drawings
Fig. 1 has shown Dectin-1 and Hsp60, the interaction of P540 in yeast.
Fig. 2 has shown Dectin-1 and Hsp60 immunoprecipitation experiment result, shows that Hsp60 can be in conjunction with Dectin-1.
Fig. 3 has shown that Hsp60 can be attached to the cell surface of expressing Dectin-1 by dosage with relying on.
Fig. 4 has shown that the Hsp60 of different concns can be attached to the fixedly chip surface of Dectin-1.
Fig. 5 shown Dectin-1 can in conjunction with and engulf fluorescently-labeled P540.
Fig. 6 has shown that Hsp60 can activate the phosphorylation of Syk Tyrosylprotein kinase.
Fig. 7 has shown that Hsp60 can promote the maturation of antigen-presenting cell by the Syk Tyrosylprotein kinase.
Embodiment
The cDNA of people Hsp60 and the cDNA of P550 are connected respectively to the pGEX-4T-1 carrier by Protocols in Molecular Biology, transform DH5 α, the positive colony that screens by penbritin, pass through plasmid purification, order-checking, the cloned sequence that proof is selected is correct, difference called after pGEX-4T/Hsp60, pGEX-4T/P540.Use pGEX-4T/Hsp60, the pGEX-4T/P540 plasmid transforms the BL21 intestinal bacteria respectively, chooses the clone and is inoculated in the LB training liquid, and 37 ℃ of cultivations when bacterium liquid reaches OD0.6-0.8, are carried out abduction delivering under the condition of IPTG concentration 0.2mM.Abduction delivering was received cell after 4 hours, cracking.According to GST purifying handbook, by GST pearl purifying target protein.Identify that by SDS-PAGE and Western blot purified product is target protein Hsp60 and P540.
Embodiment 2:
The extracellular fragment of people's Dectin-1 (aa112-247) cDNA passes through molecule clone technology, insert the pGBKT7 carrier with EcoRI and BamHI site, subsequent transformation DH5 α screens positive colony by kalamycin, select the mono-clonal bacterium colony, extract the plasmid evaluation of checking order.The result shows that the cDNA sequence of the extracellular fragment of people's Dectin-1 (aa112-247) is consistent with the frame of carrier.This carrier is referred to as pGBKT7/CTLD, and pGBKT7/CTLD and pGADT7 carrier corotation are gone into the AH109 yeast, screens on the substratum, and the result shows that pGBKT7/CTLD does not have the self activation phenomenon.
According to Clontech company yeast two-hybrid operational manual, the cDNA library of adopting pGBKT7-CTLD to screen people's thymus gland.Select and can be grown in the SD-Ade/-His/-Leu/-Trp/X-a-Gal substratum and become blue positive colony, according to operational manual carry out 3 take turns screening after, the positive colony that screens carried out plasmid separates and order-checking.Sequencing result is compared in ncbi database, finds three positive colony coding Hsp60 protein moleculars.In order to determine the calmodulin binding domain CaM of Hsp60 molecule and Dectin-1, the present invention carries out deletion mutantion with Hsp60, isolated nucleic acid molecule wherein, and it comprises a kind of of following nucleotide sequence:
The encode nucleotide sequence of isolating Mammals Dectin-1 binding domains, the Dectin-1 binding domains shown in wherein said isolating Mammals Dectin-1 binding domains and the SEQ ID NO:2 has 70% aminoacid sequence homogeny;
The encode nucleotide sequence of isolating Mammals Dectin-1 binding domains, wherein said nucleotides sequence be listed among 45 ℃ of 6 * SSC, then 50-65 ℃ down with 0.2SSC and 0.1%SDS washing one time or multipass after with the complementary sequence hybridization of the nucleotide sequence shown in the SEQ ID NO:1 of encoding D ectin-1 binding domains;
The nucleotide sequence of the isolating Mammals Dectin-1 of the coding binding domains shown in SEQ ID NO:1.
Described isolated nucleic acid molecule, wherein isolating Mammals Dectin-1 binding domains is made up of the aminoacid sequence shown in about 540-573 amino acids of SEQ ID NO:2.
Described isolated nucleic acid molecule, wherein said nucleic acid molecule encoding fusion rotein.
Constructed fusion rotein, this albumen comprise first polypeptide be made up of isolating Dectin-1 binding domains and second polypeptide of non-Hsp60.
The present invention has made up a kind of carrier, and it includes said nucleic acid molecule.
Experimental result shows, the amino acid polypeptide segment P540 (aa540-573) of Hsp60 can with the combining of Dectin-1.As shown in Figure 1, the expression of the activation reporter gene that Hsp60 and P540 can be very strong, galactosidase activity is very high, explanation is in yeast, Hsp60 and Dectin-1 interact, and activate the expression of reporter gene, are this peptide section mediations of amino acid 540-573 by Hsp60.
The cDNA of people Dectin-1 is cloned into this carrier by Protocols in Molecular Biology according to the frame of pCDNA3.1/Myc carrier.Wherein, the cDNA terminator codon of people Dectin-1 disappearance can produce the Dectin-1/Myc fusion rotein.Order-checking shows that this constructed plasmid is correct, called after pMyc-Dectin-1.With pMyc-Dectin-1 transfection HEK293T cell, collecting cell after 48 hours, cell is cracking in lysate, carries out immunoprecipitation experiment.Step is as follows:
1. the Hsp60 of prokaryotic expression was hatched 2 hours with the commentaries on classics of GST sepharose 4B pearl wheel at 4 ℃ among the embodiment 1, with cold PBS washing once.
2. the pearl of step 1 is centrifugal after 4 ℃ of wheel commentaries on classics are hatched 4 hours with above-mentioned cell pyrolysis liquid then, fully pearl is washed 3 times with cold lysis buffer, and the adding sample-loading buffer boils and carried out immune marking experiment in 10 minutes.
The result shows that Hsp60 can combine with Dectin-1, has further proved the interaction (shown in Figure 2) between them.
The Hsp60 of embodiment 1 prokaryotic expression carries out fluorescent mark according to the operational manual of pierce company, and fluorescently-labeled product packing is stored in-20 ℃.PMyc-Dectin-1 transfection HEK293T cell, collecting cell after 48 hours with cold that PBS washes 3 times, was hatched 1 hour at 4 ℃ with fluorescently-labeled Hsp60, fully washed 3 times with PBS again, carried out the cell flow cytometer showed.In the competition inhibition analysis of research receptors ligand, above-mentioned cell was hatched 30 minutes with unlabelled Hsp60 in advance, added fluorescently-labeled Hsp60 then.The result of flow cytometer showed shows the cell surface that expressing human Dectin-1 expresses that is attached to that Hsp60 can dose-dependently as shown in Figure 3, and unmarked Hsp60 can compete and suppresses fluorescently-labeled Hsp60 and be attached to the cell surface that Dectin-1 expresses.This characteristic shows that Hsp60 is the relation of a kind of acceptor and part with combining of Dectin-1.
Prokaryotic expression Dectin-1 extracellular fragment and purifying.By Biacore sensor (Biacore, Uppsala, the Sweden) interaction of direct analysis between them.On chip, fix the Dectin-1 purified product of 1000 units in advance, analyze with the Hsp60 sample introduction of different concns gradient then.Biosensor can directly be studied the interaction between the protein-protein, the result shows, Hsp60 can be attached on the biochip of Dectin-1 connection by dosage with relying on, and Kd is respectively 1.04e-6, further specifies can interact between them (Fig. 4).
Embodiment 6
HEK3T3 cell transient transfection Dectin-1 plasmid was cultivated 48 hours, after cold PBS washes 3 times, hatched 30 minutes at 4 ℃ with fluorescently-labeled P550, washed 3 times the formalin fixed with 4% 10 minutes, washing then, mounting then with cold PBS.Then continue 37 ℃ and hatch after 15 minutes more fixingly if engulf analysis, step is the same.Carry out the burnt analysis of copolymerization at last.In the immunofluorescence experiment, the green fluorescence mark be P540, red fluorescence be Dectin-1, the result show Dectin-1 can in conjunction with and engulf P550, illustrate Dectin-1 can by P540 in conjunction with and engulf hsp60 (Fig. 5).
Embodiment 7
The male BALB/c mouse in 8 ~ 10 weeks is by pre-treatment, get peritoneal macrophage, cultivate after 2 hours for 37 ℃, add syk inhibitor or contrast, add Hsp60 to 10ug/ml after 1 hour respectively, 37 ℃ hatch 10 minutes after, collecting cell carries out immune marking analysis with phosphorylation antibody behind cracking and the protein quantification.The result shows that Hsp60 can activate the phosphorylation of syk tyrosine, and can not activate Syk through the Hsp60 of heat inactivation.Prior art studies show that Hsp60 can activate Erk1/2, JNK and P38 signal path, and whether the activation that the present invention further studies them is the activation that depends on the syk Tyrosylprotein kinase.Experimental result of the present invention shows that the inhibitor piceatonnal of Syk Tyrosylprotein kinase can obviously suppress the phosphorylation of Erk1/2 and P38, but influences the phosphorylation of JNK inadequately, shows that the activation of Erk1/2 and P38 is regulated and control by Syk.The present invention has confirmed that Hsp60 can activate the Syk Tyrosylprotein kinase, and Syk has very important function (Fig. 6) in the signal conductive process of Hsp60 performance biological function.
Embodiment 8
The male BALB/c mouse in 8 ~ 10 weeks is by pre-treatment, get peritoneal macrophage, cultivate after 2 hours for 37 ℃, add syk inhibitor or contrast, add Hsp60 to 10ug/ml after 1 hour, cultivate 18 hours collecting cells, hatch respectively with different costimulatory molecules antibody and wash 3 times with PBS after 30 minutes, carry out the cell flow cytometer showed then.The result shows that Hsp60 can obviously raise antigen presenting cell surface costimulatory molecules CD80, the expression of CD86, and the Syk inhibitor can obviously suppress scavenger cell surface costimulatory molecules CD80, the expression of CD86.CD80, CD86 can provide second signal for the activation of T cell as costimulatory molecules, and the reduction scavenger cell that they express can not be effectively provides second signal for the activation of T cell.Result of the present invention confirms that Hsp60 can be delivery cell by activation antigen, effectively mediates the activation of acquired immunity.
SEQUENCE?LISTING
＜110〉Fudan University
＜120〉Dectin-1 and heat shock protein Hsp 60 polymer and coding nucleic acid thereof and application
<130>11
<160>2
<170>PatentIn?version?3.1
<210>1
<211>102
<212>DNA
＜213〉Mammals
<400>1
actacagcag?aagttgtagt?cacagaaatt?cctaaagaag?agaaggaccc?tggaatgggt
60
gcaatgggtg?gaatgggagg?tggtatggga?ggtggcatgt?tc
102
<210>2
<211>33
<212>PRT
＜213〉Mammals
<400>2
Thr?Ala?Glu?Val?Val?Val?Thr?Glu?Ile?Pro?Lys?Glu?Glu?Lys?Asp?Pro
1 5 10 15
Gly?Met?Gly?Ala?Met?Gly?Gly?Met?Gly?Gly?Gly?Met?Gly?Gly?Gly?Met
20 25 30
Phe
Claims (15)
1, Dectin-1 and heat shock protein Hsp 60 polymer is characterized in that,
The polypeptide that comprises isolating Mammals Dectin-1 binding domains, wherein said isolating Mammals Dectin-1 binding domains is by being made up of the aminoacid sequence of at least 70% homogeny with the Hsp60 Dectin-1 binding domains shown in the SEQ ID NO:2; Or,
The polypeptide that comprises the Dectin-1 binding domains, wherein said Dectin-1 binding domains is by be made up of the aminoacid sequence of 70% homogeny with the Hsp60 Dectin-1 binding domains shown in the SEQID NO:2 at least, and condition is that described polypeptide is not a total length Hsp60 polypeptide; Or,
The polypeptide that comprises the Dectin-1 binding domains shown in the SEQ ID NO:2.
2, Dectin-1 according to claim 1 and heat shock protein Hsp 60 polymer is characterized in that, and be described
The polypeptide of Dectin-1 binding domains has been done the conservative amino acid displacement.
3, Dectin-1 according to claim 1 and heat shock protein Hsp 60 polymer is characterized in that, described isolating Dectin-1 binding domains is made up of the aminoacid sequence shown in the 540-573 amino acids shown in the SEQ ID NO:2.
4, Dectin-1 according to claim 1 and heat shock protein Hsp 60 polymer, wherein said isolating Dectin-1 binding domains are regulated the purposes in the immunologic function at the maturation and the cytokine secretion of mediation antigen presenting cell.
5, a kind of isolated nucleic acid molecule, it comprises a kind of of following nucleotide sequence:
The encode nucleotide sequence of isolating Mammals Dectin-1 binding domains, the Dectin-1 binding domains shown in wherein said isolating Mammals Dectin-1 binding domains and the SEQ ID NO:2 has 70% aminoacid sequence homogeny;
The encode nucleotide sequence of isolating Mammals Dectin-1 binding domains, wherein said nucleotides sequence be listed among 45 ℃ of 6 * SSC, then 50-65 ℃ down with 0.2SSC and 0.1%SDS washing one time or multipass after with the complementary sequence hybridization of the nucleotide sequence shown in the SEQ ID NO:1 of encoding D ectin-1 binding domains;
The nucleotide sequence of the isolating Mammals Dectin-1 of the coding binding domains shown in SEQ ID NO:1.
6, isolated nucleic acid molecule according to claim 5, wherein isolating Mammals Dectin-1 binding domains is made up of the aminoacid sequence shown in the 540-573 amino acids of SEQ ID NO:2.
7, isolated nucleic acid molecule according to claim 5, wherein said isolating Mammals Dectin-1 binding domains is regulated the purposes in the immunologic function in the maturation of mediation antigen presenting cell and the secretion of cytokine.
8, isolated nucleic acid molecule according to claim 5, wherein said nucleic acid molecule encoding fusion rotein.
9, a kind of fusion rotein, this albumen comprise first polypeptide be made up of isolating Dectin-1 binding domains and second polypeptide of non-Hsp60.
10, a kind of carrier, it includes the described nucleic acid molecule of claim 5.
11, a kind of maturation and cytokine secretion that mediates antigen presenting cell, the method for adjusting immunologic function, this method comprises the activity of regulating Hsp60 polypeptide or its Dectin-1 binding domains.
12, a kind of maturation and cytokine secretion that mediates antigen presenting cell, the method for adjusting immunologic function, this method comprises the expression of regulating Hsp60 or its Dectin-1 binding domains.
13, a kind of method of authenticating compound, this compound is regulated the maturation and the cytokine secretion of the mediation antigen presenting cell of Dectin-1 binding domains, regulates the ability of immunologic function, and this method comprises:
The acellular miscellany that contains the Dectin-1 binding domains is contacted with test compounds;
Measure test compounds and regulate the ability of Dectin-1 binding domains, thereby identify the maturation and the cytokine secretion of the mediation antigen presenting cell that can regulate the Dectin-1 binding domains, regulate the compound of immunologic function.
14, a kind of method of authenticating compound, this compound is regulated the maturation and the cytokine secretion of the mediation antigen presenting cell of Dectin-1 binding domains, regulates the ability of immunologic function, and this method comprises:
The cell of expressing the Dectin-1 binding domains is contacted with test compounds;
Measure test compounds and regulate the ability of Dectin-1 binding domains, thereby identify the maturation and the cytokine secretion of the mediation antigen presenting cell that can regulate the Dectin-1 binding domains, regulate the compound of immunologic function.
15, the Dectin-1 of claim 1 and the heat shock protein Hsp 60 polymer application in preparation immunological adjuvant, adjusting immunologic function or treatment autoimmune disease medicine.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111971028A (en) * | 2017-11-21 | 2020-11-20 | 西奈山伊坎医学院 | Enhancing training immunity with therapeutic nanobiotic compositions |
| US11859021B2 (en) | 2021-03-19 | 2024-01-02 | Icahn School Of Medicine At Mount Sinai | Compounds for regulating trained immunity, and their methods of use |
-
2007
- 2007-06-05 CN CNA2007100416534A patent/CN101096386A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111971028A (en) * | 2017-11-21 | 2020-11-20 | 西奈山伊坎医学院 | Enhancing training immunity with therapeutic nanobiotic compositions |
| US11859021B2 (en) | 2021-03-19 | 2024-01-02 | Icahn School Of Medicine At Mount Sinai | Compounds for regulating trained immunity, and their methods of use |
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