CN105367658A - Human-derived anti-sialic acid-binding immunoglobulin-like lectin-9 (Siglec-9) antibody IgG and application thereof - Google Patents
Human-derived anti-sialic acid-binding immunoglobulin-like lectin-9 (Siglec-9) antibody IgG and application thereof Download PDFInfo
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- CN105367658A CN105367658A CN201510870684.5A CN201510870684A CN105367658A CN 105367658 A CN105367658 A CN 105367658A CN 201510870684 A CN201510870684 A CN 201510870684A CN 105367658 A CN105367658 A CN 105367658A
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Abstract
The invention relates to a human-derived anti-sialic acid-binding immunoglobulin-like lectin-9 (Siglec-9) antibody IgG and applications thereof. An amino acid sequence of a heavy chain variable region is represented by SEQ ID NO.2; an amino acid sequence of a light chain variable region is represented by SEQ ID NO.9; the human-derived antibody can be specifically bound with the Siglec-9. An in-vitro cell experiment result verifies that the antibody of the Siglec-9 can effectively induce apoptosis of positive cells of the Siglec-9 and inhibit secretion of cell factors, so that the monoclonal antibody provided by the invention can be applied to clinic treatment and prevention of related diseases such as severe asthma, chronic obstructive pulmonary disease, autoimmune disease, tumors and the like.
Description
Technical field
The invention belongs to monoclonal antibody drug technical field, relate to a kind of anti-sialic acid binding domain-immunoglobulin sample lectin-9 (thesurfacemoleculesialicacid-bindingimmunoglobulin-likel ectin-9, Siglec-9) full molecule human monoclonal antibody and by the DNA molecular of encoding, and the application of this full molecule human monoclonal antibody in the disease medicaments such as preparation treatment severe asthma, chronic obstructive pulmonary disease, autoimmune disorder and tumour.
Background technology
Sialic acid binding domain-immunoglobulin sample lectin (thesurfacemoleculesialicacid-bindingimmunoglobulin-likel ectin, Siglec) be the important member of immunoglobulin superfamily, by identifying containing the interaction between sialic sugar chain structure mediated cell and cell or pathogenic agent.Research display recent years, siglec family plays a significant role at cell activation, propagation and apoptosis, meanwhile, in inherent immunity and adaptive immunity, also plays important regulating and controlling effect, also can participate in the regulation and control of immunological tolerance in addition.
Siglecs is immunoglobulin superfamily member, and its molecular structure common feature has the ability in conjunction with sialic acid.Sialic acid binding domain-immunoglobulin sample lectin, is expressed in immunocyte surface, mediate both inhibitory signal.Similar to other inhibition immunity receptor families, as killer cell immunoglobulin-like receptors and leukocytic immunity albumen sample acceptor, siglecs is transmembrane molecule, immunity receptor Tyrosine Inhibitory Motifs ITIMs (immunoreceptortyrosinebasedinhibitorymotifs is comprised in cytoplasmic tail, ITIMs), outside born of the same parents, there is immunoglobulin superfamily region.Compared with other immunoglobulin superfamilies, the characteristic of siglecs is the sialylated carbohydrate of the part of its high specific, is different from other immunity receptor associated proteins of major part.The mankind have been found that 14 kinds of siglec: sialoadhesin (siglec1CD169), the glycoprotein (MAGsiglec4) that CD22 (siglec2), CD33 (siglec3) are relevant with myelin and the siglec (siglec5-11) that cd33 is recently correlated with.Except siglec4 is expressed in neurocyte, most of siglecs family is expressed in hematopoietic cell.
Sialic acid binding domain-immunoglobulin sample lectin 9 (siglec9) is I type transmembrane protein, the important member of the super race of immunoglobulin (Ig), mainly by V district and the Liang Ge C district immunoglobulin like domain of the outer N-terminal of born of the same parents, cross-film district and kytoplasm inner region are formed, the identification in V district, N-terminal immunoglobulin spline structure territory mediation sialic acid (SIA), kytoplasm inner region contains two Immuno-Tyrosine and suppresses motif (ITIM).Nearly film motif ITIM tyrosine phosphorylation, raise inhibition phosphatases, as Src homologous region 2 comprises tyrosine-phosphatase 1 (SHP-1), SHP-2 and SH2 inositol monophosphate enzyme (SHIP), thus mediate downstream tyrosine kinases phosphorylate, and in born of the same parents, transmit inhibition or lethality signal.
Siglec-9 at neutrophil leucocyte and monocyte high expression level, at NK cell, CD4
+and CD8
+the low expression of some subgroup of T cell.At the middle and advanced stage of marrow hemopoietic stem cells differentiation, the expression of Siglec-9 can be there is in myelocyte film surface, Siglec-9 participates in multiple physiological processs such as the differentiation of neutrophil leucocyte, growth and death, is the critical function acceptor regulating neutrophil leucocyte inherent immunity.Lung tissue a large amount of eosinophils companion neutrophilic leukocytosis is the important pathologic, physiologic feature of severe asthma, the replacement therapy of anti-Siglec-9 antibody takes into account both PCD, may have potential therapeutic value in the treatment of the diseases such as severe asthma, chronic obstructive pulmonary disease, autoimmune disorder, tumour.
Summary of the invention
The technical problem solved: the IgG antibody and the application thereof that the invention provides a kind of people source anti-sialic acid binding domain-immunoglobulin sample lectin-9.
Technical scheme: the IgG antibody of people source anti-sialic acid binding domain-immunoglobulin sample lectin-9, the V of described antibody
lthe complementary determining region of chain has following CDRs aminoacid sequence:
V shown in SEQIDNO.10
l-CDR1;
V shown in SEQIDNO.11
l-CDR2;
V shown in SEQIDNO.12
l-CDR3;
And the V of described antibody
hthe complementary determining region of chain has following CDRs aminoacid sequence:
V shown in SEQIDNO.3
h-CDR1;
V shown in SEQIDNO.4
h-CDR2;
V shown in SEQIDNO.5
h-CDR3.
The IgG antibody of people source anti-sialic acid binding domain-immunoglobulin sample lectin-9, the aminoacid sequence of variable region of heavy chain is as shown in SEQIDNO.2; The aminoacid sequence of variable region of light chain is as shown in SEQIDNO.9.And above-mentioned sequence is through the conservative mutation of one or more aminoacid addition, deletion, replacement, modification and the conservative variant obtained.
The IgG antibody of people source anti-sialic acid binding domain-immunoglobulin sample lectin-9, the aminoacid sequence of CH is as shown in SEQIDNO.7; The aminoacid sequence of constant region of light chain is as shown in SEQIDNO.14.And above-mentioned sequence is through the conservative mutation of one or more aminoacid addition, deletion, replacement, modification and the conservative variant obtained.
The nucleotide sequence of the IgG antibody of encoding human source anti-sialic acid binding domain-immunoglobulin sample lectin-9, the nucleotide sequence of encoding heavy chain variable region is for shown in SEQIDNO.1, and the nucleotide sequence of encoded light chain variable region is for shown in SEQIDNO.8.
The nucleotide sequence of the IgG antibody of encoding human source anti-sialic acid binding domain-immunoglobulin sample lectin-9, the nucleotide sequence of encoding heavy chain constant is for shown in SEQIDNO.6, and the nucleotide sequence of coding constant region of light chain is for shown in SEQIDNO.13.
The application of IgG antibody in preparation prevention or treatment severe asthma, chronic obstructive pulmonary disease, autoimmune disorder and/or tumour medicine of above-mentioned people source anti-sialic acid binding domain-immunoglobulin sample lectin-9.
A medicine for prevention or treatment severe asthma, effective constituent is the IgG antibody of described people source anti-sialic acid binding domain-immunoglobulin sample lectin-9.
A medicine for prevention or treatment chronic obstructive pulmonary disease, effective constituent is the IgG antibody of described people source anti-sialic acid binding domain-immunoglobulin sample lectin-9.
A medicine for prevention or treatment autoimmune disorder, effective constituent is the IgG antibody of described people source anti-sialic acid binding domain-immunoglobulin sample lectin-9.
A medicine for prevention or treatment tumour, effective constituent is the IgG antibody of described people source anti-sialic acid binding domain-immunoglobulin sample lectin-9.
Beneficial effect: the invention provides a kind of human monoclonal antibody of anti-sialic acid binding domain-immunoglobulin sample lectin-9 with high protectiveness, high specific, good affinity.Function Identification is done to this full molecule human antibody, result show this human antibody can with sialic acid binding domain-immunoglobulin sample lectin-9 specific binding; In vitro cell experiment result confirms, people source anti-sialic acid binding domain-immunoglobulin sample lectin-9 antibody effectively can induce the apoptosis of sialic acid binding domain-immunoglobulin sample lectin-9 positive cell, the secretion of the T suppression cell factor, therefore monoclonal antibody of the present invention can be applicable to clinical treatment and the prevention of the relative diseases such as severe asthma, chronic obstructive pulmonary disease, autoimmune disorder, tumour.
Accompanying drawing explanation
The SDS-PAGE of Fig. 1 purified antibodies IgG detects;
The ELISA of the anti-Siglec-9 antibody of Fig. 2 detects;
The Westernblot of the anti-Siglec-9 antibody of Fig. 3 detects;
The avidity that the anti-Siglec-9 antibody of Fig. 4 is combined with antigen detects;
The neutrophil apoptosis of the anti-Siglec-9 antibody induction of Fig. 5 detects;
Fig. 6 FACS detects the neutrophil leucocyte ROS of anti-Siglec-9 antibody induction.
Embodiment
It should be noted that, variable region of heavy chain and the variable region of light chain of this antibody are human antibody, and it are connected with the constant region of human antibody, are therefore referred to as full molecule human antibody.
The present invention for target molecule, the basis of phage antibody library technique is prepared people source anti-sialic acid binding domain-immunoglobulin sample lectin-9 IgG antibody with sialic acid binding domain-immunoglobulin sample lectin-9.Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.It should be understood that described embodiment is only exemplary, any restriction is not formed to protection scope of the present invention.It will be understood by those skilled in the art that and do not departing under spirit of the present invention and can modify to the details of technical scheme and form or replace, but these amendments or replacement all fall into protection scope of the present invention.
Embodiment 1
The screening of people source anti-sialic acid binding domain-immunoglobulin sample lectin-9 monoclonal antibody
1) use recombinant human sialic acid binding domain-immunoglobulin sample lectin-9 albumen bag of purifying by solid-phase screening elisa plate, every hole 2 μ g, washing, adds confining liquid, and washing, adds phage antibody library antibody, and unconjugated phage antibody is removed in washing.
2) add trypsinase, the phage antibody of wash-out specific binding, add helper phage VCSM13 superingection.
3) screen step more than repeating, carry out four-wheel " absorption-wash-out-amplification " enrichment isolation altogether.
4) last is taken turns screening and rise in value obtain phage dilution after be laid on add 100 μ g/mL penbritin culture plate on overnight incubation, picking 80 single bacterium colonies in Tissue Culture Plate, jolting overnight incubation.
5) from first piece of each hole of plate, distinguish transferase 45 μ L bacterium liquid to the second block plate after spending the night, jolting is cultivated.
6) add helper phage VCSM13 superingection, jolting is cultivated; Centrifugal, the resuspended precipitation of substratum, jolting overnight incubation.
7) centrifuging and taking supernatant carries out ELISA detection, measures every hole 450nm and 650nm light absorption value, calculates every hole light absorption value by A450nm ~ A650nm.When P/N value (Positive/Negative) is greater than 6, this bacterial strain is positive monoclonal phage strains.
8) positive colony is carried out nucleic acid sequence analysis, the nucleotide sequence of encoding heavy chain variable region is for shown in the SEQIDNO.1 in sequence table, the nucleotide sequence of encoded light chain variable region for shown in the SEQIDNO.8 in sequence table, thus obtains the correct Fab of a kind of gene order.
The preparation of the IgG antibody of people source anti-sialic acid binding domain-immunoglobulin sample lectin-9
1) according to the variable region sequences obtaining antibody, the primer of design InfusionPCR
According to weight, the light chain pcr amplification primer of InfusionPCR principle design antibody, this primer needs the base of the 15bp comprised on expression vector and inserts at least 15bp base of object fragment, inserts the principle design that object fragment place base designs according to general primer.
Heavy, light chain pcr amplification primer:
Heavy chain amplimer:
HF:5’-GGTGTCCACTCGCTACAGGTGCAGCTGGTGCAGTCT-3’
HR:5’-GCCCTTGGTGGATGCTGAGGAGACGGTGAC-3’
Light chain amplimer:
L5F:5’-ACAGACGCTCGCTGCTCCTATGAGCTGACTCAGCCACCT-3’
L5R：5’-GTTGGCCTTGGGCTGTAGGACGGTCAGCCT-3’
2) increase people source anti-sialic acid binding domain-immunoglobulin sample lectin-9 IgG antibody heavy chain, light chain
With the people source Fab of above-mentioned preparation for template, respectively with the full molecule human antibody heavy of upstream and downstream primer amplification of the above-mentioned heavy chain that relates to and light chain, light chain gene.
①PCR
Reaction system:
Reaction conditions:
2. 2% agarose gel electrophoresis, observes object band under ultraviolet, cut glue and reclaim.
3. glue reclaims kits target DNA fragment, deionized water wash-out.
3) double digestion IgG expression plasmid
IgG expression plasmid pFUSE-CHIg-hG1, pFUSE-CLIg-hk (purchased from Invivogen company) comprise heavy chain and light chain (Kappa) the constant region alkali yl coding sequence in IgG1 type people source, the concrete nucleotide sequence of encoding heavy chain constant is as shown in SEQIDNO.7, and the concrete nucleotide sequence of coding constant region of light chain is as shown in SEQIDNO.14.
1. the double digestion of pFUSE-CHIg-hG1, pFUSE-CLIg-hk template vector
Reaction system:
Reaction conditions: 37 DEG C of enzymes cut through night.
2. 1% agarose gel electrophoresis, ultraviolet incision glue reclaims.
3. glue reclaims kits target DNA fragment, deionized water wash-out.
4) InfusionPCR recombinant expression plasmid
Reaction system:
Reaction conditions: hatch 15min for 50 DEG C.
Get 5 μ L reaction solution transform competent bacteria, be laid on the flat board of corresponding resistant, next day chooses clone and send order-checking.Clone correct for sequencing result is preserved bacterial classification and enlarged culturing, extracts plasmid.
5) expression of the anti-sialic acid binding domain-immunoglobulin in people source sample lectin-9 IgG antibody
1. 250 μ LpFUSE-CHIg-hG1-H (i.e. 50 μ g) are got in the Opti-MEM substratum of 1mL, get 250 μ LpFUSE-CLIg-hk-L (i.e. 50 μ g) in the Opti-MEM substratum of 1mL, above-mentioned three kinds of mixed solution room temperatures, in the Opti-MEM substratum of 2.8mL, are left standstill 5min by the 293Fectin getting 200 μ L.
2., after then being mixed by two plasmid mixed solutions, the Opti-MEM substratum adding 500 μ L mixes the rear mixed solution directly adding transfection reagent 293Fectin, mixes rear standing 20min.Period process 293F cell, by resuspended with 293FExpressionMedium after 293F cell centrifugation, then count and calculates cell viability ratio with trypan blue, drawing 100 × 10
6individual cell, in culturing bottle, is 94mL with 293FExpressionMedium constant volume.
3. the mixture of DNA, 293Fectin of 6mL adds in ready 293F cell after terminating by 20min.
4. cell is placed in shaking table incubator and cultivates, culture condition 8%CO
2, 120rmp, 37 DEG C, collecting cell supernatant after 6 days.
6) purifying of the anti-sialic acid binding domain-immunoglobulin in people source sample lectin-9 IgG antibody
By the membrane filtration of the cell conditioned medium of collection with 0.22 μm, simultaneously by balance liquid and elutriant filtering membrane.Purify with AKATA the standard step that instrument purifies according to ProteinA to purify, with the speed loading of 1mL/min, with the speed wash-out of 1.5mL/min.Result successful expression IgG purification antibody.SDS-PAGE detected result is shown in Fig. 1.
The functionally active qualification of people source anti-sialic acid binding domain-immunoglobulin sample lectin-9 IgG antibody
1) euzymelinked immunosorbent assay (ELISA)
Dilute recombinant human sialic acid binding domain-immunoglobulin sample lectin-9 to 2 μ g/mL with coating buffer (0.1M carbonate buffer solution, pH9.6) to wrap by ELISA96 orifice plate, every hole adds 100 μ L, and 4 DEG C are spent the night; PBST (PBS is containing 0.5%Tween20) 5% skimmed milk-lavation buffer solution is closed, and hatches 2h for 37 DEG C; After PBST washs 5 times, in each hole, add 100 μ LPA21 antibody (2 μ g/mL initial concentrations, 14 concentration gradient dilutions), 37 DEG C of 2h; Join in hole with the anti-100 μ L/ holes of goat-anti people two of 1:2000 dilution, hatch 1h for 37 DEG C; Peroxidase substrate nitrite ion 100 μ L/ hole, room temperature is by 2M sulfuric acid stopped reaction after lower 10 minutes, and upper machine testing colorimetric adopts dual wavelength 450nm/690nm.Result such as Fig. 2 shows, people source anti-sialic acid binding domain-immunoglobulin sample lectin-9 IgG antibody can with Siglec9 protein binding.
2)Westernblot
Neutrophil leucocyte cracking supernatant is carried out 10%SDS-PAGE electrophoresis and electricity forwards on nitrocellulose membrane, this film and 2 μ g/mL antibody at room temperature are hatched 1h, and 1:2000 dilutes HRP-goat anti-human igg (Beijing Zhong Shan company) and ECL luminescence reagent box (Pierce company of the U.S.) is exposed to gel imaging system (Bio-Rad company).
Result is as shown in Figure 3: people source anti-sialic acid binding domain-immunoglobulin sample lectin-9 IgG antibody and Siglec-9 albumen have specific binding.
3) avidity detects
Optimize coupling condition according to antigen iso-electric point and according to the protocol of Biacore-X100controlsoft, slope optimized choice sodium-acetate is as coupling dilution buffer.Be coupled on CM5 chip with after this damping fluid dilution antigen samples to 25 μ g/mL.Preset the horizontal 1500RU of coupling.Then dilute antigen samples with the Runningbuffer of pH7.4, dilute a series of concentration to 0uM, 5nM, 10nM20nM, 40nM, 80nM.Arranging sample injection time is 180s, Dissociation time 10min, regeneration buffer 50mMpH2.2Gly-HCl.Examination with computer is carried out according to the protocol of BiacoreX100controlsoft.Avidity detected result is shown in Fig. 6, and KD value is 7.380 × 10
-10m.
4) detection of antibody induction neutrophil apoptosis
With Ficoll parting liquid Separation of Neutral granulocyte, GM-CSF25ng/mL pre-treatment 30 minutes, adds Siglec-9mAb10 μ g/mL (B10101) and negative control respectively.Act on after 8 hours, with Annexin-V and PI dyeing, flow cytomery.Result shows, and antibody can induce neutrophil apoptosis, and apoptosis rate reaches 21.15%.
5) ROS detected result in cell:
Take venous blood 5mL, Ficoll parting liquid Separation of Neutral granulocyte, is divided into three groups: negative control group, GM-CSF group and GM-CSF+siglec-9mAb20 μ g/mL group, acts on 2 hours respectively.Add the LPS of 100ng/mL in nutrient solution, act on 1 hour.According to working instructions, add Sigma company ROS probe, hatch 30 minutes for 37 degree, after ice PBS washed cell three times, flow cytomery.Result shows, and significantly can increase the ROS amount in neutrophil leucocyte after antibody treatment.
Above results of animal prompting, anti-human source sialic acid binding domain-immunoglobulin sample lectin-9 IgG antibody has good immunologic competence, the apoptosis of neutrophil leucocyte can be induced to increase, show that this antibody may be used for the treatment of the diseases such as severe asthma, chronic obstructive pulmonary disease, autoimmune disorder, tumour.
Sequence table
Zhu <110>, enters
The IgG antibody of <120> people source anti-sialic acid binding domain-immunoglobulin sample lectin-9 and application thereof
<130>
<160>18
<170>PatentInversion3.3
<210>1
<211>354
<212>DNA
<213> artificial sequence
<400>1
gaggtgcagctggtggagtctggggctgaggtgaagaagcctggggcctcagtgaaggtc60
tcctgcaaggcttctggatacatcttcaccggctactatatgcactgggtgcgacaggcc120
cctggacaagggcttgagtggatgggatggatcaaccctaaaagtggtggcacaaactat180
gcacagaagtttcagggcagggtcaccatgaccagggacacgtccatcagcacagcctat240
atggagctgagcaggctgagatctgacgacacggccgtgtattattgtgtgagagacttg300
ggagctacgggaggggactactggggccagggaaccctggtcaccgtctcctca354
<210>2
<211>118
<212>PRT
<213> artificial sequence
<400>2
GluValGlnLeuValGluSerGlyAlaGluValLysLysProGlyAla
151015
SerValLysValSerCysLysAlaSerGlyTyrIlePheThrGlyTyr
202530
TyrMetHisTrpValArgGlnAlaProGlyGlnGlyLeuGluTrpMet
354045
GlyTrpIleAsnProLysSerGlyGlyThrAsnTyrAlaGlnLysPhe
505560
GlnGlyArgValThrMetThrArgAspThrSerIleSerThrAlaTyr
65707580
MetGluLeuSerArgLeuArgSerAspAspThrAlaValTyrTyrCys
859095
ValArgAspLeuGlyAlaThrGlyGlyAspTyrTrpGlyGlnGlyThr
100105110
LeuValThrValSerSer
115
<210>3
<211>8
<212>PRT
<213> artificial sequence
<400>3
GlyTyrIlePheThrGlyTyrTyr
15
<210>4
<211>8
<212>PRT
<213> artificial sequence
<400>4
IleAsnProLysSerGlyGlyThr
15
<210>5
<211>11
<212>PRT
<213> artificial sequence
<400>5
ValArgAspLeuGlyAlaThrGlyGlyAspTyr
1510
<210>6
<211>990
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<213> artificial sequence
<400>6
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ggcctgtactccctgagcagcgtggtgacagtgccatccagcagcctgggcacccagacc240
tacatctgcaatgtgaaccacaagcccagcaacaccaaggtggacaagcgggtggagccc300
aagtcctgtgacaagacccacacctgccccccatgccccgcccctgagctgctgggcggc360
ccatctgtcttcctgttcccccccaagcccaaggacaccctgatgatctcccggaccccc420
gaggtgacctgtgtggtggtggatgtgagccatgaggaccccgaggtgaagttcaactgg480
tatgtggatggcgtggaggtgcacaacgccaagaccaagccccgggaggagcagtacaac540
agcacctaccgggtggtgagcgtgctgacagtgctgcatcaggactggctgaatggcaag600
gagtacaagtgcaaggtgtccaacaaggccctgcctgcccccattgagaagaccatctcc660
aaggccaagggccagccccgggagccccaggtctacaccctgcccccctcccgggaggag720
atgaccaagaaccaggtgagcctgacctgcctggtgaagggcttctaccccagcgacatt780
gctgtggagtgggagagcaacggccagcctgagaacaactacaagaccaccccccctgtg840
ctggactctgatggctccttcttcctgtacagcaagctgacagtggacaagagccggtgg900
cagcagggcaatgtcttctcctgctctgtgatgcatgaggccctgcacaaccactacacc960
cagaagagcctgtccctgtcccccggcaag990
<210>7
<211>330
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<213> artificial sequence
<400>7
AlaSerThrLysGlyProSerValPheProLeuAlaProSerSerLys
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SerThrSerGlyGlyThrAlaAlaLeuGlyCysLeuValLysAspTyr
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PheProGluProValThrValSerTrpAsnSerGlyAlaLeuThrSer
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GlyValHisThrPheProAlaValLeuGlnSerSerGlyLeuTyrSer
505560
LeuSerSerValValThrValProSerSerSerLeuGlyThrGlnThr
65707580
TyrIleCysAsnValAsnHisLysProSerAsnThrLysValAspLys
859095
ArgValGluProLysSerCysAspLysThrHisThrCysProProCys
100105110
ProAlaProGluLeuLeuGlyGlyProSerValPheLeuPheProPro
115120125
LysProLysAspThrLeuMetIleSerArgThrProGluValThrCys
130135140
ValValValAspValSerHisGluAspProGluValLysPheAsnTrp
145150155160
TyrValAspGlyValGluValHisAsnAlaLysThrLysProArgGlu
165170175
GluGlnTyrAsnSerThrTyrArgValValSerValLeuThrValLeu
180185190
HisGlnAspTrpLeuAsnGlyLysGluTyrLysCysLysValSerAsn
195200205
LysAlaLeuProAlaProIleGluLysThrIleSerLysAlaLysGly
210215220
GlnProArgGluProGlnValTyrThrLeuProProSerArgGluGlu
225230235240
MetThrLysAsnGlnValSerLeuThrCysLeuValLysGlyPheTyr
245250255
ProSerAspIleAlaValGluTrpGluSerAsnGlyGlnProGluAsn
260265270
AsnTyrLysThrThrProProValLeuAspSerAspGlySerPhePhe
275280285
LeuTyrSerLysLeuThrValAspLysSerArgTrpGlnGlnGlyAsn
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ValPheSerCysSerValMetHisGluAlaLeuHisAsnHisTyrThr
305310315320
GlnLysSerLeuSerLeuSerProGlyLys
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<211>321
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<213> artificial sequence
<400>8
gagctcgtgctgactcagccaccttcagtgtcagtgtccccaggacaaacggccagggtc60
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caggcccctatactggtcatctatggggacaacaaacgaccctccgggatccctgagaga180
ttctctggctccagctcagggacaatggccacgttgaccatcagtggggcccaggtggag240
gatgaagccgactactactgttactcaatagacaggagtggtaatcgggtattcggcgga300
gggaccaggctgaccgtccta321
<210>9
<211>107
<212>PRT
<213> artificial sequence
<400>9
GluLeuValLeuThrGlnProProSerValSerValSerProGlyGln
151015
ThrAlaArgValThrCysSerGlyAspAlaLeuProLysLysTyrAla
202530
TyrTrpTyrGlnGlnLysSerGlyGlnAlaProIleLeuValIleTyr
354045
GlyAspAsnLysArgProSerGlyIleProGluArgPheSerGlySer
505560
SerSerGlyThrMetAlaThrLeuThrIleSerGlyAlaGlnValGlu
65707580
AspGluAlaAspTyrTyrCysTyrSerIleAspArgSerGlyAsnArg
859095
ValPheGlyGlyGlyThrArgLeuThrValLeu
100105
<210>10
<211>6
<212>PRT
<213> artificial sequence
<400>10
AlaLeuProLysLysTyr
15
<210>11
<211>3
<212>PRT
<213> artificial sequence
<400>11
GlyAspAsn
1
<210>12
<211>10
<212>PRT
<213> artificial sequence
<400>12
TyrSerIleAspArgSerGlyAsnArgVal
1510
<210>13
<211>315
<212>DNA
<213> artificial sequence
<400>13
cagcccaaggccaaccccaccgtgaccctgttccccccatcttctgaggagctgcaagcc60
aacaaggccaccctggtgtgcctgatctctgacttctaccctggcgctgtgacagtggcc120
tggaaggctgatggctctcctgtgaaggctggcgtggagaccaccaagccatctaagcag180
tctaacaacaagtatgctgcctcttcttacctgtctctgacccctgagcagtggaagagc240
caccggtcttactcttgccaggtgacccatgagggctctacagtggagaagacagtggcc300
cccacagagtgctct315
<210>14
<211>105
<212>PRT
<213> artificial sequence
<400>14
GlnProLysAlaAsnProThrValThrLeuPheProProSerSerGlu
151015
GluLeuGlnAlaAsnLysAlaThrLeuValCysLeuIleSerAspPhe
202530
TyrProGlyAlaValThrValAlaTrpLysAlaAspGlySerProVal
354045
LysAlaGlyValGluThrThrLysProSerLysGlnSerAsnAsnLys
505560
TyrAlaAlaSerSerTyrLeuSerLeuThrProGluGlnTrpLysSer
65707580
HisArgSerTyrSerCysGlnValThrHisGluGlySerThrValGlu
859095
LysThrValAlaProThrGluCysSer
100105
<210>15
<211>36
<212>DNA
<213> artificial sequence
<400>15
ggtgtccactcgctacaggtgcagctggtgcagtct36
<210>16
<211>30
<212>DNA
<213> artificial sequence
<400>16
gcccttggtggatgctgaggagacggtgac30
<210>17
<211>39
<212>DNA
<213> artificial sequence
<400>17
acagacgctcgctgctcctatgagctgactcagccacct39
<210>18
<211>30
<212>DNA
<213> artificial sequence
<400>18
gttggccttgggctgtaggacggtcagcct30
Claims (10)
1. the IgG antibody of the anti-sialic acid binding domain-immunoglobulin in people source sample lectin-9, is characterized in that: the V of described antibody
lthe complementary determining region of chain has following CDRs aminoacid sequence:
V shown in SEQIDNO.10
l-CDR1;
V shown in SEQIDNO.11
l-CDR2;
V shown in SEQIDNO.12
l-CDR3;
And the V of described antibody
hthe complementary determining region of chain has following CDRs aminoacid sequence:
V shown in SEQIDNO.3
h-CDR1;
V shown in SEQIDNO.4
h-CDR2;
V shown in SEQIDNO.5
h-CDR3.
2. the IgG antibody of the anti-sialic acid binding domain-immunoglobulin in people source sample lectin-9, is characterized in that: the aminoacid sequence of variable region of heavy chain is as shown in SEQIDNO.2; The aminoacid sequence of variable region of light chain is as shown in SEQIDNO.9.
3. the IgG antibody of the anti-sialic acid binding domain-immunoglobulin in people source sample lectin-9 according to claim 1 or 2, is characterized in that: the aminoacid sequence of CH is as shown in SEQIDNO.7; The aminoacid sequence of constant region of light chain is as shown in SEQIDNO.14.
4. the nucleotide sequence of the IgG antibody of people source anti-sialic acid binding domain-immunoglobulin sample lectin-9 described in claim 2 of encoding, it is characterized in that the nucleotide sequence of encoding heavy chain variable region is for shown in SEQIDNO.1, the nucleotide sequence of encoded light chain variable region is for shown in SEQIDNO.8.
5. the nucleotide sequence of the IgG antibody of people source anti-sialic acid binding domain-immunoglobulin sample lectin-9 described in claim 3 of encoding, it is characterized in that the nucleotide sequence of encoding heavy chain constant is for shown in SEQIDNO.6, the nucleotide sequence of coding constant region of light chain is for shown in SEQIDNO.13.
6. the application of IgG antibody in preparation prevention or treatment severe asthma, chronic obstructive pulmonary disease, autoimmune disorder and/or tumour medicine of the anti-sialic acid binding domain-immunoglobulin in people source sample lectin-9 described in claim 1.
7. a medicine for prevention or treatment severe asthma, is characterized in that effective constituent is the IgG antibody of people source according to claim 1 anti-sialic acid binding domain-immunoglobulin sample lectin-9.
8. a medicine for prevention or treatment chronic obstructive pulmonary disease, is characterized in that effective constituent is the IgG antibody of people source according to claim 1 anti-sialic acid binding domain-immunoglobulin sample lectin-9.
9. a medicine for prevention or treatment autoimmune disorder, is characterized in that effective constituent is the IgG antibody of people source according to claim 1 anti-sialic acid binding domain-immunoglobulin sample lectin-9.
10. a medicine for prevention or treatment tumour, is characterized in that effective constituent is the IgG antibody of people source according to claim 1 anti-sialic acid binding domain-immunoglobulin sample lectin-9.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106831989A (en) * | 2017-01-04 | 2017-06-13 | 中国人民解放军***军事医学研究所 | The IgG antibody of the anti-sialic acid binding domain-immunoglobulin sample agglutinin 7 in people source and its application |
| CN112672757A (en) * | 2018-09-25 | 2021-04-16 | 中央研究院 | Antibodies to sialic acid binding immunoglobulin-like lectins, pharmaceutical compositions comprising the same, and uses thereof |
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| CN1371916A (en) * | 2001-02-28 | 2002-10-02 | 第二军医大学免疫学研究所 | Human siali acid conjugated immunoglobulin-like agglutinant, its coding sequence and use |
| CN101035807A (en) * | 2004-06-09 | 2007-09-12 | 泰勒公司 | Diagnosis and treatment of SIGLEC-6 associated diseases |
| CN102803293A (en) * | 2009-10-06 | 2012-11-28 | 阿莱斯亚生物疗法股份有限公司 | SIGLEC 15 antibodies in treating bone loss-related disease |
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| CN1371916A (en) * | 2001-02-28 | 2002-10-02 | 第二军医大学免疫学研究所 | Human siali acid conjugated immunoglobulin-like agglutinant, its coding sequence and use |
| CN101035807A (en) * | 2004-06-09 | 2007-09-12 | 泰勒公司 | Diagnosis and treatment of SIGLEC-6 associated diseases |
| CN102803293A (en) * | 2009-10-06 | 2012-11-28 | 阿莱斯亚生物疗法股份有限公司 | SIGLEC 15 antibodies in treating bone loss-related disease |
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| JIQUAN Q. ZHANG等: "Siglec-9, a Novel Sialic Acid Binding Member of the Immunoglobulin Superfamily Expressed Broadly on Human Blood Leukocytes", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
| TAKASHI ANGATA等: "Cloning, Characterization, and Phylogenetic Analysis of Siglec-9,a New Member of the CD33-related Group of Siglecs", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106831989A (en) * | 2017-01-04 | 2017-06-13 | 中国人民解放军***军事医学研究所 | The IgG antibody of the anti-sialic acid binding domain-immunoglobulin sample agglutinin 7 in people source and its application |
| CN112672757A (en) * | 2018-09-25 | 2021-04-16 | 中央研究院 | Antibodies to sialic acid binding immunoglobulin-like lectins, pharmaceutical compositions comprising the same, and uses thereof |
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|---|---|
| CN105367658B (en) | 2020-06-16 |
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