CN101885769A - Fully human antibody capable of specifically bonding CD4 and use thereof - Google Patents
Fully human antibody capable of specifically bonding CD4 and use thereof Download PDFInfo
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- CN101885769A CN101885769A CN2009100510465A CN200910051046A CN101885769A CN 101885769 A CN101885769 A CN 101885769A CN 2009100510465 A CN2009100510465 A CN 2009100510465A CN 200910051046 A CN200910051046 A CN 200910051046A CN 101885769 A CN101885769 A CN 101885769A
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Abstract
The invention provides a fully human anti-CD4 antibody. The heavy chain variable region of the antibody has an amino acid sequence shown by an amino acid sequence SEQ ID No.2, the light chain variable region of the antibody has an amino acid sequence shown by an amino acid sequence SEQ ID No.4, the molecule of the antibody can be bonded with a surface antigen cluster 4 (CD4 antigen) on an auxiliary T cell membrane specifically, and the affinity is more than 10<-7>M. The invention also discloses a DNA molecule for coding the antibody. The antibody has potential use in the treatment or diagnosis of diseases such as lymphocytoma, Aids, rheumatoid arthritis, allogenic organ transplant rejection and the like.
Description
Technical field
The present invention relates to the antibody technique field, relate in particular to antibody and the application of specificity in conjunction with CD4.
Background technology
The CD4 molecule mainly is present in T lymphocyte and most thymocytes surface, and mononuclear macrophage, neutrophil leucocyte, B lymphoblast all have the CD4 developed by molecule.CD4 molecule on the human t helper cell film is the strand glycoprotein of 55KD, comprises extracellular region, strides film district and cytoplasmic domain 3 parts, its cytoplasmic domain afterbody and tyrosine protein kinase coupling.Anti-CD4 monoclonal antibody combines with the CD4 molecule of T surface of cell membrane and causes enzyme activation, and calcium ion concn changes in the endochylema, and conduction negativity inhibition signal, causes stronger immunosuppressive effect.
Rheumatoid arthritis (rheumatoid arthritis RA) is a kind of to involve chronic the disabling property inflammatory autoimmunization disease of synovium of joint, thinks that RA is antigen presenting cell (APC) and the synergistic result of CD4+ cell more.So at multiple link in the immunologic process and multiple composition, intervene, regulate their immunocompetences in RA, and reach therapeutic purpose as T cell, cytokine, adhesion molecule and MHC molecule etc.
The anti-CD4 monoclonal antibody of full humanization (zanolimumab, Hum Max-CD4) by the exploitation of Medarex/Genmab/Seono company enters III clinical trial (www.clinicaltrial.gov) in the U.S. as the T-cell lymphoma,cutaneous medicine.Americanism diseases caused by dampness academic year in 1991 can on reported with anti-CD4McAb treatment rheumatoid arthritis (RA).For example the Clenoliximab and the Keliximab of the development of IDEC Pharma Inc. are exactly the chimeric CD4 monoclonal antibody of people-monkey (IgG
4-IgG
1), be used for rheumatoid arthritis treatment, entered the clinical phase (Sharma A, Davis CB, Tobia LA, et al.J Pharmacol Ex p Ther, 2000,293 (1): 33).The anti-CD4 chimeric antibody of Centocor company research has also entered II phase clinical verification at treatment rheumatoid arthritis, multiple sclerosis.Other has the anti-CD4 McAb of report also to be used for the treatment of HIV infection (Antimicrob Agents Chemother 2006; 50:2231-2233).The purposes that other has anti-CD4McA to can be used as organ transplant rejection also is confirmed (Chinese microbiology and Journal of Immunology, Zhu Zhigang etc., 2002 the 22nd the 2nd phases of volume).
This area presses for the anti-CD 4 antibodies in the total man source with high-affinity, is used for diagnosis and treatment CD4 relative disease such as lymphocytoma, acquired immune deficiency syndrome (AIDS), rheumatoid arthritis, allogeneic organ transplant rejection etc.
Summary of the invention
The purpose of this invention is to provide the antibody of a specific specificity in conjunction with T4 antigen, total man's resource monoclonal antibody especially is to satisfy the needs of diagnosing and/or treating the CD4 relative disease clinically.
A first aspect of the present invention provides the total man source antibody of a specific specificity in conjunction with T4 antigen, and the variable region of heavy chain of this total man source antibody comprises following complementary determining region:
CDR1 shown in the SEQ ID NO:5,
CDR2 shown in the SEQ ID NO:6,
CDR3 shown in the SEQ ID NO:7.
In preferred scheme, the variable region of heavy chain of this total man source antibody has the aminoacid sequence shown in the SEQ ID NO:2.
A second aspect of the present invention provides the total man source antibody of a specific specificity in conjunction with T4 antigen, and the variable region of light chain of this total man source antibody comprises following complementary determining region:
CDR1 shown in the SEQ ID NO:8,
CDR2 shown in the SEQ ID NO:9,
CDR3 shown in the SEQ ID NO:10.
In preferred scheme, the variable region of light chain of this total man source antibody has the aminoacid sequence shown in the SEQ ID NO:4.
A third aspect of the present invention provides the total man source antibody of a specific specificity in conjunction with T4 antigen, and the variable region of heavy chain of this total man source antibody and variable region of light chain have the aminoacid sequence shown in SEQ ID NO:2 and the SEQ ID NO:4 respectively.
A fourth aspect of the present invention provides the total man source antibody fragment of a specific specificity in conjunction with T4 antigen, and this total man source antibody fragment comprises the aminoacid sequence shown in aminoacid sequence shown in the SEQ ID NO:2 and/or the SEQ ID NO:4.This total man source antibody fragment comprises single-chain antibody (scFv), Fab, Fab ', F (ab ')
2Etc. form.
A fifth aspect of the present invention provides fusion rotein or the polypeptide of a specific specificity in conjunction with T4 antigen, and this fusion rotein or polypeptide comprise the aminoacid sequence shown in aminoacid sequence shown in the SEQ ID NO:2 and/or the SEQ ID NO:4.
A sixth aspect of the present invention provides a kind of activated protein of small molecules mark, and this activated protein comprises the aminoacid sequence shown in aminoacid sequence shown in the SEQ ID NO:2 and/or the SEQ ID NO:4.
A seventh aspect of the present invention provides a kind of specificity of encoding in conjunction with the dna sequence dna of the variable region of heavy chain of the total man source antibody of T4 antigen, the polypeptide shown in this dna sequence encoding SEQ ID NO:2.
In a preferred scheme, this dna sequence dna is shown in SEQ ID NO:1.
A eighth aspect of the present invention provides a kind of specificity of encoding in conjunction with the dna sequence dna of the variable region of light chain of the total man source antibody of T4 antigen, the polypeptide shown in this dna sequence encoding SEQ ID NO:4.
In a preferred scheme, this dna sequence dna is shown in SEQ ID NO:3.
A ninth aspect of the present invention provides the application in diseases such as treatment or diagnosis lymphocytoma, acquired immune deficiency syndrome (AIDS), rheumatoid arthritis, allogeneic organ transplant rejection of the total man source antibody of specificity of the present invention in conjunction with T4 antigen, antibody fragment, fusion rotein, small molecules mark activated protein.
A tenth aspect of the present invention provides a kind of pharmaceutical composition, comprises the specificity of the present invention of safe and effective amount in conjunction with the total man source antibody of T4 antigen, antibody fragment, fusion rotein, small molecules mark activated protein and acceptable carrier pharmaceutically.
Description of drawings
The active detected result of the ELISA of 11 strain anti-CD 4 antibodies positive colony bacterium of Fig. 1: embodiment 4 preparations.Envelope antigen is the solubility T4 antigen (1ug/ml) of 100ul, and the open column shape body is for adding the test group of 100ul positive colony bacterium (anti-CD 4 antibodies), and the column of band oblique line is the control group of the BSA (100ug/ml) of adding 100ul.The highest wherein active positive colony bacterium is the 2nd strain, is numbered 4-91
#
Fig. 2: 4-91
#The doubling dilution detected result of the anti-CD 4 antibodies that bacterium produces.
Embodiment
Term used herein " antibody " is to pass through at least one antigen recognition site, and the immunoglobulin (Ig) of target molecule (comprising sugar, Polynucleotide, lipid, polypeptide etc.) specific combination.Complete antibody is the about 150000 daltonian different four glycan albumen that the same structure feature is arranged, and it is made up of with two identical heavy chains (H) two identical light chains (L).Every light chain links to each other with heavy chain by a covalent disulfide bonds, and the heavy interchain disulfide linkage number difference of different immunoglobulin (Ig) isotypes.Every heavy chain and light chain be the intrachain disulfide bond at regular interval also.One end of every heavy chain has variable region (VH), is thereafter a plurality of constant regions.One end of every light chain has variable region (VL), and the other end has constant region; First constant region of the constant region of light chain and heavy chain is relative, and the variable region of light chain is relative with the variable region of heavy chain.Special amino-acid residue forms the interface between the variable region of the heavy chain of light chain.
Term used herein " monoclonal antibody " is meant and comprises the same antibody population that participates in a certain antigen amino acid structure of selective binding (nature or through reconstruction).Monoclonal antibody has high degree of specificity, at a certain single antigen site.
Some part of variable region is different on sequence in term used herein " variable region " the expression antibody, and it has formed combination and the specificity of various specific antibodies to its specific antigen.Yet mutability is not evenly distributed in the whole variable region.It concentrates in three fragments that are called in light chain and the variable region of heavy chain in complementary determining region (CDR) or the hypervariable region.The conservative part in variable region is called framework region (FR).Comprise four FR districts in the variable region of natural heavy chain and light chain separately, they are the beta sheet configuration haply, are linked to each other by three CDR districts that form shack, can form part βZhe Die structure in some cases.CDR in every chain closely be close together by the FR district and with the CDR of another chain form antibody antigen-binding site (referring to Kabat etc., NIH Publ.No.91-3242, volume I, 647-669 page or leaf (1991).Constant region is not participated in antibody directly and is combined with antigenic, but they show different effector functions, for example participates in the cytotoxicity that depends on antibody of antibody.
" light chain " of vertebrates antibody (immunoglobulin (Ig)) can be classified as the class in visibly different two classes (being called κ and λ) according to the aminoacid sequence of its constant region.According to the aminoacid sequence of its CH, immunoglobulin (Ig) can be divided into different kinds, mainly contains 5 immunoglobulin like protein: IgA, IgD, IgE, IgG and IgM, some of them also can be further divided into subclass (of the same type), as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.Be called α, β, ε, μ, γ corresponding to different immunoglobulin heavy chain constant regions.
The subunit structure and the 3-d modelling of different immunoglobulin (Ig)s are well-known
" total man source antibody " used herein is meant the human antibody in antibody gene source.
Herein, so-called " antibody " not only comprises complete polyclone or monoclonal antibody, also comprise various antibody fragments (as Fab, Fab ', F (ab ') 2, Fv), single-chain antibody (ScFv), disome, by the multi-specificity antibody that antibody fragment forms, contain the fusion rotein of antibody fragment, and any through transforming but comprise the immunoglobulin molecules in required specific recognition site.The source of antibody or preparation method and unrestricted, for example by hybridoma, phage select, recombinant expressed, transgenic animal etc.
" mark " that suits comprises radionuclide, enzyme, substrate, cofactor, inhibitor, fluorescence part, chemiluminescent moiety, magnetic-particle etc.
Describe the present invention in further detail below in conjunction with embodiment, should be appreciated that, enumerate these embodiment just for the present invention is described, rather than be used for limiting the present invention.The concentration that does not specialize in the following example is mass percent concentration; The experimental technique of unreceipted actual conditions, usually according to normal condition, people such as Sambrook for example, " molecular cloning experiment guide " (New York:Cold Spring Harbor LaboratoryPress, 1989) condition described in, or the condition of advising according to manufacturer.
The structure of embodiment 1. total man source antibody libraries
The structure in storehouse, total man source single-chain antibody (scFv) is people [He et al.Methods Mol Biol.2004 such as employing He; 248:177-189] the reported method structure, the mRNA that extracts from the mixing peripheral blood of 83 routine tumour patients, adopt following primer, with the method amplification in vitro heavy chain of RT-PCR and the variable region gene of light chain, two fragment genes are dressed up complete antibody library sequence by one section Linker sequence set with the method for overlapping extension PCR (Overlap-extension PCR).
The heavy chain primer:
(1)HuVH/B：5′-CAGGT(c/g)CAGCTGG(t/a)G(c/g)AGTC(t/c)GG-3′
(2)HuJH/F：5′-TGAGGAGACGGTGACCA(t/g)GGTCCC-3′
The catenation sequence primer
(3)Linker/B：5′-GGGACC(a/c)TGGTCACCGTCTCCTCAGGCGGTGGCTC
TGG-3′
(4)Linker/F：5′-TTGCCACCGCCAGAACC-3′
κ light chain primer
(5)HuVκ/B：5′-GGTTCTGGCGGTGGCAAAGA(a/c)AT(c/t)(g/a)TG(a/c)TGA
C(c/g/t)CAGTCTCC-3′
(6)HuCκ/F：5′-TGAGGAGACGGTGACCA(t/g)GGTCCC-3′
The lambda light chain primer
(7)HuVλ/B：5′-GGTTCTGGCGGTGGCAAACAG(t/g)CTGTGCTGACTCAG
CC(g/c)CC-3′
(8)HuCλ/F：5′-AGATTCTGTAGGGGCCACTGTC-3′
Utilize a pair of primer 5 '-ACCACCATGGAGGTSCASCTCGAGSAGTCTGG-3 ' and 5 '-GCGAATTCTTAGCAGCCACCCAGGTGATGGTGATGGTGATGAGATGGTGCAGCCAC AG-3 ' introduces Nco I and two restriction enzyme sites of EcoR I respectively in the both sides of antibody library.The PCR library of scFv is cut with Nco I and EcoR I enzyme, pET22b cuts with Nco I and EcoR I enzyme, reclaim the fragment after enzyme is cut respectively, scFv library after enzyme is cut is connected the back and transforms the Rosetta competence with carrier, coating LB solid plate, picking the single bacterium colonies of 1500 strains, adhere to the clone with filter paper and show blue reaction method (Colony filter method) and carry out functional screening cloning son.
Xerox the bacterium colony that grows on the flat board with the pvdf membrane that a methyl alcohol was handled, bacterium colony one faces up and is tiled on the agar plate that contains 1mM IPTG and 50ug/ml of ampicillin afterwards, and 25-30 ℃ leaves standstill cultivation 4 hours.Wash plate hole 3 times with the PBS lavation buffer solution that contains 0.05%Tween 20, each 5 minutes, then with the PBS room temperature sealing that contains 0.05%Tween 20 and 1%BSA 1 hour.Wash plate hole 3 times with the PBS lavation buffer solution that contains 0.05%Tween20, each 5 minutes, hatched two hours with pvdf membrane after Anti-His HRP (Sigma product) was diluted to working fluid with the PBS that contains 0.05%Tween 20 and 1%BSA by 1: 5000, with the PBS damping fluid washing that contains 0.05%Tween 20 3 times, use chemical illuminating reagent (Pierce company product) that film is exposed again, obtained the positive bacterium colony of 420 strains.
The detection (chemoluminescence method) of embodiment 3. antigen-antibody effects
With 80ul PBS with solubility T4 antigen (name of product: sCD4 antigen; R﹠amp; D, product article No.: 514-CD-050) be diluted to the concentration of 10ug/ml, be coated on the pvdf membrane that methyl alcohol handled 4 ℃ of overnight incubation.With the PBS washing that contains 0.05%Tween three times, each 5 minutes.Carry out room temperature sealing 1 hour with Superblock buffer in PBS (Thermo company).With the PBS washing that contains 0.05%Tween three times, each 5 minutes.The periplasm protein of the positive bacterium colony of 420 strains that extracting is good point sample respectively carries out specific action with antigen, incubated at room 1 hour on pvdf membrane.The PBS of 0.05%Tween washing is three times again, each 5 minutes.Anti-HisHRP (Sigma aldrich) with 8000 times of the PBS dilutions that contains 0.05%Tween and 1%BSA, is layered on the film room temperature light shaking 2 hours equably.The PBS of 0.05%Tween washing is three times again, each 5 minutes.With chemical illuminating reagent Supersignal west Pico (Pierce company) incubated at room 5 minutes, in the darkroom of chemoluminescence imaging system Chemidoc (Bio-rad company), expose, filter out the darker positive colony bacterium of about 11 strains and CD4 specific combination and spot.
The 11 strain positive colony bacterium that the last step was screened are with the LB substratum that contains 0.5% glucose, and wherein adding final concentration is the penbritin of 100ug/ml and the paraxin of 64ug/ml, in 37 ℃ of overnight incubation.Add the IPTG that final concentration is 1mM morning next day, continue 25-30 ℃ and cultivated after 4 hours 5000g centrifugal 5 minutes.Ratio according to the heavy corresponding 5ml of every gram bacterium adds lavation buffer solution (300mM NaCl, 50mMNaH
2PO
4With the 10mM imidazoles, PH 8.0), ultrasonic disruption cell on the ice bath (ultrasonic 4s is 5s at interval, 99 circulations), 12000g is centrifugal 40 minutes afterwards.Get supernatant and mix with the affine filler of lavation buffer solution equilibrated 1mlNi-NTA before, 4 ℃ of concussions were carried on the purification column in 1 hour, the lavation buffer solution washing of 30 column volumes.Elution buffer (300mM NaCl, 50mM NaH with 1ml
2PO
4With the 250mM imidazoles) wash-out, collect the target elutriant.The sample of collecting is carried out conventional ELISA experiment, the results are shown in Figure 1, select the bacterial strain of OD450 absorption value maximum: 4-91
#Bacterium.
4-91
#The employing SDS-PAGE electrophoresis of antibody behind the Ni-affinitive layer purification that bacterium produces, with QualityOne analysis software purity greater than 80%, reach the basic purity of ELISA avidity detection method, the concentration of sample adopts the Bradford method to measure, and is about 160ug/ml.
Embodiment 5.ELISA detects the avidity of antigen-antibody
Solubility T4 antigen coated elisa plate with the 1ug/ml of 100ul detects hole, 4 ℃ of overnight incubation.Wash plate hole 3 times with the PBS lavation buffer solution that contains 0.05%Tween 20, each 5 minutes, afterwards with the PBS room temperature sealing that contains 0.05%Tween 20 and 1%BSA 1 hour.Wash plate hole 3 times with the PBS lavation buffer solution that contains 0.05%Tween 20, each 5 minutes.The antibody dilution that obtains the generation of 4-91# bacterium after adopting doubling dilution with affinitive layer purification is a gradient, and get 100ul successively and join above-mentioned being coated with in the antigenic hole, negative control adopts the albumen of the Rosetta bacterium extraction that does not transform plasmid, hatches 1 hour for 37 ℃.Wash plate hole 3 times with the PBS lavation buffer solution that contains 0.05%Tween 20, each 5 minutes.Anti-HisHRP (Sigma product) was hatched two hours by being diluted at 1: 5000 in the hole of hatching sample before being added in behind the working fluid with the PBS that contains 0.05%Tween 20 and 1%BSA, with the PBS damping fluid washing that contains 0.05%Tween 20 3 times.In the hole, add TMB solution, hatched 10 minutes for 37 ℃, add the reaction of 50ul 1N HCl color development stopping, use the ELISA microplate reader in the 450nm wavelength readings.By dilution method detect its to CD4 in conjunction with dependency, the results are shown in Figure 2.
According to formula, calculating antibody and antigenic avidity are 3.5 * 10
-7M.
The order-checking of embodiment 6.CD4 monoclonal antibody gene
To the 4-91 that screens
#The antibody gene that bacterium produces checks order, and the result is in sequence table.Wherein SEQID NO:1 and SEQ ID NO:3 are the variable region of heavy chain of the highly active monoclonal antibody that obtains of the present invention and the dna sequence dna of variable region of light chain, and SEQ ID NO:2 and SEQ ID NO:4 infer the aminoacid sequence that according to above-mentioned dna sequence dna.Complementary determining region CDR1～the CDR3 of the variable region of heavy chain that is inferred in SEQ ID NO:5～7th; Complementary determining region CDR1～the CDR3 of the variable region of light chain that is inferred in SEQ ID NO:8～10th.
The foregoing description is not that the present invention is defined in above-mentioned scope just for the present invention is described.The foregoing description may be done a lot of changes, and after having read content disclosed by the invention, those skilled in the art can make various changes and modification to the present invention, and these equivalent form of values fall within the protection domain of claims of the present invention equally.
Sequence table
＜110〉Sichuan Inst. of Antibiotic Industry, China Medicine Group Corp.
＜120〉specific specificity is in conjunction with total man source antibody and the application of CD4
<160>20
<170>PatentIn?version?3.1
<210>1
<211>351
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
<221>misc_feature
<222>(1)..(351)
＜223〉variable region of heavy chain
<400>1
gaggtgcagc?tcgaggagtc?tgggggagac?ttagttcgac?ctgggggatc?cctgagactc 60
tcctgtacag?cctctggatc?cgccttcaac?aattatgtct?tcaactgggt?ccgccaggct 120
ccagggaagg?ggctggagtg?gattggccgt?actaaaagta?aagatgatgg?tgagacaata 180
cagtacgctg?cgcccgtgaa?aggcagattc?gtcatctcaa?gagatgattc?aaaaaagaca 240
gtgtatctgg?aaatgaacac?cctgaaaacc?gaggacacag?ccgtatacta?ttgtaccaca 300
ggaagctaca?attaccactg?gggccacggg?accctggtca?ccgtctcctc?a 351
<210>2
<211>117
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
<221>misc_feature
<222>(1)..(117)
＜223〉variable region of heavy chain
<400>2
Glu?Val?Gln?Leu?Glu?Glu?Ser?Gly?Gly?Asp?Leu?Val?Arg?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Thr?Ala?Ser?Gly?Ser?Ala?Phe?Asn?Asn?Tyr
20 25 30
Val?Phe?Asn?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Ile
35 40 45
Gly?Arg?Thr?Lys?Ser?Lys?Asp?Asp?Gly?Glu?Thr?Ile?Gln?Tyr?Ala?Ala
50 55 60
Pro?Val?Lys?Gly?Arg?Phe?Val?Ile?Ser?Arg?Asp?Asp?Ser?Lys?Lys?Thr
65 70 75 80
Val?Tyr?Leu?Glu?Met?Asn?Thr?Leu?Lys?Thr?Glu?Asp?Thr?Ala?Val?Tyr
85 90 95
Tyr?Cys?Thr?Thr?Gly?Ser?Tyr?Asn?Tyr?His?Trp?Gly?His?Gly?Thr?Leu
100 105 110
Val?Thr?Val?Ser?Ser
115
<210>3
<211>327
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
<221>misc_feature
<222>(1)..(327)
＜223〉variable region of light chain
<400>3
gacattgtaa?tgactcagtc?tccaggcacc?ctgtctctgt?ctccagggga?aagagccacc 60
ctctcctgca?gggccagtca?gagtgttagc?agcagctact?tagcctggta?ccagcagaaa 120
cctggccagg?ctcccaggct?cctcatctat?ggtgcatcca?ccagggccac?tggtacccca 180
gcccggttca?gtggcagtgg?gtctgggaca?gacttcactc?tcaccatcag?cagcctgcag 240
tctgaagatt?ttgcagttta?ttactgtcag?caatataata?actggcctct?cacttttggc 300
caggggacca?agctggagat?caaacga 327
<210>4
<211>109
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
<221>misc_feature
<222>(1)..(109)
＜223〉variable region of light chain
<400>4
Asp?Ile?Val?Met?Thr?Gln?Ser?Pro?Gly?Thr?Leu?Ser?Leu?Ser?Pro?Gly
1 5 10 15
Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg?Ala?Ser?Gln?Ser?Val?Ser?Ser?Ser
20 25 30
Tyr?Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Ala?Pro?Arg?Leu?Leu
35 40 45
Ile?Tyr?Gly?Ala?Ser?Thr?Arg?Ala?Thr?Gly?Thr?Pro?Ala?Arg?Phe?Ser
50 55 60
Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln
65 70 75 80
Ser?Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys?Gln?Gln?Tyr?Asn?Asn?Trp?Pro
85 90 95
Leu?Thr?Phe?Gly?Gln?Gly?Thr?Lys?Leu?Glu?Ile?Lys?Arg
100 105
<210>5
<211>5
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(5)
＜223〉variable region of heavy chain CDR1
<400>5
Asn?Tyr?Val?Phe?Asn
1 5
<210>6
<211>19
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(19)
＜223〉variable region of heavy chain CDR2
<400>6
Arg?Thr?Lys?Ser?Lys?Asp?Asp?Gly?Glu?Thr?Ile?Gln?Tyr?Ala?Ala?Pro
1 5 10 15
Val?Lys?Gly
<210>7
<211>6
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(6)
＜223〉variable region of heavy chain CDR3
<400>7
Gly?Ser?Tyr?Asn?Tyr?His
1 5
<210>8
<211>12
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(12)
＜223〉variable region of light chain CDR1
<400>8
Arg?Ala?Ser?Gln?Ser?Val?Ser?Ser?Ser?Tyr?Leu?Ala
1 5 10
<210>9
<211>7
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(7)
＜223〉variable region of light chain CDR2
<400>9
Gly?Ala?Ser?Thr?Arg?Ala?Thr
1 5
<210>10
<211>9
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(9)
＜223〉variable region of light chain CDR3
<400>10
Gln?Gln?Tyr?Asn?Asn?Trp?Pro?Leu?Thr
1 5
<210>11
<211>23
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(23)
＜223〉primer
<400>11
caggtscagc?tggwgsagtc?ygg 23
<210>12
<211>24
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(24)
＜223〉primer
<400>12
tgaggagacg?gtgaccakgg?tccc 24
<210>13
<211>38
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(38)
＜223〉primer
<400>13
gggaccmtgg?tcaccgtctc?ctcaggcggt?ggctctgg 38
<210>14
<211>17
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(17)
＜223〉primer
<400>14
ttgccaccgc?cagaacc 17
<210>15
<211>41
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(41)
＜223〉primer
<400>15
ggttctggcg?gtggcaaaga?matyrtgmtg?acbcagtctc?c 41
<210>16
<211>24
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(24)
＜223〉primer
<400>16
tgaggagacg?gtgaccakgg?tccc 24
<210>17
<211>41
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(41)
＜223〉primer
<400>17
ggttctggcg?gtggcaaaca?gkctgtgctg?actcagccsc?c 41
<210>18
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(22)
＜223〉primer
<400>18
agattctgta?ggggccactg?tc 22
<210>19
<211>32
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(32)
＜223〉primer
<400>19
accaccatgg?aggtscasct?cgagsagtct?gg 32
<210>20
<211>48
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(48)
＜223〉primer
<400>20
gcgaattctt?agcagccacc?caggtgatgg?tgatggtgat?gagatggtgc?agccacag 48
Claims (15)
1. a specific specificity is characterized in that in conjunction with the total man source antibody of T4 antigen, and the variable region of heavy chain of described total man source antibody comprises following complementary determining region:
CDR1 shown in the SEQ ID NO:5,
CDR2 shown in the SEQ ID NO:6,
CDR3 shown in the SEQ ID NO:7.
2. according to the total man source antibody of claim 1, it is characterized in that the variable region of heavy chain of described total man source antibody has the aminoacid sequence shown in the SEQ ID NO:2.
3. a specific specificity is characterized in that in conjunction with the total man source antibody of T4 antigen, and the variable region of light chain of described total man source antibody comprises following complementary determining region:
CDR1 shown in the SEQ ID NO:8,
CDR2 shown in the SEQ ID NO:9,
CDR3 shown in the SEQ ID NO:10.
4. according to the human antibody of claim 3, it is characterized in that the variable region of light chain of described total man source antibody has the aminoacid sequence shown in the SEQ ID NO:4.
5. a specific specificity is characterized in that in conjunction with the total man source antibody of T4 antigen, and the variable region of heavy chain of described human antibody and variable region of light chain have the aminoacid sequence shown in SEQ ID NO:2 and the SEQ ID NO:4 respectively.
6. a specific specificity is characterized in that in conjunction with the total man source antibody fragment of T4 antigen, and described total man source antibody fragment comprises the variable region of heavy chain of the described antibody of claim 1 and/or the variable region of light chain of the described antibody of claim 3.
7. according to the total man source antibody fragment of claim 6, it is characterized in that described antibody fragment comprises single-chain antibody (scFv), Fab, Fab ', F (ab ')
2Form.
8. a specific specificity is characterized in that in conjunction with the fusion rotein or the polypeptide of T4 antigen, and described fusion rotein or polypeptide comprise the variable region of heavy chain of the described antibody of claim 1 and/or the variable region of light chain of the described antibody of claim 3.
9. a specific specificity is characterized in that in conjunction with the activated protein of T4 antigen, and described activated protein comprises the variable region of heavy chain of the described antibody of claim 1 and/or the variable region of light chain of the described antibody of claim 3, and this activated protein has the small molecules mark.
10. a dna molecular is characterized in that, the variable region of heavy chain shown in the described dna molecule encode SEQ ID NO:2.
11. the dna molecular according to claim 10 is characterized in that, described dna molecular has the nucleotide sequence shown in the SEQID NO:1.
12. a dna molecular is characterized in that, the variable region of light chain shown in the described dna molecule encode SEQ ID NO:4.
13. the dna molecular according to claim 12 is characterized in that, described dna molecular has the nucleotide sequence shown in the SEQ ID NO:3.
14. according to each described antibody, antibody fragment, fusion rotein among the claim 1-9 or the application of activated protein in the medicine of preparation treatment or diagnosis lymphocytoma, acquired immune deficiency syndrome (AIDS), rheumatoid arthritis or allogeneic organ transplant rejection disease that have a small molecules mark.
15. a pharmaceutical composition is characterized in that, comprises each described antibody, antibody fragment, the fusion rotein among the claim 1-9 of safe and effective amount or has the activated protein of small molecules mark and acceptable carrier pharmaceutically.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102854306A (en) * | 2011-12-23 | 2013-01-02 | 中国人民解放军第三〇九医院 | Kit for early warning rejection reaction after kidney transplant and use method of kit |
| CN102854305A (en) * | 2011-12-23 | 2013-01-02 | 中国人民解放军第三〇九医院 | Prewarning kit for transplant kidney rejection and use method thereof |
| CN102854323A (en) * | 2011-12-23 | 2013-01-02 | 中国人民解放军第三〇九医院 | Transplant kidney rejection reaction early diagnosis kit and use method of kit |
| CN110702916A (en) * | 2019-04-15 | 2020-01-17 | 四川大学华西医院 | Quantitative analysis method for integrating proteome and glycoprotein |
| CN116948032A (en) * | 2023-09-19 | 2023-10-27 | 汇智生物技术(苏州)有限公司 | Humanized CD4 monoclonal antibody and preparation method and application thereof |
| CN116990504A (en) * | 2023-09-27 | 2023-11-03 | 苏州旭光科星抗体生物科技有限公司 | Double antibody sandwich ELISA kit for detecting human soluble CD4 |
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102854306A (en) * | 2011-12-23 | 2013-01-02 | 中国人民解放军第三〇九医院 | Kit for early warning rejection reaction after kidney transplant and use method of kit |
| CN102854305A (en) * | 2011-12-23 | 2013-01-02 | 中国人民解放军第三〇九医院 | Prewarning kit for transplant kidney rejection and use method thereof |
| CN102854323A (en) * | 2011-12-23 | 2013-01-02 | 中国人民解放军第三〇九医院 | Transplant kidney rejection reaction early diagnosis kit and use method of kit |
| CN102854306B (en) * | 2011-12-23 | 2013-08-07 | 中国人民解放军第三〇九医院 | Kit for early warning rejection reaction after kidney transplant and use method of kit |
| CN102854323B (en) * | 2011-12-23 | 2013-08-07 | 中国人民解放军第三〇九医院 | Transplant kidney rejection reaction early diagnosis kit and use method of kit |
| CN102854305B (en) * | 2011-12-23 | 2013-08-07 | 中国人民解放军第三〇九医院 | Prewarning kit for transplant kidney rejection and use method thereof |
| CN110702916A (en) * | 2019-04-15 | 2020-01-17 | 四川大学华西医院 | Quantitative analysis method for integrating proteome and glycoprotein |
| CN110702916B (en) * | 2019-04-15 | 2022-06-21 | 四川大学华西医院 | Quantitative analysis method for integrating proteome and glycoprotein |
| CN116948032A (en) * | 2023-09-19 | 2023-10-27 | 汇智生物技术(苏州)有限公司 | Humanized CD4 monoclonal antibody and preparation method and application thereof |
| CN116948032B (en) * | 2023-09-19 | 2023-12-12 | 汇智生物技术(苏州)有限公司 | Humanized CD4 monoclonal antibody and preparation method and application thereof |
| CN116990504A (en) * | 2023-09-27 | 2023-11-03 | 苏州旭光科星抗体生物科技有限公司 | Double antibody sandwich ELISA kit for detecting human soluble CD4 |
| CN116990504B (en) * | 2023-09-27 | 2023-12-01 | 苏州旭光科星抗体生物科技有限公司 | Double antibody sandwich ELISA kit for detecting human soluble CD4 |
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