CN108593912A - A kind of detection method of solubility CD38 concentration - Google Patents
A kind of detection method of solubility CD38 concentration Download PDFInfo
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- CN108593912A CN108593912A CN201810312462.5A CN201810312462A CN108593912A CN 108593912 A CN108593912 A CN 108593912A CN 201810312462 A CN201810312462 A CN 201810312462A CN 108593912 A CN108593912 A CN 108593912A
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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Abstract
The present invention relates to a kind of detection methods of solubility CD38 concentration.Filtering out can be in combination with the single domain antibody pair of CD38, i.e. single domain antibody 1053 and 551, and wherein single domain antibody 551, which is used as, captures antibody, and the fusion protein of single domain antibody 1053 and Fluc mutant Fluc2 are as detection antibody.Single domain antibody 551 is coated on elisa plate bottom to capture the CD38 in solution, the luminous signal of the catalysis luciferins of the Fluc2 in antibody is detected to assess the concentration of CD38, develops the soluble CD38 detection methods of a species specificity hypersensitivity.
Description
Technical field
The invention belongs to biomedical or biopharmaceutical technologies, are related to a kind of detection side of solubility CD38 concentration
Method.
Background technology
CD38 is the glycoprotein of a single pass transmembrane, is often used as the mark molecule of cell differentiation.It 1996, finds for the first time
Soluble CD38 (soluble CD38, sCD38), it is considered as the film extracellular portion of overall length CD38 molecules by cutting release
Product.They are present in a variety of physiology and pathological body liquids and tumor cell culture supernatant, remain CD38 antigenic characteristic and
Catalytic activity1.It is subsequent the study found that solubility CD38 can be used as a kind of signaling molecule, there is induced cell proliferation and move
It moves2, extend the memory antibody service life3With adjust maternal-fetal tolerance etc.4Etc. functions.
Overexpression is presented in a variety of blood cancer cells, such as multiple myeloma cells surface in CD385, and normal
Low expression state is on lymphocyte, bone marrow cell and some non-hematopoietic cells.Therefore, CD38 is considered as a treatment
The target of Huppert's disease, multiple therapeutic monoclonal antibodies for it are just in R＆D process6.Cell surface CD38's
Expression quantity is a diagnosis index of Huppert's disease, but the acquisition of patient myeloma cell needs to do bone marrow aspiration, tool
There is invasive, and the load that can not reflect patient's entirety myeloma is extracted in the part of marrow, so Blood diagnosis is with excellent
More property.
Since patient's blood plasma is a complex system, soluble CD38 contents are very low, need highly sensitive method that could carry out
Accurate detection.The highest a CD38 of detection sensitivity detects ELISA kit currently on the market, is consolidated using conventional antibodies
Phase sandwich ELISA, the nominal 93.75pg/mL of detection sensitivity.Disadvantage：1) detection sensitivity is still limited；2) it examines
It is not strong to survey specificity, is not suitable for detection of complex sample；3) of high cost, 5000 yuan of a kit is only capable of doing 96 sample/tests.
Bibliography
1 Funaro,A.et al.Identification and characterization of an active
soluble form of human CD38in normal and pathological fluids.International
immunology 8,1643-1650(1996).
2 Deaglio,S.et al.CD38/CD31interactions activate genetic pathways
leading to proliferation and migration in chronic lymphocytic leukemia
cells.Molecular medicine 16,87-91,doi:10.2119/molmed.2009.00146(2010).
3 Liu,X.Q.,Hart,D.N.,MacPherson,G.G.,Good,M.F.&Wykes,M.N.Soluble
CD38significantly prolongs the lifespan of memory B-cell responses.Immunology
125,14-20,doi:10.1111/j.1365-2567.2008.02914.x(2008).
4 Kim,B.J.et al.Seminal CD38is a pivotal regulator for fetomaternal
tolerance.Proceedings of the National Academy of Sciences of the United
States of America 112,1559-1564,doi:10.1073/pnas.1413493112(2015).
5 Lin,P.,Owens,R.,Tricot,G.&Wilson,C.S.Flow cytometric
immunophenotypic analysis of 306cases of multiple myeloma.American journal of
clinical pathology 121,482-488,doi:10.1309/74R4-TB90-BUWH-27JX(2004).
6 van de Donk,N.,Richardson,P.G.&Malavasi,F.CD38antibodies in
multiple myeloma:back to the future.Blood 131,13-29,doi:10.1182/blood-2017-
06-740944(2018).
Invention content
Technical problem to be solved by the invention is to provide the detection solubility CD38's of a kind of hypersensitivity, specificity
Method, and high-throughput can detect.
To achieve the above object, the first aspect of the present invention provides the detection method of soluble CD38, i.e. sandwich presss from both sides
Heart method, including capture antibody, detection antibody.
Second aspect of the present invention, provides a kind of capture antibody, a kind of detection antibody, i.e. CD38 single domain antibodies and CD38 is mono-
Domain antibodies Luciferase fusion albumen, including CD38 single domain antibodies part and Fluc part.
Third aspect present invention provides two kinds of DNA moleculars, it encodes capture antibody of the present invention and detection is anti-
Body or CD38 single domain antibodies Luciferase fusion albumen of the present invention.
Fourth aspect present invention provides two kinds of expression vectors, it includes CD38 single domain antibodies gene order and firefly
The gene order of luciferase mutant.
Fifth aspect present invention, provides two kinds of host cells, it contains previously described expression vector.
The CDS sequences of the encoding gene of CD38 single domain antibodies 1053 of the present invention, cDNA sequence overall length are 546bp
(sequence 1), particular sequence is as follows：
ATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCGGCCCAGCCGGCCATGGCCGAT
GTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTACAGGCTCAGGACG
CACCTTCAGGAACTATCCCATGGCCTGGTTCCGCCAGGCTCCAGGAAAGGAGCGTGAGTTTGTAGCAGGTATTACCT
GGGTCGGTGCTAGCACACTCTATGCAGACTTCGCGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACG
GTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATAGTTGTGCAGCAGGTCGCGGTATAGTGGC
TGGTAGGATCCCAGCTGAGTATGCCGACTGGGGCCAGGGCACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAA
AACCACAACCAGCGGCCGCACATCATCATCACCATCACGGGGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCTG
AATGGGGCCGCATAG
Coding generates the albumen that length is 181 amino acid (end terminator is not included in, i.e., * * * are not included in sequence)
Matter sequence (sequence 2), particular sequence is as follows：
The CDS sequences of the encoding gene of CD38 single domain antibodies 551 of the present invention, cDNA sequence overall length are 558bp (sequences
Row 3), particular sequence is as follows：
ATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCGGCCCAGCCGGCCATGGCCGAT
GTGCAGCTGCAGGAGTCAGGAGGAGGATTGGTGCAGGCTGGACACTCTCTGAGACTCTCCTGTGTAGGCTCCGGTAG
CAGATTCGATAACTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAATTTGTAGCCGCTATTAGCT
GGAGTAGTGGCACTACGCGCTATTTAGACACCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAGTACG
GTATATCTTCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCTCGATATCAGCCGAGGTA
CTACGACTCAGGGGATATGGATGGATATGAGTATGACAACTGGGGTCAGGGGACCCAGGTCACCGTCTCCTCAGAAC
CCAAGACACCAAAACCACAACCAGCGGCCGCACATCATCATCACCATCACGGGGCCGCAGAACAAAAACTCATCTCA
GAAGAGGATCTGAATGGGGCCGCATAG
Coding generates the albumen that length is 185 amino acid (end terminator is not included in, i.e., * * * are not included in sequence)
Matter sequence (sequence 4), particular sequence is as follows：
The CDS sequences of the encoding gene of detection antibody of the present invention, cDNA sequence overall length are 2085bp (sequence 5),
Particular sequence is as follows：
TCCGGTACCATGGATGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTG
TACAGGCTCAGGACGCACCTTCAGGAACTATCCCATGGCCTGGTTCCGCCAGGCTCCAGGAAAGGAGCGTGAGTTTG
TAGCAGGTATTACCTGGGTCGGTGCTAGCACACTCTATGCAGACTTCGCGAAGGGCCGATTCACCATCTCCAGAGAC
AACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATAGTTGTGCAGCAGG
TCGCGGTATAGTGGCTGGTAGGATCCCAGCTGAGTATGCCGACTGGGGCCAGGGCACCCAGGTCACCGTCTCCTCAG
AACCCAAGACACCAAAACCACAACCAGCGGAGCTCCCGGGGGCGGCCGCCTGCAGAATGGAAGACGCCAAAAACATA
AAGAAAGGCCCGGCGCCATTCTATCCGCTGGAAGATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGAGATA
CGCCCTGGTTCCTGGAACAATTGCTTTTACAGATGCACATATCGAGGTGGACATCACTTACGCTGAGTACTTCGAAA
TGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTGAATACAAATCACAGAATCGTCGTATGCAGTGAAAAC
TCTCTTCAATTCTTTATGCCGGTGTTGGGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAACGACATTTATAA
TGAACGTGAATTGCTCAACAGTATGGGCATTTCGCAGCCTACCGTGGTGTTCGTTTCCAAAAAGGGGTTGCAAAAAA
TTTTGAACGTGCAAAAAAAGCTCCCAATCATCCAAAAAATTATTATCATGGATTCTAAAACGGATTACCAGGGATTT
CAGTCGATGTACACGTTCGTCACATCTCATCTACCTCCCGGTTTTAATGAATACGATTTTGTGCCAGAGTCCTTCGA
TAGGGACAAGACAATTGCACTGATCATGAACTCCTCTGGATCTACTGGTCTGCCTAAAGGTGTCGCTCTGCCTCATA
GAACTGCCTGCGTGAGATTCTCGCATGCCAGAGATCCTATTTTTGGCAATCAAATCATTCCGGATACTGCGATTTTA
AGTGTTGTTCCATTCCATCACGGTTTTGGAATGTTTACTACACTCGGATATTTGATATGTGGATTTCGAGTCGTCTT
AATGTATAGATTTGAAGAAGAGCTGTTTCTGAGGAGCCTTCAGGATTACAAGATTCAAAGTGCGCTGCTGGTGCCAA
CCCTATTCTCCT TCTTCGCCAAAAGCACTCTGATTGACAAATACGATTTATCTAATTTACACGAAATTGCTTCTGG
TGGCGCTCCCCTCTCTAAGGAAGTCGGGGAAGCGGTTGCCAAGAGGTTCCATCTGCCAGGTATCAGGCAAGGATATG
GGCTCACTGAGACTACATCAGCTATTCTGATTACACCCGAGGGGGATGATAAACCGGGCGCGGTCGGTAAAGTTGTT
CCATTTTTTGAAGCGAAGGTTGTGGATCTGGATACCGGGAAAACGCTGGGCGTTAATCAAAGAGGCGAACTGTGTGT
GAGAGGTCCTATGATTATGTCCGGTTATGTAAACAATCCGGAAGCGACCAACGCCTTGATTGACAAGGATGGATGGC
TACATTCTGGAGACATAGCTTACTGGGACGAAGACGAACACTTCTTCATCGTTGACCGCCTGAAGTCTCTGATTAAG
TACAAAGGCTATCAGGTGGCTCCCGCTGAATTGGAATCCATCTTGCTCCAACACCCCAACATCTTCGACGCAGGTGT
CGCAGGTCTTCCCGACGATGACGCCGGTGAACTTCCCGCCGCCGTTGTTGTTTTGGAGCACGGAAAGACGATGACGG
AAAAAGAGATCGTGGATTACGTCGCCAGTCAAGTAACAACCGCGAAAAAGTTGCGCGGAGGAGTTGTGTTTGTGGAC
GAAGTACCGAAAGGTCTTACCGGAAAACTCGACGCAAGAAAAATCAGAGAGATCCTCATAAAGGCCAAGAAGGGCGG
AAAGTGA
Coding generates the albumen that length is 694 amino acid (end terminator is not included in, i.e., * * * are not included in sequence)
Matter sequence (sequence 6), particular sequence is as follows：
Implement the present invention, has the advantages that：The present invention provides a kind of hypersensitivity, the high-throughput inspections of specificity
The method for surveying solubility CD38, sensitivity reach 10pg/mL, are higher than commercial ELISA kits；Importantly, relative to
Kit, DepID methods can effectively detect the content of CD38 in complex samples (such as plasma sample).
Description of the drawings
In order to illustrate the technical solution of the embodiments of the present invention more clearly, making letter to the attached drawing used in embodiment description
Singly introduce.In attached drawing：
Fig. 1 is CD38 single domain antibodies 1053,551 and the SDS-PAGE analysis charts of recombinant protein 1053-Fluc2；
Fig. 2 is the competition of single domain antibody 1053,551,375, NbGFP and label single domain antibody 551-AF48 combinations CD38
Figure；
Fig. 3 is the competition of single domain antibody 1053,551,375, NbGFP and label single domain antibody 1053-AF488 combinations CD38
Figure；
Fig. 4 is the detection principle diagram of DepID methods；
Fig. 5 is the standard curve of DepID methods detection CD38；
Fig. 6 is DepID methods detection specificity figure；
Fig. 7 is the healthy volunteer of DepID detections compared with solubility CD38 levels in myelomatosis multiplex human plasma.
Specific implementation mode
The embodiment of the present invention is specifically described below in conjunction with attached drawing.
Since soluble CD38 derives from the CD38 of cell surface, the concentration of plasma soluble CD38 can reflect multiple
Fall off enzyme (Sheddase) activated state in the proliferation and tumor microenvironment of property myeloma cell, can be used as diagnosing and monitoring multiple
Property myeloma bone disease development marker.
The present invention describes a kind of special, superelevation developed in the single domain antibody of CD38 different epitopes based on two basic change
Sensitive solubility CD38 detection methods --- double neoepitope Western method of identifications (Dual epitopes proteinIDEntification, DepID), and show the correlation of soluble CD38 and myelomatosis multiplex journey.
A series of CD38 single domain antibodies that the present invention obtains screening, by the method for flow cytometry, filtering out can
In combination with the single domain antibody pair of CD38, i.e. single domain antibody 1053 and 551, wherein single domain antibody 551 is as capture antibody, single domain
The fusion protein of antibody 1053 and Fluc mutant Fluc2 are as detection antibody.Single domain antibody 551 is coated on
Elisa plate bottom captures the CD38 in solution, detects the luminous signal of the catalysis luciferins of the Fluc2 in antibody to assess CD38's
Concentration (as shown in Figure 4) develops the soluble CD38 detection methods DepID of a species specificity hypersensitivity.
Present invention will be further explained below with reference to specific examples.
Embodiment 1：The structure of antibody expression vector is detected for DepID
(1) gene order of PCR amplification CD38 single domain antibodies 1053 (is purchased from using restriction enzyme KpnI and SacI
Thermo Scientific) digestion pRHSUL2 carriers and 1053 gene orders, it is used in combination T4DNA ligases (public purchased from TAKARA
Department) two segments of connection, build pRHSUL2-1053.
(2) restriction enzyme SacI and PstI digestion pRHSUL2-1053 carriers are used, SacI and PstI viscosity is contained
End and 2 × G4Two DNA of S sequences are single-stranded annealed, using T4DNA ligases by 2 × G4S is inserted into pRHSUL2-1053,
Build pRHSUL2-1053-G4S。
(3) gene order of PCR amplification Fluc mutant Fluc2, using restriction enzyme PstI and
III digestion pRHSUL2-1053-G of Hind4S and Fluc2 gene orders connect two segments, structure with T4DNA ligases
pRHSUL2-1053-G4S-Fluc2, i.e. DepID detect the expression vector of antibody.
Embodiment 2：The expression and purifying of CD38 single domain antibodies and DepID detection antibody 1053-Fluc2
(1) CD38 single domain antibody expression vector pHEN2-1053, pHEN2-551 and sequencing are identified into correct recombinant plasmid
pRHSUL2-1053-G4S-Fluc2 is transformed into expressive host bacterium Rosetta2 (DE3), and 37 DEG C expand culture to OD600Reach
0.6-0.8 adds 18 DEG C of induced expressions of 1mM IPTG, collects thalline ultrasonication, and high speed centrifugation collects supernatant, for further
Purifying.
(2) CD38 single domain antibodies are purified through Ni-NTA affinity chromatographys and anion-exchange chromatography, the albumen purified.
(3) DepID detects antibody through Ni-NTA after purification, the use of sumo proteinase excision includes 6 × His
Sumo-tag, then through a Ni-NTA reversed phase chromatography, collect efflux and carry out Q columns displacement chromatography to get recombinant protein 1053-
Fluc2.SDS-PAGE results such as Fig. 1 of single domain antibody 1053,551 and recombinant protein 1053-Fluc2.
Embodiment 3：CD38 single domain antibodies mark
(1) solution replacement is such as PBS of the buffer solution without Tris by CD38 single domain antibodies ultrafiltration；
(2) the fluorescent dye Alexa Fluor that can be reacted with aminoTM488 succinyl Asia ester (Thermo
Scientific it) is dissolved with DMSO；
(3) the above-mentioned fluorescent dye of about 5 times of molal quantitys is added in CD38 single domain antibodies, 4 DEG C are protected from light rotation mixing overnight；
(4) ultrafiltration, will be unmarked in CD38 single domain antibody solution on fluorescent dye removal.
Embodiment 4：CD38 single domain antibodies are to selection
(1) multiple myeloma cell line LP-1 is counted, and cell density is adjusted to 5 × 105The PBS of/mL, precooling (contain
1mg/mL BSA) it washes twice；
(2) add the single domain antibody of the single domain antibody and gradient concentration of 0.5 μ g/mL labels, 4 DEG C are protected from light incubation 30min；
(3) PBS (BSA containing 1mg/mL) being pre-chilled is washed twice, and 100 μ L PBS (BSA containing 1mg/mL) are resuspended；
(4) Flow cytometry, testing result is as shown in Figures 2 and 3, and single domain antibody 1053 and 551 can mutually not
It influences in combination in the different epitopes of CD38.
Embodiment 5：DepID method and steps
(1) single domain antibody 551 is diluted to 10 μ g/mL with PBS, is coated with elisa plate per 100 μ L of hole, 4 DEG C overnight；
(2) PBS board-washings are three times；
(3) 1%BSA is diluted in 0.1%PBST (PBS, 0.1%Tween 20), and per 100 μ L of hole, room temperature closes 1h；
(4) 0.1%PBST is washed three times；
(5) CD38 standard items or 20 μ L of sample and 0.5 μ g/mL 1053-Fluc2,1mg/mL BSA is taken to be diluted in 0.1%
PBST to 100 μ L is added to elisa plate, is incubated at room temperature 1h；
(6) 0.1%PBST is washed three times；
(7) add luciferin substrate dynamic detection, take initial 15s mean value calculations, detect standard curve such as Fig. 5 of CD38,
Its sensitivity is up to 10pg/mL.
Embodiment 6：DepID specific detections
(1) CD38 single domain antibody pearls are taken out, PBS is washed twice；
(2) CD38 standard items or plasma sample, each sample average are divided into two parts, and CD38 single domain antibodies are added in a copy of it
After 4 DEG C of pearl is incubated 3h altogether, supernatant is taken out in centrifugation；
CD38 single domain antibodies pearl treated sample and untreated sample, is detected according to embodiment 5, specificity
As a result after seeing that Fig. 6, single domain antibody pearl remove the CD38 in sample, detection signal is reduced to background level.Detect health simultaneously
Volunteer and myelomatosis multiplex human plasma sample, the results are shown in Figure 7, and patient's plasma soluble CD38 is apparently higher than normally
It is horizontal.In addition, in relatively DepID methods and commercial ELISA kits, while patient's plasma sample is detected, table 1 is DepID methods
Detect the horizontal results contrasts of patient plasma sample solubility CD38 simultaneously with commercial ELISA kits.
Table 1
N.D.：It is not detected (not detectable).
The results show that DepID can effectively detect the concentration of plasma soluble CD38, and commercial ELISA kits
Signal is not detected, fully shows the specificity of DepID methods, the detection especially suitable for complex samples.
The embodiment of the present invention is described with above attached drawing, but the invention is not limited in above-mentioned specific
Embodiment, the above mentioned embodiment is only schematical, rather than restrictive, those skilled in the art
Under the inspiration of the present invention, without breaking away from the scope protected by the purposes and claims of the present invention, it can also make very much
Form, all of these belong to the protection of the present invention.
Sequence table
<110>Shenzhen Graduate School of Peking University
<120>A kind of detection method of solubility CD38 concentration
<130>
<160> 6
<210> 1
<211> 546
<212> DNA
<213>Artificial sequence
<400> 1
ATGAAATACC TATTGCCTAC GGCAGCCGCT GGATTGTTAT TACTCGCGGC CCAGCCGGCC 60
ATGGCCGATG TGCAGCTGCA GGAGTCTGGA GGAGGCTTGG TGCAGGCTGG GGGCTCTCTG 120
AGACTCTCCT GTACAGGCTC AGGACGCACC TTCAGGAACT ATCCCATGGC CTGGTTCCGC 180
CAGGCTCCAG GAAAGGAGCG TGAGTTTGTA GCAGGTATTA CCTGGGTCGG TGCTAGCACA 240
CTCTATGCAG ACTTCGCGAA GGGCCGATTC ACCATCTCCA GAGACAACGC CAAGAACACG 300
GTGTATCTGC AAATGAACAG CCTGAAACCT GAGGACACGG CCGTTTATAG TTGTGCAGCA 360
GGTCGCGGTA TAGTGGCTGG TAGGATCCCA GCTGAGTATG CCGACTGGGG CCAGGGCACC 420
CAGGTCACCG TCTCCTCAGA ACCCAAGACA CCAAAACCAC AACCAGCGGC CGCACATCAT 480
CATCACCATC ACGGGGCCGC AGAACAAAAA CTCATCTCAG AAGAGGATCT GAATGGGGCC 540
GCATAG 546
<210> 2
<211> 181
<212> PRT
<213>Artificial sequence
<400> 2
Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala 10
Gly Leu Leu Leu Leu Ala Ala Gln Pro Ala 20
Met Ala Asp Val Gln Leu Gln Glu Ser Gly 30
Gly Gly Leu Val Gln Ala Gly Gly Ser Leu 40
Arg Leu Ser Cys Thr Gly Ser Gly Arg Thr 50
Phe Arg Asn Tyr Pro Met Ala Trp Phe Arg 60
Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 70
Ala Gly Ile Thr Trp Val Gly Ala Ser Thr 80
Leu Tyr Ala Asp Phe Ala Lys Gly Arg Phe 90
Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr 100
Val Tyr Leu Gln Met Asn Ser Leu Lys Pro 110
Glu Asp Thr Ala Val Tyr Ser Cys Ala Ala 120
Gly Arg Gly Ile Val Ala Gly Arg Ile Pro 130
Ala Glu Tyr Ala Asp Trp Gly Gln Gly Thr 140
Gln Val Thr Val Ser Ser Glu Pro Lys Thr 150
Pro Lys Pro Gln Pro Ala Ala Ala His His 160
His His His His Gly Ala Ala Glu Gln Lys 170
Leu Ile Ser Glu Glu Asp Leu Asn Gly Ala 180
Ala 181
<210> 3
<211> 558
<212> DNA
<213>Artificial sequence
<400> 3
ATGAAATACC TATTGCCTAC GGCAGCCGCT GGATTGTTAT TACTCGCGGC CCAGCCGGCC 60
ATGGCCGATG TGCAGCTGCA GGAGTCAGGA GGAGGATTGG TGCAGGCTGG ACACTCTCTG 120
AGACTCTCCT GTGTAGGCTC CGGTAGCAGA TTCGATAACT ATGCCATGGG CTGGTTCCGC 180
CAGGCTCCAG GGAAGGAGCG TGAATTTGTA GCCGCTATTA GCTGGAGTAG TGGCACTACG 240
CGCTATTTAG ACACCGTGAA GGGCCGATTC ACCATCTCCA GAGACAACGC CAAGAGTACG 300
GTATATCTTC AAATGAACAG CCTGAAACCT GAGGACACGG CCGTTTATTA CTGTGCAGCT 360
CGATATCAGC CGAGGTACTA CGACTCAGGG GATATGGATG GATATGAGTA TGACAACTGG 420
GGTCAGGGGA CCCAGGTCAC CGTCTCCTCA GAACCCAAGA CACCAAAACC ACAACCAGCG 480
GCCGCACATC ATCATCACCA TCACGGGGCC GCAGAACAAA AACTCATCTC AGAAGAGGAT 540
CTGAATGGGG CCGCATAG 558
<210> 4
<211> 185
<212> PRT
<213>Artificial sequence
<400> 4
Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala 10
Gly Leu Leu Leu Leu Ala Ala Gln Pro Ala 20
Met Ala Asp Val Gln Leu Gln Glu Ser Gly 30
Gly Gly Leu Val Gln Ala Gly His Ser Leu 40
Arg Leu Ser Cys Val Gly Ser Gly Ser Arg 50
Phe Asp Asn Tyr Ala Met Gly Trp Phe Arg 60
Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 70
Ala Ala Ile Ser Trp Ser Ser Gly Thr Thr 80
Arg Tyr Leu Asp Thr Val Lys Gly Arg Phe 90
Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr 100
Val Tyr Leu Gln Met Asn Ser Leu Lys Pro 110
Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala 120
Arg Tyr Gln Pro Arg Tyr Tyr Asp Ser Gly 130
Asp Met Asp Gly Tyr Glu Tyr Asp Asn Trp 140
Gly Gln Gly Thr Gln Val Thr Val Ser Ser 150
Glu Pro Lys Thr Pro Lys Pro Gln Pro Ala 160
Ala Ala His His His His His His Gly Ala 170
Ala Glu Gln Lys Leu Ile Ser Glu Glu Asp 180
Leu Asn Gly Ala Ala 185
<210> 5
<211> 2085
<212> DNA
<213>Artificial sequence
<400> 5
TCCGGTACCA TGGATGTGCA GCTGCAGGAG TCTGGAGGAG GCTTGGTGCA GGCTGGGGGC 60
TCTCTGAGAC TCTCCTGTAC AGGCTCAGGA CGCACCTTCA GGAACTATCC CATGGCCTGG 120
TTCCGCCAGG CTCCAGGAAA GGAGCGTGAG TTTGTAGCAG GTATTACCTG GGTCGGTGCT 180
AGCACACTCT ATGCAGACTT CGCGAAGGGC CGATTCACCA TCTCCAGAGA CAACGCCAAG 240
AACACGGTGT ATCTGCAAAT GAACAGCCTG AAACCTGAGG ACACGGCCGT TTATAGTTGT 300
GCAGCAGGTC GCGGTATAGT GGCTGGTAGG ATCCCAGCTG AGTATGCCGA CTGGGGCCAG 360
GGCACCCAGG TCACCGTCTC CTCAGAACCC AAGACACCAA AACCACAACC AGCGGAGCTC 420
CCGGGGGCGG CCGCCTGCAG AATGGAAGAC GCCAAAAACA TAAAGAAAGG CCCGGCGCCA 480
TTCTATCCGC TGGAAGATGG AACCGCTGGA GAGCAACTGC ATAAGGCTAT GAAGAGATAC 540
GCCCTGGTTC CTGGAACAAT TGCTTTTACA GATGCACATA TCGAGGTGGA CATCACTTAC 600
GCTGAGTACT TCGAAATGTC CGTTCGGTTG GCAGAAGCTA TGAAACGATA TGGGCTGAAT 660
ACAAATCACA GAATCGTCGT ATGCAGTGAA AACTCTCTTC AATTCTTTAT GCCGGTGTTG 720
GGCGCGTTAT TTATCGGAGT TGCAGTTGCG CCCGCGAACG ACATTTATAA TGAACGTGAA 780
TTGCTCAACA GTATGGGCAT TTCGCAGCCT ACCGTGGTGT TCGTTTCCAA AAAGGGGTTG 840
CAAAAAATTT TGAACGTGCA AAAAAAGCTC CCAATCATCC AAAAAATTAT TATCATGGAT 900
TCTAAAACGG ATTACCAGGG ATTTCAGTCG ATGTACACGT TCGTCACATC TCATCTACCT 960
CCCGGTTTTA ATGAATACGA TTTTGTGCCA GAGTCCTTCG ATAGGGACAA GACAATTGCA 1020
CTGATCATGA ACTCCTCTGG ATCTACTGGT CTGCCTAAAG GTGTCGCTCT GCCTCATAGA 1080
ACTGCCTGCG TGAGATTCTC GCATGCCAGA GATCCTATTT TTGGCAATCA AATCATTCCG 1140
GATACTGCGA TTTTAAGTGT TGTTCCATTC CATCACGGTT TTGGAATGTT TACTACACTC 1200
GGATATTTGA TATGTGGATT TCGAGTCGTC TTAATGTATA GATTTGAAGA AGAGCTGTTT 1260
CTGAGGAGCC TTCAGGATTA CAAGATTCAA AGTGCGCTGC TGGTGCCAAC CCTATTCTCC 1320
TTCTTCGCCA AAAGCACTCT GATTGACAAA TACGATTTAT CTAATTTACA CGAAATTGCT 1380
TCTGGTGGCG CTCCCCTCTC TAAGGAAGTC GGGGAAGCGG TTGCCAAGAG GTTCCATCTG 1440
CCAGGTATCA GGCAAGGATA TGGGCTCACT GAGACTACAT CAGCTATTCT GATTACACCC 1500
GAGGGGGATG ATAAACCGGG CGCGGTCGGT AAAGTTGTTC CATTTTTTGA AGCGAAGGTT 1560
GTGGATCTGG ATACCGGGAA AACGCTGGGC GTTAATCAAA GAGGCGAACT GTGTGTGAGA 1620
GGTCCTATGA TTATGTCCGG TTATGTAAAC AATCCGGAAG CGACCAACGC CTTGATTGAC 1680
AAGGATGGAT GGCTACATTC TGGAGACATA GCTTACTGGG ACGAAGACGA ACACTTCTTC 1740
ATCGTTGACC GCCTGAAGTC TCTGATTAAG TACAAAGGCT ATCAGGTGGC TCCCGCTGAA 1800
TTGGAATCCA TCTTGCTCCA ACACCCCAAC ATCTTCGACG CAGGTGTCGC AGGTCTTCCC 1860
GACGATGACG CCGGTGAACT TCCCGCCGCC GTTGTTGTTT TGGAGCACGG AAAGACGATG 1920
ACGGAAAAAG AGATCGTGGA TTACGTCGCC AGTCAAGTAA CAACCGCGAA AAAGTTGCGC 1980
GGAGGAGTTG TGTTTGTGGA CGAAGTACCG AAAGGTCTTA CCGGAAAACT CGACGCAAGA 2040
AAAATCAGAG AGATCCTCAT AAAGGCCAAG AAGGGCGGAA AGTGA 2085
<210> 6
<211> 694
<212> PRT
<213>Artificial sequence
<400> 6
Ser Gly Thr Met Asp Val Gln Leu Gln Glu 10
Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 20
Ser Leu Arg Leu Ser Cys Thr Gly Ser Gly 30
Arg Thr Phe Arg Asn Tyr Pro Met Ala Trp 40
Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 50
Phe Val Ala Gly Ile Thr Trp Val Gly Ala 60
Ser Thr Leu Tyr Ala Asp Phe Ala Lys Gly 70
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys 80
Asn Thr Val Tyr Leu Gln Met Asn Ser Leu 90
Lys Pro Glu Asp Thr Ala Val Tyr Ser Cys 100
Ala Ala Gly Arg Gly Ile Val Ala Gly Arg 110
Ile Pro Ala Glu Tyr Ala Asp Trp Gly Gln 120
Gly Thr Gln Val Thr Val Ser Ser Glu Pro 130
Lys Thr Pro Lys Pro Gln Pro Ala Glu Leu 140
Pro Gly Ala Ala Ala Cys Arg Met Glu Asp 150
Ala Lys Asn Ile Lys Lys Gly Pro Ala Pro 160
Phe Tyr Pro Leu Glu Asp Gly Thr Ala Gly 170
Glu Gln Leu His Lys Ala Met Lys Arg Tyr 180
Ala Leu Val Pro Gly Thr Ile Ala Phe Thr 190
Asp Ala His Ile Glu Val Asp Ile Thr Tyr 200
Ala Glu Tyr Phe Glu Met Ser Val Arg Leu 210
Ala Glu Ala Met Lys Arg Tyr Gly Leu Asn 220
Thr Asn His Arg Ile Val Val Cys Ser Glu 230
Asn Ser Leu Gln Phe Phe Met Pro Val Leu 240
Gly Ala Leu Phe Ile Gly Val Ala Val Ala 250
Pro Ala Asn Asp Ile Tyr Asn Glu Arg Glu 260
Leu Leu Asn Ser Met Gly Ile Ser Gln Pro 270
Thr Val Val Phe Val Ser Lys Lys Gly Leu 280
Gln Lys Ile Leu Asn Val Gln Lys Lys Leu 290
Pro Ile Ile Gln Lys Ile Ile Ile Met Asp 300
Ser Lys Thr Asp Tyr Gln Gly Phe Gln Ser 310
Met Tyr Thr Phe Val Thr Ser His Leu Pro 320
Pro Gly Phe Asn Glu Tyr Asp Phe Val Pro 330
Glu Ser Phe Asp Arg Asp Lys Thr Ile Ala 340
Leu Ile Met Asn Ser Ser Gly Ser Thr Gly 350
Leu Pro Lys Gly Val Ala Leu Pro His Arg 360
Thr Ala Cys Val Arg Phe Ser His Ala Arg 370
Asp Pro Ile Phe Gly Asn Gln Ile Ile Pro 380
Asp Thr Ala Ile Leu Ser Val Val Pro Phe 390
His His Gly Phe Gly Met Phe Thr Thr Leu 400
Gly Tyr Leu Ile Cys Gly Phe Arg Val Val 410
Leu Met Tyr Arg Phe Glu Glu Glu Leu Phe 420
Leu Arg Ser Leu Gln Asp Tyr Lys Ile Gln 430
Ser Ala Leu Leu Val Pro Thr Leu Phe Ser 440
Phe Phe Ala Lys Ser Thr Leu Ile Asp Lys 450
Tyr Asp Leu Ser Asn Leu His Glu Ile Ala 460
Ser Gly Gly Ala Pro Leu Ser Lys Glu Val 470
Gly Glu Ala Val Ala Lys Arg Phe His Leu 480
Pro Gly Ile Arg Gln Gly Tyr Gly Leu Thr 490
Glu Thr Thr Ser Ala Ile Leu Ile Thr Pro 500
Glu Gly Asp Asp Lys Pro Gly Ala Val Gly 510
Lys Val Val Pro Phe Phe Glu Ala Lys Val 520
Val Asp Leu Asp Thr Gly Lys Thr Leu Gly 530
Val Asn Gln Arg Gly Glu Leu Cys Val Arg 540
Gly Pro Met Ile Met Ser Gly Tyr Val Asn 550
Asn Pro Glu Ala Thr Asn Ala Leu Ile Asp 560
Lys Asp Gly Trp Leu His Ser Gly Asp Ile 570
Ala Tyr Trp Asp Glu Asp Glu His Phe Phe 580
Ile Val Asp Arg Leu Lys Ser Leu Ile Lys 590
Tyr Lys Gly Tyr Gln Val Ala Pro Ala Glu 600
Leu Glu Ser Ile Leu Leu Gln His Pro Asn 610
Ile Phe Asp Ala Gly Val Ala Gly Leu Pro 620
Asp Asp Asp Ala Gly Glu Leu Pro Ala Ala 630
Val Val Val Leu Glu His Gly Lys Thr Met 640
Thr Glu Lys Glu Ile Val Asp Tyr Val Ala 650
Ser Gln Val Thr Thr Ala Lys Lys Leu Arg 660
Gly Gly Val Val Phe Val Asp Glu Val Pro 670
Lys Gly Leu Thr Gly Lys Leu Asp Ala Arg 680
Lys Ile Arg Glu Ile Leu Ile Lys Ala Lys 690
Lys Gly Gly Lys 694
Claims (6)
1. a kind of detection method of solubility CD38 concentration, which is characterized in that include the following steps：
S1, using single domain antibody 551 as capture antibody, be coated on elisa plate bottom to capture the CD38 in sample solution；
S2, using the fusion protein of single domain antibody 1053 and luciferase as detection antibody, specifically bind CD38；
S3, detection luciferase are catalyzed the luminous signal of luciferin to assess the concentration of CD38.
2. the detection method of solubility CD38 concentration according to claim 1, which is characterized in that the luciferase is firefly
Noctiluca luciferase mutant Fluc2.
3. the detection method of solubility CD38 concentration according to claim 1, which is characterized in that the single domain antibody 551
Amino acid sequence such as sequence table in shown in sequence 4, in encoding gene such as sequence table shown in sequence 3.
4. the detection method of solubility CD38 concentration according to claim 1, which is characterized in that the single domain antibody 1053
Amino acid sequence such as sequence table in shown in sequence 2, in encoding gene such as sequence table shown in sequence 1.
5. the detection method of solubility CD38 concentration according to claim 1, which is characterized in that the ammonia of the detection antibody
In base acid sequence such as sequence table shown in sequence 6, in encoding gene such as sequence table shown in sequence 5.
6. the detection method of solubility CD38 concentration according to claim 1, which is characterized in that the table of the detection antibody
Up to carrier, building process includes the following steps：
The gene order of S201, PCR amplification CD38 single domain antibodies 1053 use restriction enzyme KpnI and SacI digestion
PRHSUL2 carriers and 1053 gene orders are used in combination T4 DNA ligases to connect two segments, build pRHSUL2-1053；
S202, using restriction enzyme SacI and PstI digestion pRHSUL2-1053 carriers, contain SacI and PstI viscosity end
End and 2 × G4Two DNA of S sequences are single-stranded annealed, using T4 DNA ligases by 2 × G4S is inserted into pRHSUL2-1053,
Build pRHSUL2-1053-G4S；
The gene order of S203, PCR amplification Fluc mutant Fluc2, using restriction enzyme PstI and
III digestion pRHSUL2-1053-G of Hind4S and Fluc2 gene orders connect two segments, structure with T4 DNA ligases
pRHSUL2-1053-G4S-Fluc2, i.e., the expression vector of the described detection antibody.
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Cited By (1)
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| CN112457401A (en) * | 2020-10-21 | 2021-03-09 | 上海交通大学医学院附属仁济医院 | Novel molecular imaging probe for diagnosing multiple myeloma |
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