CN107674122A - A kind of single domain antibody for identifying human serum albumins - Google Patents
A kind of single domain antibody for identifying human serum albumins Download PDFInfo
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- CN107674122A CN107674122A CN201611230995.6A CN201611230995A CN107674122A CN 107674122 A CN107674122 A CN 107674122A CN 201611230995 A CN201611230995 A CN 201611230995A CN 107674122 A CN107674122 A CN 107674122A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention discloses a kind of single domain antibody of identification human serum albumins, and plays CDR1, CDR2 and the CDR3 sequence of specific recognition；A kind of fusion protein, using listed amino acid sequence with treating the peptide molecule of function or the fusion protein of fragment preparation including at least a tool；A kind of more special or polyfunctional molecules, single domain antibody are more special or polyfunctional molecule parts；A kind of nucleic acid molecules, encode above-mentioned amino acid sequence；A kind of carrier, above-mentioned nucleotide sequence are a part for the carrier；A kind of protein or polypeptide, above-mentioned carrier are expressed by expression system；A kind of host cell, comprising any of the above-described single domain antibody, fusion protein, more special or polyfunctional molecule, nucleotide sequence, power carrier or the host cell of protein or polypeptide can be expressed.The present invention obtains the single domain antibody with high-affinity by screening, can identify specific human serum albumins, extends bio-pharmaceutical half-life period, is advantageous to the treatment to tumour.
Description
Technical field
The invention belongs to antibody technique field, more particularly to a kind of single domain antibody for identifying human serum albumins.
Background technology
At present, the conventional antibody of the neoplasm targeted therapy based on antibody is single-chain antibody or Antibody Fab fragment, with them
Compare, a variable region of the single domain antibody only containing heavy chain of antibody, be the antigen-binding fragment of minimum fully functioning, immunogene
Property is weaker；It is more easy to by vascular wall, due to no Fc sections, it is impossible to combined with the Fc acceptors of non-target cell, can more focus on and reach
Lesions position, therefore easily carry out genetic engineering operation.
Pharmaceutical grade protein is successfully widely used in clinical treatment, and many pharmaceutical grade proteins provide effectively for some diseases
With the therapeutic effect of uniqueness.But the half-life period of most of albumen and polypeptide is very short, first, because pharmaceutical grade protein can be by body
Interior proteasome degradation and quickly remove, two be due to that the filtration of glomerulus is medicine of the molecular weight less than 60KD in kidney
Easily it is eliminated in metabolism.In order to reach effective therapeutic effect, often using high dose or repeatedly, long-time medication, such one
Aspect can increase its toxic side effect, while also bring great inconvenience to patient medication.
In order to extend the half-life period of pharmaceutical grade protein in vivo, so as to strengthen the therapeutic effect of its medicine, it is desirable to
Delay degraded scavenging action of the body to pharmaceutical grade protein.Someone modifies pharmaceutical grade protein molecule in itself first, so as to
Change identification and proteases for decomposing effect of the body protein degraded removing system to pharmaceutical grade protein.Pass through the structure of mutant
Sensitiveness of the polypeptide to hydrolase can be generally reduced, the effective half-life period for extending polypeptide bio-pharmaceutical, but many half
The phase longer mutant that declines can change the activity of medicine.PEG modifications can reduce while the stability of protein drug is improved
Its immunogenicity, but have an impact the potentially possible of the bioactivity of polypeptide bio-pharmaceutical.
It is a kind of practicable extension drug half-life by directly being merged with the human body protein with relatively long half-life
Effective way.Human serum albumins is the major protein in blood of human body, is the albumen that content is most in human recycle system,
And the albumen that people's Half-life in vivo is most long, long half time was up to 19 days.It can be combined with some drug molecules, to drug molecule
Protective effect is provided, avoids being disposed by body premature breakdown, therefore, it is a kind of preferably pharmaceutical carrier molecule.
By the albumin function land (albumin binding domain, ABD) of staphylococcal protein G and Fc acceptors
It is merged composition fusion protein.The fusion protein can be specifically bound with the albumin in blood, and Fc acceptors are in body
Interior half-life period greatly prolongs.Human epidermal growth factor receptor 2 (HER2) is made into fusion protein together with ABD also identical
Effect.But the amalgamation mode of HSA- polypeptides considerably increases the molecular weight of polypeptide bio-pharmaceutical, come to production and zone of transformation
Many problems, the increase of molecular weight can also change some pharmacokinetic properties of medicine, it may be made to be difficult to reach target spot
Position, and then influence drug effect.
The characteristics of being combined using antigen and antibody specific, by protein drug and the knot of the specific antibody of human serum albumins
Conjunction functional areas, which are merged, is made fusion protein, can effectively improve the half-life period of Protein and peptide drugs.This patent utilizes
Display technique of bacteriophage, in the single domain antibody storehouse of people source screening obtain more plants can be with the antibody of HSA specific recognitions.Single domain resists
A variable region of the body only containing heavy chain of antibody, be the antigen-binding fragment of minimum fully functioning, compared to single-chain antibody or
Antibody Fab fragment molecular weight is smaller, immunogenicity is weaker, and extension biological medicament is played when specific binding can occurs with HSA in it
The function of thing half-life period.
The content of the invention
The present invention is for technical problem present in prior art, there is provided a kind of single domain for identifying human serum albumins resists
Body, it can reach and the single domain antibody with high-affinity is obtained by screening, specific human serum albumins can be identified, extend life
Thing drug half-life, be advantageous to the beneficial effect of biological medicament in the treatment.
In order to solve the above technical problems, the technical solution adopted by the present invention is：
A kind of immunoglobulin for identifying human serum albumins, the immunoglobulin amino acid sequence include
Complementary determining region 1 (CDR1) amino acid sequence SEQ ID NO.1, (CDR2) amino acid sequence of complementary determining region 2 SEQ
ID NO.2, and complementary determining region 3 (CDR3) amino acid sequence SEQ ID NO.3；
Complementary determining region 1 (CDR1) amino acid sequence SEQ ID NO.4, (CDR2) amino acid sequence of complementary determining region 2 SEQ
ID NO.5, and complementary determining region 3 (CDR3) amino acid sequence SEQ ID NO.6；
Complementary determining region 1 (CDR1) amino acid sequence SEQ ID NO.7, (CDR2) amino acid sequence of complementary determining region 2 SEQ
ID NO.8, and complementary determining region 3 (CDR3) amino acid sequence SEQ ID NO.9.
The single domain antibody is single domain antibodies, heavy chain becomes area and light chain becomes any of area.
The single domain antibody amino acid sequence includes SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12.
Single domain antibody or any cdr amino acid sequence of single domain antibody and FC fusion protein.
The single domain antibody include one or two described in claim 1 in complementary determining region (CDR) more than
Amino acid sequence, and at least with an amino acid sequence is minimum has 79% homology.
The amino acid sequence of the single domain antibody include described in claim 3 in framework (FR) one or two with
On amino acid sequence, and at least with an amino acid sequence is minimum has 90% homology.
The fusion protein is that any listed amino acid sequence is treated with one or more tools in usage right requirement 1
The peptide molecule or segment composition of function prepared gained together.
The fusion protein can directly be merged by each functional polypeptide, and what can also be connected by joint peptide contains 1 or 1
Treatment functional polypeptide molecule fusion protein above.
A kind of nucleic acid, codified go out the amino acid sequence of above-mentioned any single domain antibody.
A kind of carrier, include the nucleic acid sequence corresponding to above-mentioned single domain antibody, fusion protein, more special or polyfunctional molecules
Row, because genetic codon has degenerate, the nucleotide sequence can be different according to different application purposes.
A kind of protein or polypeptide, the protein or polypeptide can as the nucleotide sequence corresponding to above-mentioned amino acid sequence and
Above-mentioned nucleotide sequence or small part sequence can be expressed by suitable expression system to obtain phase；Above-mentioned expression system
System includes bacterium, saccharomycete, filamentous fungi, eucaryon mammalian cell, insect cell, plant cell, or acellular expression system
System.
A kind of host cell, it is characterised in that comprising above-mentioned single domain antibody, fusion protein, how special or more work(can be expressed
Can molecule, nucleotide sequence, carrier or the host cell of protein or polypeptide.
Compared with prior art, beneficial effect possessed by the present invention is：The present invention obtains having height affine by screening
The single domain antibody of power, specific human serum albumins can be identified, extend bio-pharmaceutical half-life period, be advantageous to control tumour
Treat.
Brief description of the drawings
Fig. 1 is ELISA detections and data analysis figure after three-wheel elutriation of the invention；
A：The equal envelope antigen HSA in all holes of ELISA；Antibody is different in per hole.
B：Data analysis ordinate is absorbance value of each hole under 650nm, and abscissa is 96 holes, wherein, 1-8 is
A1, B1, C1, D1, E1, F1, G1, H1,9-16 A2, B2, C2, D2, E2, F2, G2, H2, the like, 89-96 A12,
B12、C12、D12、F12、F12、G12、H12。
Fig. 2 is the HSA single domain antibodies of the present invention to HSA and IgG specific affinity data analysis figures；
Select 33 positive colonies to be ELISA from Fig. 1 and detect affinity of each single domain antibody to HSA and IgG, number
According to arrangement such as Fig. 2.Wherein, ordinate is the absorbance value under 650nm, and abscissa is 33 single domain antibodies.Each two is adjacent
In bar chart, that the left side represents antigen coat is HSA, and that the right represents antigen coat is IgG.
The SDS-PAGE figures that Fig. 3 is the fusion protein HB5-FC expressed in the pET22b of the present invention；
Marker band is followed successively by 14,25,30,40,50,70,100,120,160KD from small to large.Line1 is reduction
State HB5-FC, about 43KD.
Fig. 4 is the data analysis figure of fusion protein HB5-FC of the invention to HSA specific recognition；
Ordinate is the absorbance value under 650nm in figure, and abscissa is two fusion antibodies after purification, HB5 HB5-
FC antibody, Blank are a kind of non-HSA antibody.
Fig. 5 is the data analysis figure that the fusion protein HF11-FC of the present invention identifies to different antigentic specificities；
Figure is the HF11-FC fusion proteins of ELISA detections after purification to human serum albumins (HSA), bovine serum albumin(BSA)
(BSA), the affinity of four kinds of antigen of lowlenthal serum and horse serum.Ordinate be 650nm under absorbance value, abscissa four
Kind antigen, sample HF11-FC (-) represent to be not added with primary antibody HF11-FC control；HF11-FC (+), that is, represent to add primary antibody HF11-
FC。
Fig. 6 is single domain antibody amalgamation and expression FC in pET22b of present invention plasmid map schematic diagram；
Between single domain antibody and FC the restricted digestions of Xho I are used after Not I restriction enzyme sites and linker (G4S), FC.
Fig. 7 is single domain antibody amalgamation and expression FC in pcDNA3.1 of present invention plasmid map schematic diagram.
Introduced between single domain antibody and FC and pass through BamH I restriction enzyme sites, it is restricted interior using Hind III before single domain antibody
Enzyme cutting, the restricted digestions of Xba I are used after FC.There are signal peptide and kozak sequences before single domain antibody.
Embodiment
To make those skilled in the art be better understood from technical scheme, below in conjunction with the accompanying drawings and specific embodiment
The present invention is elaborated.
Embodiment of the invention discloses that a kind of single domain antibody for identifying human serum albumins, by three-wheel biopanning,
Screening obtains the higher antibody sequence of affinity from bacteriophage single domain library；The antibody sequence that screening obtains is cloned into original
In core/carrier for expression of eukaryon, with people source FC amalgamation and expressions, transfection host cell, obtain antibody and carry out testing for external affinity again
Card.
It is a kind of identify human serum albumins single domain antibody, the antibody sequence, its amino acid sequence as shown in sequence table,
Including amino acid sequence SEQ ID NO.10, SEQ ID NO.11, SEQID NO.12.
A kind of protein or polypeptide, amino acid sequences more than one or two in the framework region, and at least with
One amino acid sequence is minimum to have 90% homology.
A kind of protein or polypeptide, comprising amino acid sequences more than one or two in the complementary determining region,
And at least with an amino acid sequence is minimum has 79% homology.
A kind of nucleic acid molecules, one of the nucleic acid with may be encoded as each sequence in sequence table, can be with by genetic codon
The particular sequence of the nucleic acid molecules is obtained at any time, and because genetic codon has degenerate, the nucleotide sequence can be according to difference
Application purpose and it is different.
A kind of protein or polypeptide, the protein or polypeptide be able to can be passed through by nucleotide sequence or at least part sequence
Suitable expression system is expressed to obtain corresponding protein or polypeptide, and above-mentioned expression system includes bacterium, saccharomycete, silk
Shape fungi, zooblast, insect cell, plant cell, or Cell free expression system.
A kind of carrier, a part for the carrier can be above-mentioned nucleotide sequence.
Fusion protein can also be that single domain antibody and the polypeptide of other one or more tool treatment functions or identification function melt
Close the fusion protein prepared；Amalgamation mode can be merged directly, can also be connected by joint peptide.
Nucleic acid molecules can be to encode each complementary determining region or the nucleotides of single domain antibody or fusion protein amino acid sequence
Sequence, because genetic codon has degenerate, the nucleotide sequence can be different according to different application purposes.The nucleic acid molecules
Can be in bacterium, saccharomycete, filamentous fungi, mammalian cell, insect cell, plant cell, or Cell free expression system etc.
Protein is given expression in expression system.
A kind of host cell, it is characterised in that comprising above-mentioned single domain antibody, fusion protein, how special or more work(can be expressed
Can molecule, nucleotide sequence, carrier or the host cell of protein or polypeptide.
Embodiment 1 screens HSA single domain antibody
It is prepared by 1.1 single domain antibody phage libraries
1.1.1 the preparation of helper phage (BM13)
By M13KE bacteriophages (being purchased from NEB#N0316S) replicon AlwnI and AfeI (being purchased from NEB) double digestion, simultaneously
Synthetic gene fragment also uses AlwnI and AfeI double digestions, is then linked together with T4 ligases.TG1 is transfected after connection
Obtain helper phage BM13.In this way, in former replicon
tctggtggtggttctggtggcggctctgagggtggtggctctgagggtggcggttctgagggtggcggctctgaggg
Aggcggttccggtggtggctct sequences are substituted by synthetic gene sequence, i.e., in bacteriophage GIII coding regions, add
Add trypsase to cut sequence, when helper phage is used as, increased Trypsin Induced step, reduced without fusion mesh
Gene protein bacteriophage number.
Synthetic gene sequence is as follows：
CCA GCC GGC CTT TCT GAG GGG TCG ACT ATA GAA GGA CGA GGG GCC CAC GAA
GGAGGT GGG GTA CCC GGT TCC GAG GGT
1.1.2 phage library is built
1.1.2.1 vector construction
By pUC19 (being purchased from NEB) HindIII and NdeI (being purchased from NEB) double digestion, addition is based on DP47 antibody sequences
(ITomlinson et al：JMB 227：776,1992) the artificial single domain antibody sequence of heavy chain.In single domain antibody expression framework,
Single domain antibody and GIII protein fusions, centre add Myc and VSV-G labels, for purifying or identifying, are built into phage display
Carrier pBG3.
1.1.2.2ssDNA prepared by template
Coli strain CJ236 (being purchased from NEB) lacks feature dUTPase and uracil-N glycosylase, can
To produce uracil single-stranded DNA templates.PBG3 plasmid transfections are entered into CJ236 and are coated on containing Carbenicillin (50 μ
G/ml) and chloramphenicol (on (15 μ g/ml) agar plate, overnight incubation.Select the single bacterium filtered out on flat board
Fall in 3ml 2 × TY broth bouillons (containing the dual anti-of above-mentioned same concentration), overnight incubation under the conditions of 37 DEG C of 250rpm.Next day
Take 0.3ml overnight cultures to add in the fresh 2 × TY broth bouillons of 30ml (Carbenicillin50 μ g/ml), continue 3-4h
Culture, make OD600=0.4-0.6, add helper phage (pressing bacterium: bacteriophage number adds than 1: 10), 37 DEG C of 150rpm
1h, centrifugation, bacterial precipitation is resuspended in the dual anti-culture mediums of 2 × TY of 60ml, and 25 DEG C of 250rpm cultivate 22h, and precipitation is abandoned in centrifugation.
Supernatant containing bacteriophage is precipitated with 5%PEG (PEG800 and 300mM NaCl are adjusted to concentration as 5%), is then resuspended in PBS
In, prepare ssDNA using QIAprep Spin M13 kits (being purchased from Qiagen).
1.1.2.3 prepared by library
Prepared by library prepares (KunKel TA with KunKel methods：PNAS 82：488,1985) CDR mutant oligonucleotides chain
According to the form below synthesizes：
Olig_CDR1
CTGCGTCTCTCCTGTGCAGCCTCCGGAKWTANSNTTANCNMTNASDHTRSCNNTTGGGTCCGCCAGGCT
CCAGGGAA
Olig_CDR2
GGGTCTAGAGTGGGTATCARSCNNKRNSVVWCGTAGCGGTAGCACATACTACGCAGACTCCGTG
Olig_CDR3a
GACACCGCGGTATATTATTGCGCGGRSWNVSTHTNNKNNKNNKNNKNNKNNKNNKNNKNNKNNKNNKNN
KNNKNNKTGGGGTCAGGGAACCCTGGTCACCGTC
Olig_CDR3b
CGAGGACACCGCGGTATATTATTGCGCGGRSWNVSTHTNNKNNKNNKNNKNNKNNKNNKNNKNNKTGGG
GTCAGGGAACCCTGGTCACCGTCACC
The phosphorylation of oligonucleotide chain
Artificial synthesized oligonucleotide chain is added into following 100ul 50mM Tris-HCl pH7.5, and (Tris alkali is purchased from rope
Lay is precious, and PH7.5 is adjusted to hydrochloric acid) contain in following composition system：5U T4 polynucleotide kinases, 10mM MgCl2；1mM ATP and
After 5mM DTT, 37 DEG C of 1h, the good oligonucleotide chain of phosphorylation is purified with PCR purification kits (being purchased from Tiangeng).
Phosphorylated oligonucleotide and ssDNA annealed combinations
By phosphorylated oligonucleotide and Uracilated ssDNA (phosphorylated oligonucleotides: ssDNA=3: 1, ssDNA are
1.1.2.2 obtained by middle preparation) it is dissolved in containing 10mM, in MgCl2 50mM Tris-HCl, pH7.5 buffer solutions, 90 DEG C after 2 minutes,
It is down to 25 DEG C (1 DEG C/min of reduction per minute).
DsDNA is synthesized
Materials described below is added in the phosphorylated oligonucleotide and ssDNA compounds of annealed combination：0.55mM ATP；
0.8mM dNTPs；5mM DTT；15u T4DNA synzyme and 15u T7DNA synzyme,
After 20 DEG C of 3h, purified with Qiaquick PCR purification kits (being purchased from Qiagen).DNA after purification is to be transformed
Turn to electricity in competence TG1.
1.2BM13 helper phage and the increment of phage library
The increment of BM13 helper phages
From the fresh single bacterium colony of one e. coli tg1 of picking on basic agar medium flat board (being purchased from the vast spirit in Wuhan),
It is inoculated into 20ml 2 × TY culture mediums, gentle to shake, 37 DEG C of cultures to OD600 are about 0.8 standby.By what is prepared in 1.1.1
Original BM13 helper phages prepare a series of BM13 bacteriophages of 10 times of dilutions with 2 × TY culture mediums.Each dilution factor takes respectively
10 μ l and 200 μ l TG1 (OD600=0.8) bacterium solutions mix, and oscillator gently shakes 3s, and is mixed with top-layer agar culture medium,
It is poured on the TY flat boards of pre-balance room temperature.Pivotal plate is to ensure that thalline and top-layer agar are evenly distributed.Treat that upper strata is cultivated
After base solidification, in 37 DEG C of biochemical culture carton upside down overnight incubations.
Next day selects the good single bacteriophage of separation and is inoculated in the 2 × TY for containing 25 μ g/ml kanamycins equipped with 2~3ml
In the 15ml culture tubes of culture medium.37 DEG C, 12~16h of 250rpm concussion and cultivates.It is sterile micro- that infection supernatant is transferred to 1.5ml
Centrifuge tube is measured, on microcentrifuge, with maximum (top) speed, 4 DEG C of centrifugation 2min.Supernatant is transferred to 4 DEG C of preservations in new pipe.
The increment of phage library
The phage library prepared is seeded in 2 × TY culture mediums of the 100ml containing 60 μ g/ml ampicillins, 37
DEG C, when 250rpm concussion and cultivates to OD600 are 0.8, addition BM13 to concentration is 2 × 107pfu/ml.37 DEG C, 300rpm cultures
1h, 25 μ g/ml kanamycins are added, 37 DEG C are continued 14~18h of culture.Bacterium solution is centrifuged, supernatant is precipitated with 5%PEG, then
It is resuspended in standby in 5%MPBS.
1.3 screening HSA single domain antibody
1.3.1 biopanning
By the immune pipe of HSA direct coateds, room temperature 2h.PBST and PBS is washed 2~3 times.MPBS closings 2h, PBST and PBS wash 2
~3 times.Phage library is added in immune pipe, is incubated at room temperature 2h.
Uncombined library is removed, immune pipe is respectively washed 10 times with PBST and PBS.It is molten that 0.01% pancreatin is added into immune pipe
Liquid, it is incubated at room temperature 1h.Obtain pancreatin eluent.
Pancreatin eluent is added in 30ml TG1 (OD600=0.5), text is eluted for the first time by method increment in 1.2
Storehouse.
So carry out the second wheel, third round elutriation.The single bacterium of gained is dropped down onto in 96 orifice plates after picking three-wheel elutriation.By 1.2
Chinese library value adding method prepares culture supernatants.
1.3.2ELISA identification
A certain amount of supernatant is taken to do ELISA identifications.See Figure 1A.
ELISA steps are as follows：Known antigens are diluted to 1~10 μ g/ml with coating buffer solution, add 0.1ml per hole, 4 DEG C
Overnight；Next day washs 3 times；The measuring samples 0.1ml necessarily diluted is added to put 37 DEG C of incubations in the above-mentioned reacting hole being coated with
1h, washing；Add diluted fresh ELIAS secondary antibody (horseradish peroxidase HRP mark antiphagin secondary antibody, 1: 5000)
0.1ml, 37 DEG C of incubation 60min, washing；Last time is washed with DDW.The tmb substrate of Extemporaneous is added in each reacting hole
Solution 0.1ml, 37 DEG C of 10~30min.With advanced plate reading machine under 650nm wavelength read plate.
Each hole absorbance value such as Figure 1B.
Wherein, A：The equal envelope antigen HSA in all holes of ELISA；Antibody is different in per hole.B：Data analysis ordinate is
Absorbance value of each hole under 650nm, abscissa are 96 holes, wherein, 1-8 A1, B1, C1, D1, E1, F1, G1, H1,9-16
For A2, B2, C2, D2, E2, F2, G2, H2, the like, 89-96 A12, B12, C12, D12, E12, F12, G12, H12.
Select 33 positive colonies to be ELISA from Fig. 1, detect affinity of each single domain antibody to HSA and IgG,
Data preparation such as Fig. 2.Wherein, ordinate is the absorbance value under 650nm, and abscissa is 33 single domain antibodies.Blue bar shaped resists
Primordial covering is HSA, and green bar shaped antigen coat is IgG.
It is higher to HSA affinity in selection Fig. 2, and the clone of IgG affinity low (A650≤0.3) is sequenced, obtain
To multiple different amino acid sequences, SEQ ID NO.10 (HB5), SEQ ID NO.11 (HE9), SEQ ID NO.12
(HF11)。
The expression of embodiment 2, fusion protein HB5-FC
Single domain antibody gene is linked together by NotI and linker (G4S) and people source FC, it is front and rear to use Nco I respectively
It is cloned into Xho I restriction enzymes on pET22b, plasmid map is shown in Fig. 6.
The carrier built is converted into E.coli/DE3, chooses within second day monoclonal, 37 DEG C of 220rpm concussion and cultivates are extremely
During OD600 about 0.5, after adding IPTG (working concentration 1mM), 18 DEG C of 220rpm induced expressions 20h.After collecting thalline, PBS is used
(PH7.4) ultrasonication after being resuspended uniformly.Ultrasonication condition：600W, ultrasonic 2sec, interval 6sec, common 10min, 16 DEG C.It is super
4 DEG C of 12000rpm centrifugations 10min after sound.
Wherein, HB5-FC ultrasonic clear liquid runs SDS-PAG after purification with ProteinA, sees Fig. 3.Marker band is from small
To being followed successively by 14,25,30,40,50,70,100,120,160KD greatly.Line1 is the HB5-FC, about 43KD of reduction-state.
HB5-FC after purification is done into ELISA and detects its binding ability to HSA.Wherein, ELISA antigens are coated with HSA,
Primary antibody sample selects HB5-FC and a kind of non-HSA antibody (Blank), secondary antibody Goat anti human IgG 1: 5000 respectively.ELISA Plate
Add after TMB 20min with advanced plate reading machine read plate, data preparation such as Fig. 4 under 650nm wavelength.Ordinate is under 650nm
Absorbance value, abscissa are two samples.
As a result show, the fusion antibody HB5-FC of pET22b expression can combine HSA.
The fusion protein HF11-FC of embodiment 3 identifies HSA specific detection
In order to prepare stable antibody, single domain antibody Gene Fusion people source FC, and Kozak sequences are previously incorporated in antibody
And signal peptide rear clone, on pcDNA3.1, plasmid map is shown in Fig. 7.Introduced between single domain antibody and FC and pass through BamHI digestions
Site, HindIII restriction enzymes are used before single domain antibody, the restricted digestions of XbaI are used after FC.
The recombinant plasmid is transiently transfected into 293F, culture centrifuges after 4 days, collects supernatant, and pure with ProteinA
Change.
The HF11-FC fusion proteins of ELISA detections after purification are to human serum albumins (HSA), bovine serum albumin(BSA)
(BSA), the affinity of lowlenthal serum (albumin containing lowlenthal serum) and horse serum (albumin containing horse serum).ELISA data
Arrange such as Fig. 5.Wherein, HF11-FC (-) represents to be not added with primary antibody, only adds secondary antibody；HF11-FC (+) is represented plus primary antibody HF11-FC melts
Hop protein, add secondary antibody.
As a result showing, the fusion antibody HF11-FC expressed in pcDNA3.1 can specifically bind HSA, and not combine BSA,
Lowlenthal serum is not also combined, there is the combination of weak degrees to horse serum.
Some terms that the present invention is described have following implication：
Homology：Two or more amino acid sequence similarity degrees, first amino acid sequence and second amino are described
The percentage of homology can pass through formula between acid sequence：(in first amino acid sequence with second amino acid sequence
The quantity of the amino acid sequence identical amino acid residue of corresponding position)/(amino acid sum in first amino acid sequence) *
100% calculates, wherein the missing of some amino acid that second amino acid sequence is merely able to, insertion, replacement or addition are (with the
One amino acid is compared) it is considered as to have difference.Percent homology can also utilize the known calculating for being used for sequences match
Machine operation program such as NCBI Blast are obtained.Codon, also known as triplet codon, refer to the nucleotides corresponding to certain amino acid
Triplet.The position of polypeptide chain in this kind of amino acid insertion growth is determined during translation.
The present invention is described in detail above by embodiment, but the content is only the exemplary implementation of the present invention
Example, it is impossible to be considered as the practical range for limiting the present invention.Protection scope of the present invention is defined by the claims.All utilizations
Technical solutions according to the invention, or those skilled in the art is under the inspiration of technical solution of the present invention, in the reality of the present invention
In matter and protection domain, design similar technical scheme and reach above-mentioned technique effect, or application range is made
Equivalent change and improvement etc., the patent that all should still belong to the present invention cover within protection domain.
SEQUENCE LISTING
<110>Tianjin Tian Rui bio tech ltd
<120>A kind of single domain antibody for identifying human serum albumins
<160> 12
<210> 1
<211> 9
<212> PRT
<213>Artificial sequence
<400> 1
Val Lys Ala Ile Asn His Thr Met Gly
1 5
<210> 2
<211> 6
<212> PRT
<213>Artificial sequence
<400> 2
Ala Ile Arg Thr Pro Asp
1 5
<210> 3
<211> 15
<212> PRT
<213>Artificial sequence
<400> 3
Ser Val Gly Asp Asn Ala Gly Ser Ser Ser Pro Pro Phe Ala Ser
1 5 10 15
<210> 4
<211> 9
<212> PRT
<213>Artificial sequence
<400> 4
Phe Met Val Thr Ser Glu Ile Met Gly
1 5
<210> 5
<211> 6
<212> PRT
<213>Artificial sequence
<400> 5
Thr Ile Val Ala Asn Asn
1 5
<210> 6
<211> 16
<212> PRT
<213>Artificial sequence
<400> 6
Ser Pro Pro Arg Arg Thr Gly Val Asp Gly Thr Glu Ser Met His Phe
1 5 10 15
<210> 7
<211> 9
<212> PRT
<213>Artificial sequence
<400> 7
Val Lys Ile Ser Tyr Glu Ser Met Ala
1 5
<210> 8
<211> 6
<212> PRT
<213>Artificial sequence
<400> 8
Ser Ile Leu Met Gln Asn
1 5
<210> 9
<211> 12
<212> PRT
<213>Artificial sequence
<400> 9
Thr Thr Arg Val Arg Arg Pro Ala Asn Met Lys Tyr
1 5 10
<210> 10
<211> 123
<212> PRT
<213>Artificial sequence
<400> 10
Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala
1 5 10 15 20
Ala Ser Gly Val Lys Ala Ile Asn His Thr Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
25 30 35 40 45
Trp Val Ser Ala Ile Arg Thr Pro Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60 65
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
70 75 80 85 90
Val Tyr Tyr Cys Ala Ser Val Gly Asp Asn Ala Gly Ser Ser Ser Pro Pro Phe Ala Ser Trp Gly Gln
95 100 105 110 115
Gly Thr Leu Val Thr Val Ser Ser
120
<210> 11
<211> 124
<212> PRT
<213>Artificial sequence
<400> 11
Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala
1 5 10 15 20
Ala Ser Gly Phe Met Val Thr Ser Glu Ile Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
25 30 35 40 45
Trp Val Ser Thr Ile Val Ala Asn Asn Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60 65
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
70 75 80 85 90
Val Tyr Tyr Cys Ala Ser Pro Pro Arg Arg Thr Gly Val Asp Gly Thr Glu Ser Met His Phe Trp Gly
95 100 105 110 115
Gln Gly Thr Leu Val Thr Val Ser Ser
120
<210> 12
<211> 120
<212> PRT
<213>Artificial sequence
<400> 12
Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala
1 5 10 15 20
Ala Ser Gly Val Lys Ile Ser Tyr Glu Ser Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
25 30 35 40 45
Trp Val Ser Ser Ile Leu Met Gln Asn Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60 65
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
70 75 80 85 90
Val Tyr Tyr Cys Ala Thr Thr Arg Val Arg Arg Pro Ala Asn Met Lys Tyr Trp Gly Gln Gly Thr Leu
95 100 105 110 115
Val Thr Val Ser Ser
120
Claims (12)
1. a kind of single domain antibody for identifying human serum albumins, it is characterised in that the amino acid sequence of the single domain antibody includes
Three complementary determining regions, respectively CDR1, CDR2 and CDR3, wherein：
CDR1 is SEQ ID NO.1, and CDR2 is that SEQ ID NO.2, CDR3 are SEQ ID NO.3；
Or CDR1 is SEQ ID NO.4, CDR2 is that SEQ ID NO.5, CDR3 are SEQ ID NO.6；
Or CDR1 is SEQ ID NO.7, CDR2 is that SEQ ID NO.8, CDR3 are SEQ ID NO.9.
2. a kind of single domain antibody for identifying human serum albumins according to claim 1, it is characterised in that the single domain resists
Body is single domain antibodies, heavy chain becomes area and light chain becomes any of area.
3. a kind of single domain antibody, it is characterised in that the single domain antibody amino acid sequence includes SEQ ID NO.10, SEQ ID
NO.11, SEQ ID NO.12.
4. a kind of single domain antibody for identifying human serum albumins according to claim 1, it is characterised in that the single domain resists
Body include one or two described in claim 1 in complementary determining region (CDR) more than amino acid sequence, and at least with
One amino acid sequence is minimum to have 79% homology.
5. a kind of single domain antibody for identifying human serum albumins according to claim 1, it is characterised in that the single domain resists
The amino acid sequence of body is included described in claim 3 in framework (FR, i.e. single domain antibody remove the unexpected amino acid sequences of CDR)
Amino acid sequence more than one or two, and at least with an amino acid sequence is minimum has 90% homology.
A kind of 6. fusion protein, it is characterised in that in fusion protein usage right requirement 1 listed amino acid sequence with least
The peptide molecule of function or the fusion protein of fragment preparation are treated comprising a tool.
7. a kind of fusion protein according to claim 7, it is characterised in that the fusion protein can be by each functional polypeptide
Directly merge, also can contain 1 or 1 function above peptide molecule fusion protein by what joint peptide connected.
8. a kind of more special or polyfunctional molecules, it is characterised in that the single domain antibody described in claim 1 is how special or more for this
A part for functional molecular.
A kind of 9. nucleic acid molecules, it is characterised in that the amino acid sequence described in coding claim 1-8.
10. a kind of carrier, it is characterised in that nucleotide sequence described in claim 9 is a part for the carrier, due to genetic code
Son has degenerate, and the nucleotide sequence can be different according to different application purposes.
11. a kind of protein or polypeptide, it is characterised in that carrier is expressed by expression system described in claim 10, institute
Obtain corresponding protein or polypeptide；The expression system includes bacterium, saccharomycete, filamentous fungi, mammalian cell, insect
Cell, plant cell, or Cell free expression system.
12. a kind of host cell, it is characterised in that resist comprising the single domain that can be expressed in claim 1-5 described in any claim
How special or polyfunctional molecules described in fusion protein, claim 8 described in body, claim 6-7, described in claim 9
Nucleotide sequence, the carrier described in claim 10 or protein or the host cell of polypeptide described in claim 11.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611230995.6A CN107674122A (en) | 2016-12-28 | 2016-12-28 | A kind of single domain antibody for identifying human serum albumins |
| PCT/CN2017/118281 WO2018121473A1 (en) | 2016-12-28 | 2017-12-25 | Single-domain antibody capable of recognizing human serum albumin |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201611230995.6A CN107674122A (en) | 2016-12-28 | 2016-12-28 | A kind of single domain antibody for identifying human serum albumins |
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| CN201611230995.6A Pending CN107674122A (en) | 2016-12-28 | 2016-12-28 | A kind of single domain antibody for identifying human serum albumins |
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| CN116023487A (en) * | 2022-07-11 | 2023-04-28 | 南方医科大学第三附属医院（广东省骨科研究院） | Nanobody combined with various serum albumin and preparation method and application thereof |
| WO2024239515A1 (en) * | 2023-05-19 | 2024-11-28 | Next Medicine Co., Ltd. | Anti-hsa single-domain antibodies and uses thereof for assessing renal function |
| WO2024051383A1 (en) * | 2023-07-28 | 2024-03-14 | 上海洛启生物医药技术有限公司 | Anti-trop2 antibody, conjugate comprising said antibody, and use thereof |
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| CN120173100A (en) * | 2025-05-20 | 2025-06-20 | 中国科学院动物研究所 | Anti-human serum albumin nanoantibody and its application |
| CN120173100B (en) * | 2025-05-20 | 2025-08-15 | 中国科学院动物研究所 | Anti-human serum albumin nano antibody and application thereof |
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| WO2018121473A1 (en) | 2018-07-05 |
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