CN106831981B - A kind of single domain antibody peptide backbone and preparation method thereof - Google Patents
A kind of single domain antibody peptide backbone and preparation method thereof Download PDFInfo
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- CN106831981B CN106831981B CN201611104480.1A CN201611104480A CN106831981B CN 106831981 B CN106831981 B CN 106831981B CN 201611104480 A CN201611104480 A CN 201611104480A CN 106831981 B CN106831981 B CN 106831981B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/005—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies constructed by phage libraries
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- 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]
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- 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
- C07K2317/567—Framework region [FR]
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- 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
- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
Abstract
The invention discloses a kind of single domain antibody peptide backbones and preparation method thereof.Its single domain antibody peptide backbone sequence composition is followed successively by FR1, CDR1, FR2, CDR3 and FR3 areas, wherein FR1, FR2, FR3 area are fixed amino acid sequence, CDR1 and CDR3 areas determine area for complementary antibody, its amino acid sequence is variable, CDR1 and CDR3 areas determine and different antigen bindings.The single domain antibody derives from Upeneus bensari.The molecular weight of the single domain antibody peptide backbone only has 13kD or so, while the species relationship difference bigger of Upeneus bensari and people, can generate the single domain antibody of more high-affinity.Upeneus bensari is convenient for raising simultaneously.
Description
Technical field
The present invention relates to biotechnologies more particularly to a kind of single domain antibody peptide backbone and preparation method thereof.
Background technology
Antibody drug is the important component of biological medicine.Compared with traditional small-molecule drug, antibody drug due to
Its targeting is good, Small side effects, and antibody drug becomes bio-pharmaceutical industry with the fastest developing speed, is that several years new drug will be ground from now on
The Main way of hair, economic benefit are particularly evident.It is counted according to Reichert, the market value of antibody class drug in 2011 reaches 48,000,000,000
Dollar, the biological species drug for taking the lead in race all, it is contemplated that even more reached 68,000,000,000 dollars or more by 2016.
The research progress of antibody is looked back, the preparation of antibody is always one of core and bottleneck of entire industry.In antibody medicine
In the industrialization process of object, conventional antibody molecule expression quantity in mammalian cell is low, and holds in prokaryotic cell system
The problems such as aggregation easily occur and can not glycosylating so that antibody producing is of high cost, causes research and development and cost of use high, just limits
Its further application is made.Moreover, conventional antibodies since its relative molecular mass is big, cause antibody molecule in tumor tissues and
The penetrability of vascular barrier is poor, and the effective concentration especially in entity tumor is lower, greatly limits the validity of antibody drug.
Therefore, antibody miniaturization has become the main trend of antibody drug research.
Antibody molecule of the heavy chain antibody as natural deletions light chain, relative to traditional IgG antibody in genetic engineering transformation
Molecule has the advantage of bigger.By the single domain antibody (single domain antibody) of heavy chain antibody transformation natural
There is the ability with antigen binding under state, compared with the single domain antibody in people source or mouse source, affinity retains more preferably, due to
Its relative molecular mass is far smaller than conventional antibodies, and antibody size only has Nano grade to be otherwise known as nano antibody
(nanobody).Relative to traditional genetic engineering small molecular antibody, single domain antibody has various advantages：1) molecular weight is small
And the flexibility of CDR3 increases, and single domain antibody is allow to identify hiding antigen site, the traditional antibody of these epitopes can not
Identification, such as the activated centre of enzyme, can be as the inhibitor of enzyme or the diagnostic reagent of trypanosomiasis；2) since single domain antibody exists
Only there are one the zones of action under natural conditions, and it is more convenient to carry out genetic manipulation to it；3) single domain antibody, which only has, identifies antigen
Variable region is without constant region, so as to increase the range of functional areas；4) because hinge area is more flexible changeable, and without weight
The mispairing of chain variable region is more prone to the generation of multivalent antibody；5) due to single-stranded natural quality and hydrophilic increase
Folding efficiency is caused to improve, so as to good physical and chemical stability；6) since hydrophily increases, improve its dissolubility；7)
Molecular weight is small, therefore with good tissue permeability, and the antibody for being conducive to carry the substances such as toxin reaches tumor tissues；8)
Efficiently folding can help it to express well；9) molecular weight is small, thus weak to the immunogenicity of human body；10) in terms of production,
Because conventional antibodies will play biological function, it is often necessary to suitable glycosylation, so most antibody can only be in lactation
It is expressed in animal system, relative to protokaryon and Yeast system, mammlian system is more expensive.And single domain antibody is due to dividing
Son amount is small, and does not need to be glycosylation modified and can play biological function, is suitble in expression in escherichia coli.Due to have with
Upper advantage, in recent years single domain antibody attracted the huge research enthusiasm of people, explore antigen receptor origin, research and development vaccine, control
It treats drug, diagnostic reagent and biotechnology research tool etc. and achieves considerable progress.It is ground in terms of single domain antibody at present
It is camel single domain antibody that it is best, which to study carefully exploitation, wherein Ablynx biopharmaceutical companys of Belgium have had developed multiple camel single domains
Antibody, wherein antithrombotic single domain antibody have been enter into III phases clinic, and also 4 autoimmune disease camel single domain antibodies enter II
Phase is clinical.1 anticancer camel single domain antibody enters I phases clinic.But camel, which is raised and difficulty is immunized, causes hunchbacked source single domain antibody
R＆D costs it is too high, and the research of hunchbacked source single domain antibody is too many in the world at present, should for multiple target spots of a variety of diseases
With all by patent protection.Therefore the single domain antibody in other sources of the exploitation in addition to camel is of great significance.
After camel finds single domain antibody, Greenberg etc. is found that similar out of the selachians body such as nurse shark in succession again
The immunoglobulin neoantigen receptor (Ig new antigen receptor, IgNAR) of structure.As shown in Figure 1, IgNAR albumen
It is a kind of dimer, every chain is made of 1 variable region (vNAR) and 5 constant regions (cNAR).There is no L chains or other any eggs
This white adjoint dimer.Electronic Speculum shows that the V areas of IgNAR are different from traditional Ig and TCR, does not form dimer, but quite solely
Vertical flex region.The antigen binding flexible variable area of IgNAR can be expressed as independent soluble egg using technique for gene engineering
In vain, referred to as shark single domain antibody, is represented with vNAR.VNAR is divided into I, II and III according to the development period of disulfide bond and selachian and is total to
3 types, I type exist only in nurse shark, and III type is primarily present in shark embryo, before being the immune response maturation that antigen drives
To antiviral the first line of defence.For 3 types containing 4 conservative framework regions and 2 complementary determining regions, CDR1 is very short,
Diversity is limited；CDR3 longer, about 5-23 amino acid residue, diversity are high；Lack traditional CDR2, relevant position is by molecule
The short β at middle part-corner substitutes, entitled (the hypervariable region of hypervariable region 2 of annular section that should be located in FR2-CDR2
2, HV2)；There are one the cyclic annular knots of entitled hypervariable region 4 (hypervariable region 4, HV4) between HV2 and CDR3
Structure, it is homologous with the HV4 of T cell receptor.HV2 and HV4 may participate in antigen binding.II type forms 1 between CDR1 and CDR3
Additional disulfide bond；I type forms 1 additional disulfide bond between FR2 and FR4, and 1~2 two sulphur is formed in CDR3
Key.Compared with camel single domain antibody, shark source single domain antibody has the advantage that：1) there is 350mM in the blood of shark
Urea.Which results in the albumen in all shark blood innately to have very strong stability.The thermostabilization of shark source single domain antibody
Property is more preferable than hunchbacked source, can keep biological activity in the presence of higher temperature and denaturant；2) shark single domain resists
Body only has 13kD or so than the smaller in hunchbacked source, therefore with better tissue permeability；3) the species relationship difference of shark and people
Bigger can generate the single domain antibody of more high-affinity.
It is considerably less to the research application of shark single domain antibody at present, and focus primarily upon grinding for nurse's shark single domain antibody
Study carefully, and nurse shark individual is big, the immune period is long, is unfavorable for large-scale breeding and generation single domain antibody is immunized.
Invention content
The purpose of the present invention is to provide a kind of single domain antibody peptide backbones.
To achieve the above object, the present invention provides a kind of single domain antibody peptide backbone, which is characterized in that its sequence composition according to
Secondary is FR1, CDR1, FR2, CDR3 and FR3 areas, wherein FR1, FR2, FR3 area are fixed amino acid sequence, CDR1 and CDR3 areas are
Complementary antibody determines area, and amino acid sequence is variable, CDR1 and CDR3 areas determine and different antigen bindings.
Further, the amino acid sequence in the FR1 areas such as SEQ ID NO:Shown in 22, the amino acid sequence such as SEQ in FR2 areas
ID NO:Shown in 23, the amino acid sequence such as SEQ ID NO in FR3 areas:Shown in 24.
Further, the single domain antibody derives from Upeneus bensari.
Further, the CDR1 is 8 amino acid, and wherein second is cysteine, and the 7th is threonine；It is preferred that
, the amino acid sequence of CDR1 is SEQ ID NO:Any one in 25-32.
Further, the sequence of CDR3 is 10-25 amino acid sequence；Preferably, the amino acid sequence of CDR3 is SEQ ID
NO:Any one in 33-49.
The present invention also provides a kind of preparation methods of the single domain antibody peptide backbone, which is characterized in that and step is,
1) Upeneus bensari is immunized；
2) Upeneus bensari vNAR single domain antibodies library is built；
3) it screens and obtains Upeneus bensari vNAR single domain antibodies.
Further, the Upeneus bensari, which is immunized, is,
A. using antigen and complete Freund's adjuvant 1:1 volume ratio mixed immunity Upeneus bensari is for the first time；
B.4 after week, using antigen and non-fully complete Freund's adjuvant 1:1 volume ratio mixed immunity Upeneus bensari
It is secondary；
C.4 after week, Upeneus bensari third time is immunized using the direct tail vein injection of antigen；
D.4 Upeneus bensari is immunized the 4th time using the direct tail vein injection of antigen after week；
E.4 Upeneus bensari is immunized the 5th time using the direct tail vein injection of antigen after week.
Further, the Upeneus bensari vNAR single domain antibodies library is configured to,
The acquisition of A.cDNA：CDNA is obtained in the peripheral blood and spleen of the Upeneus bensari being immunized from antigen；
B. the clone of target gene and library construction：Primer, PCR amplification are designed according to Upeneus bensari vNAR conserved sequences
It obtains vNAR area's encoding genes and connects into suitable Vector for Phage Display, make vNAR areas gene and bacteriophage coat protein
PIII amalgamation and expressions, and pass through electricity and turn the library that TG1 Escherichia coli are built.
Further, it is described to screen and to obtain Upeneus bensari vNAR single domain antibodies be the spy that expanded again using bacteriophage
Property, by affine absorption-elution-amplification, washed in a pan from the library built be sieved to the antibody that can be combined with antigentic specificity and its
Gene is Upeneus bensari vNAR single domain antibodies.
The present invention extracts RNA and using inverse using Upeneus bensari peripheral blood and spleen as starting material, using TRIZOL methods
The reverse transcription of transcript reagent box is cDNA.Designing primer according to the conserved sequence in Upeneus bensari vNAR areas, (sense primer, sequence is such as
SEQ ID NO:Shown in 1, specially CAACGG GTTGAACAAACACC；Downstream primer, sequence such as SEQ ID NO:Shown in 2, tool
Body is TTTCACAGTCAGAATGGTGC), it is expanded by PCR methods and obtains full set Upeneus bensari vNAR areas coding.Striped mottled bamboo
Shark (Chiloscyllium plagiosum Bennett) vNAR represents sequence (its protein sequence such as SEQ ID NO:Shown in 3,
Can also be SEQ ID NO:Any one in 3-17) it is represented with speckle wobbegong (Orectolobus Maculatus) vNAR
Sequence (Gene bank sequence number AAP86761) compares CDR1, CDR3 area for determining Upeneus bensari vNAR.Randomly select 15
(SEQ ID NO are sequenced in Upeneus bensari vNAR region sequences:3-17), CDR1 areas and CDR3 areas amino acid are found by comparison
Sequence height change (see Fig. 2 and Fig. 3 A and Fig. 3 B).1 represents Upeneus bensari (Chiloscyllium plagiosum in Fig. 2
Bennett) vNAR represents sequence, sequence number SEQ ID NO:3；2 represent speckle wobbegong (Orectolobus Maculatus)
VNAR represents sequence, Gene bank sequence numbers AAP86761.Upeneus bensari vNAR is can be seen that from Fig. 2, Fig. 3 A and Fig. 3 B
Area is a CDR1 area and CDR3 region amino acid sequences height change and all metastable sequence in FR1, FR2, FR3 area.This hair
The sequence of bright peptide backbone is to include invariant region and Variable Area, invariant region be by FR1, FR2, FR3 district's groups into it is steady
Fixed amino acid sequence, complementary antibody determine that area is CDR1 and CDR3 areas, and the length of CDR1 is 8 amino acid, wherein second
For cysteine (Cys, C), the 7th is threonine (Thr, T).The length of CDR3 is 9-25 amino acid.
Single domain antibody peptide backbone of the present invention, it is as follows with advantage：1) small only 13kD or so of molecular weight, than hunchbacked source
Smaller, 2) the species relationship difference bigger of Upeneus bensari and people, the single domain antibody of more high-affinity can be generated.Striped simultaneously
Mottled bamboo shark is convenient for raising.
The Upeneus bensari (Chiloscyllium plagiosum Bennett) of the present invention belongs to Elasmobranchii
(Elasmobranchii) wobbegong mesh (Orectolobiformes) Orectolobidae (Orectolobldae) mottled bamboo shark category
(Chiloscyllium).Research and utilization to Upeneus bensari single domain antibody is in the world also in blank.
The present inventor is found that the possible new way towards application from a simple Upeneus bensari.It is expected to
Upeneus bensari is realized from kitchen to laboratory, then the Value Transformation to application.
Description of the drawings
Fig. 1 is immunoglobulin neoantigen receptor (IgNAR) and single domain antibody structure chart.
Fig. 2 is that Upeneus bensari (Chiloscyllium plagiosum Bennett) vNAR represents sequence (with SEQ ID
NO:For 3) with speckle wobbegong (Orectolobus Maculatus) vNAR represent sequence (Gene bank sequence numbers
AAP86761 sequence alignment figure).
Fig. 3 A are 15 different Upeneus bensari vNAR region sequence comparison charts.
Fig. 3 B are 15 different Upeneus bensari vNAR region sequence comparison charts.
Fig. 4 is Upeneus bensari vNAR areas PCR product electrophoretogram.
Fig. 5 is that ELISA detections screen vNAR single domain antibodies and the combination datagram of antigen HSA.
Fig. 6 is Upeneus bensari vNAR areas PCR product electrophoretogram.
Fig. 7 is that ELISA detections screen vNAR single domain antibodies and the combination datagram of antigen original TNF-α.
Specific embodiment
The embodiment of the present invention is described below in detail, the example of the embodiment is shown in the drawings, wherein from beginning to end
Same or similar label represents same or similar element or the element with same or like function.Below with reference to attached
The embodiment of figure description is exemplary, it is intended to for explaining the present invention, and is not considered as limiting the invention.Embodiment
In particular technique or condition person is not specified, according to the described technology of document in the art or condition or according to the description of product
Book carries out.Reagents or instruments used without specified manufacturer, being can be with conventional products that are commercially available.
Embodiment 1：The screening of the anti-HSA single domain antibodies in Upeneus bensari source
1. Upeneus bensari is immune.
A. Upeneus bensari is selected to carry out the screening of following Upeneus bensari source single domain antibody as immunization.
B. 50 micrograms antigen HSA (Sigma-Aldrich, 70024-90-7, below herewith) and complete Freund's adjuvant are used
(Sigma-Aldrich, F5881) 1:1 volume ratio mixed immunity Upeneus bensari is for the first time；
C.4 after week, using 50 micrograms antigen HSA and non-fully complete Freund's adjuvant (Sigma-Aldrich, F5606) 1:1
Volume ratio mixed immunity Upeneus bensari second；
D.4 Upeneus bensari third time is immunized using the 50 direct tail vein injections of micrograms antigen HSA after week；
E.4 Upeneus bensari is immunized the 4th time using the 50 direct tail vein injections of micrograms antigen HSA after week；
F.4 Upeneus bensari is immunized the 5th time using the 50 direct tail vein injections of micrograms antigen HSA after week；
2. structure Upeneus bensari source phage antibody library and the naughty sieve for carrying out specific antigen HSA single domain antibodies.
A. the peripheral blood and spleen for the Upeneus bensari being immunized using above-mentioned HSA antigens is starting materials, using TRIZOL
(Thermo, 15596018) method extraction RNA diseases are cDNA using Reverse Transcriptase kit (TAKARA, 6210B) reverse transcription.
Remove genome reaction
42 DEG C of reaction 2min.
Obtain the RNA of removal genome.
Reverse transcription reaction
37 DEG C, 15min；85 DEG C, 5sec.
Obtain cDNA.
B. the clone of target gene.
Primer is designed according to Upeneus bensari vNAR conserved sequences, primer both ends carry and clone into pComb3XSS carriers
SfiI restriction enzyme sites, the amplification of PCR methods obtain vNAR areas encoding gene.
Build library sense primer (SEQ ID NO:18)：
TCGCTACCGTGGCCCAGGCGGCCCAACGGGTTGAACAAACACC wherein underscores are SfiI restriction enzyme site sequences
Row.
Build library downstream primer (SEQ ID NO:19)：
TGATGGTGCTGGCCGGCCTGGCCTTTCACAGTCAGAATGGTGC wherein underscores are SfiI restriction enzyme site sequences
Row.
PCR reaction systems (TAKARA,HS,R040Q)：
Reaction condition：
As a result it is molecule Marker to see Fig. 4, wherein swimming lane M, and swimming lane 2 and 3 is Upeneus bensari vNAR areas PCR product.From
It can be seen from the figure that, Upeneus bensari vNAR areas PCR product size are 300bp or so, and size is correct.
C. library construction.The a full set of single domain antibody encoding gene segment and pComb3XSS carriers that are cloned into are used into SfiI
VNAR areas genetic fragment is connected to pComb3XSS carriers with T4 ligases after digestion is complete, makes vNAR areas gene and phagocytosis by digestion
External glutelin pIII amalgamation and expressions.And pass through electricity and turn TG1 Escherichia coli and build library.
PComb3XSS endonuclease reaction systems：
50 DEG C of digestions are stayed overnight.
VNAR areas PCR product endonuclease reaction system：
50 DEG C of digestions are stayed overnight.
T4 ligase coupled reaction systems：
16 DEG C of connections are overnight.
D. sieve is washed in a pan in library.The characteristic that can be expanded again using bacteriophage, by HSA by biotin labeling and with
Streptactin beads, which are combined, to be fixed to above pearl, by the affine absorption-elution-amplification of three-wheel, is washed in a pan and is sieved to energy and antigen
The antibody and its gene of specific binding.
Embodiment 2ELISA detects the combination for screening vNAR single domain antibodies and antigen HSA
1. coating：Antigen protein HSA is diluted to content as 1 μ g/ml.Add in the reacting hole of each polystyrene board
Antigen protein after 0.1ml dilutions, 4 DEG C overnight.Next day discards solution in hole, is washed 3 times, every time 3 minutes with washing buffer.
2. the infection of bacteriophage：Its OD600 is made to grow to 0.5 TG1 (Beijing Hua Yue ocean biology) amplification, add in what is screened
1 gained of bacteriophage, that is, embodiment, and filter out monoclonal in amp resistant panels.
3. infection amplification：Picking monoclonal makes its OD600 grow to 0.5, adds in helper phage M13KO7 and (is purchased from
NEB companies), 37 DEG C of incubation 30-60min.4 DEG C of centrifugation 10min of 3200g.Precipitation is collected, (is contained with 500ml 2 × YT culture mediums
0.1%glucose, 100 μ g/ml ampicillin, 50 μ g/ml kanamycin) 25 DEG C, 250rpm cultures 20h.3200g 4
DEG C centrifugation 10min, collect supernatant.2 × YT culture medium prescriptions (1L):Peptone 16g, yeast extract 10g, sodium chloride
4ml, pH are to 7.0.
4. sample-adding：Add the bacteriophage supernatant that step 3 is collected into the above-mentioned reacting hole being coated with, it is small to put 37 DEG C of incubations 1
When.It is washed out.(while doing blank control wells, blank control wells are other than being not added with antigen, other all sames).
5. enzyme labeling antibody：In each reacting hole, His enzyme labelled antibodies (the Santa Cruz sc- of diluted fresh are added in
8036HRP, 1:2500 dilutions) 0.1ml.37 DEG C are incubated 0.5~1 hour, washing.
6. add substrate solution colour developing：Add in the tmb substrate solution 0.1ml of Extemporaneous in each reacting hole, 37 DEG C 10~30
Minute.Tmb substrate solution formula：TMB (10mg/5ml absolute ethyl alcohols) 0.5ml；Substrate buffer solution 10ml；0.75% hydrogen peroxide
32ul；Wherein 50ml substrate buffer solutions:0.2M disodium hydrogen phosphates 25.7ml；0.1M citric acid 24.3ml, distilled water.
7. terminate reaction：2M sulfuric acid 0.05ml is added in each reacting hole.
8. result judgement：On microplate reader instrument, each hole OD values are surveyed at 450nm, as a result see Fig. 5.Wherein control is blank
Control is not plus HSA antigens, 2C1-2C11 are the different clones of screening.From fig. 5, it can be seen that the ELISA OD of No. 2C5 clone
Reading is 1.0 or so, and compares only 0.1 or so, and No. 2C5 clone can be very good to be combined with HSA.
Striped spot is obtained 9. choosing positive colony 2C5 and (Xiamen Bo Rui Bioisystech Co., Ltd is sequenced) being sequenced
The anti-HSA single domain antibodies sequence in bamboo shark source, such as SEQ ID NO:Shown in 20.
SEQ ID NO:20：Wherein lower stroke of single line is CDR1 areas, and lower stroke of two-wire is CDR3 areas.
QRVEQTPTTTTKEAGESLTINCVLKGSSCPMSNTYWYFTKKGATKKASLSTGG
RYSDTKNTASKSFSLRISDLRVEDSGTYHCEAGGGTILTVK
ELISA results show that No. 2C5 clone can be very good to be combined with HSA, it was demonstrated that can be with by the way that Upeneus bensari is immunized
Obtain the single domain antibody in Upeneus bensari source, sequencing result is also shown, the single domain antibody skeleton of acquisition and be sequenced 15
A clone (SEQ ID NO:Skeleton 3-17) is consistent, and sequence, which forms, is followed successively by FR1, CDR1, FR2, CDR3 and FR3 areas,
FR1, FR2, FR3 district's groups into stabilization amino acid sequence, complementary antibody determines that area is CDR1 and CDR3 areas, and the length of CDR1 is
8 amino acid, wherein second are cysteine (Cys, C), and the 7th is threonine (Thr, T).The length of CDR3 is 16
Amino acid.
The screening of 3. Upeneus bensari source anti-tnf-alpha single domain antibody of embodiment
1. Upeneus bensari is immune.
A. Upeneus bensari is selected to carry out the screening of following Upeneus bensari source single domain antibody as immunization.
B. 50 micrograms antigen TNF-α (Sigma-Aldrich, T6674, below herewith) and complete Freund's adjuvant are used
(Sigma-Aldrich, F5881) 1:1 volume ratio mixed immunity Upeneus bensari is for the first time；
C.4 after week, using 50 micrograms antigen TNF-α and non-fully complete Freund's adjuvant (Sigma-Aldrich, F5606)
1:Second of 1 volume ratio mixed immunity Upeneus bensari；
D.4 Upeneus bensari third time is immunized using the 50 direct tail vein injections of micrograms antigen TNF-α after week；
E.4 Upeneus bensari is immunized the 4th time using the 50 direct tail vein injections of micrograms antigen TNF-α after week；
F.4 Upeneus bensari is immunized the 5th time using the 50 direct tail vein injections of micrograms antigen TNF-α after week.
2. structure Upeneus bensari source phage antibody library and the naughty sieve for carrying out specific antigen TNF-α single domain antibody.
A. the peripheral blood and spleen for the Upeneus bensari being immunized using TNF-α antigen is starting materials, using TRIZOL
(Thermo, 15596018) method extraction RNA diseases are cDNA using Reverse Transcriptase kit (TAKARA, 6210B) reverse transcription.
Remove genome reaction
42 DEG C of reaction 2min.
Obtain the RNA of removal genome.
Reverse transcription reaction
37 DEG C, 15min；85 DEG C, 5sec.
Obtain cDNA.
B. the clone of target gene.
Primer is designed according to Upeneus bensari vNAR conserved sequences, primer both ends carry and clone into pComb3XSS carriers
SfiI restriction enzyme sites, the amplification of PCR methods obtain vNAR areas encoding gene.
Build library sense primer (SEQ ID NO:18)：
TCGCTACCGTGGCCCAGGCGGCCCAACGGGTTGAACAAACACC wherein underscores are SfiI restriction enzyme site sequences
Row.
Build library downstream primer (SEQ ID NO:19)：
TGATGGTGCTGGCCGGCCTGGCCTTTCACAGTCAGAATGGTGC wherein underscores are SfiI restriction enzyme site sequences
Row.
PCR reaction systems (TAKARA,HS,R040Q)：
Reaction condition：
The result shows that Upeneus bensari vNAR areas PCR product size is 300bp or so, size is correct.
C. library construction.The a full set of single domain antibody encoding gene segment and pComb3XSS carriers that are cloned into are used into SfiI
VNAR areas genetic fragment is connected to pComb3XSS carriers by digestion after digestion is complete with T4 ligases, makes vNAR areas gene with biting
Phage coat protein pIII amalgamation and expressions.And pass through electricity and turn TG1 Escherichia coli and build library.
PComb3XSS endonuclease reaction systems：
50 DEG C of digestions are stayed overnight.
VNAR areas PCR product endonuclease reaction system：
50 DEG C of digestions are stayed overnight.
T4 ligase coupled reaction systems：
16 DEG C of connections are overnight.
D. sieve is washed in a pan in library.The characteristic that can be expanded again using bacteriophage, by TNF-α by biotin labeling with
Streptactin beads, which are combined, to be fixed to above pearl, by the affine absorption-elution-amplification of three-wheel, is washed in a pan and is sieved to energy and antigen
The antibody and its gene of specific binding.
Embodiment 4.ELISA detects the combination for screening vNAR single domain antibodies and antigen TNF-α
1. coating：Antigen protein TNF-α is diluted to content as 5 μ g/ml.Add in the reacting hole of each polystyrene board
0.1ml, 4 DEG C overnight.Next day discards solution in hole, is washed 3 times, every time 3 minutes with washing buffer.
2. the infection of bacteriophage：Its OD600 is made to grow to 0.5 TG1 (Beijing Hua Yue ocean biology) amplification, add in what is screened
3 gained of bacteriophage, that is, embodiment, and filter out monoclonal in amp resistant panels.
3. infection amplification：Picking monoclonal makes its OD600 grow to 0.5, adds in helper phage M13KO7 and (is purchased from
NEB companies), 37 DEG C of incubation 30-60min.4 DEG C of centrifugation 10min of 3200g.Precipitation is collected, (is contained with 500ml 2x YT culture mediums
0.1%glucose, 100 μ g/ml ampicillin, 50 μ g/ml kanamycin) 25 DEG C, 250rpm cultures 20h.3200g 4
DEG C centrifugation 10min, collect supernatant.
4. sample-adding：Add the bacteriophage that step 3 is collected into the above-mentioned reacting hole being coated with, put 37 DEG C and be incubated 1 hour.So
After wash.(while doing blank control wells, blank control wells are other than being not added with antigen, other all sames).
5. enzyme labeling antibody：In each reacting hole, His enzyme labelled antibodies 0.1ml (the Santa Cruz of diluted fresh are added in
Sc-8036HRP, 1:2500 dilutions).37 DEG C are incubated 0.5~1 hour, washing.
6. add substrate solution colour developing：Add in the tmb substrate solution 0.1ml of Extemporaneous in each reacting hole, 37 DEG C 10~30
Minute.
7. terminate reaction：2M sulfuric acid 0.05ml is added in each reacting hole.
8. result judgement：In microplate reader, each hole OD values are surveyed at 450nm, as a result see Fig. 7.Wherein control is is not added with resisting
Former TNF-α, 3D1-3D11 are the different clones of screening.From figure 7 it can be seen that the ELISA OD readings of No. 3D2 clone are 1.1
Left and right, and only 0.1 or so is compareed, No. 3D2 clone can be very good to be combined with TNF-α.
9. it chooses positive colony 3D2 to be sequenced to obtain Upeneus bensari source anti-tnf-alpha single domain antibody sequence, such as SEQ
ID NO:Shown in 21.
SEQ ID NO:21：Wherein lower stroke of single line is CDR1 areas, and lower stroke of two-wire is CDR3 areas.
QRVEQTPTTTTKEAGESLTINCVLKGSSCALGSTYWYFTKKGATKKASLSTGG
RYSDTKNTASKSFSLRISDLRVEDSGTYHCEAGGGTILTVK
ELISA results show that No. 3D2 clone can be very good to be combined with TNF-α, it was demonstrated that can by immune Upeneus bensari
To obtain the single domain antibody in Upeneus bensari source, sequencing result is also shown, and the single domain antibody skeleton of acquisition and has been sequenced
15 clone (SEQ ID NO:Skeleton 3-17) is consistent, and sequence, which forms, is followed successively by FR1, CDR1, FR2, CDR3 and FR3 areas,
FR1, FR2, FR3 district's groups into stabilization amino acid sequence, complementary antibody determines that area is CDR1 and CDR3 areas, and the length of CDR1 is
8 amino acid, wherein second are cysteine (Cys, C), and the 7th is threonine (Thr, T).The length of CDR3 is 12
Amino acid.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is impossible to limitation of the present invention is interpreted as, those of ordinary skill in the art are not departing from the principle of the present invention and objective
In the case of can make changes, modifications, substitutions and variations to the above described embodiments within the scope of the invention.
SEQUENCE LISTING
<110>State Oceanic Administration Bureau The Third Oceanography Institute
<120>A kind of single domain antibody peptide backbone and preparation method thereof
<130> HYSS-16001-CNI
<160> 49
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213>It is artificial synthesized
<400> 1
caacgggttg aacaaacacc 20
<210> 2
<211> 20
<212> DNA
<213>It is artificial synthesized
<400> 2
tttcacagtc agaatggtgc 20
<210> 3
<211> 110
<212> PRT
<213>Upeneus bensari
<400> 3
Gln Arg Val Glu Gln Thr Pro Thr Thr Thr Thr Lys Glu Ala Gly Glu
Ser Leu Thr Ile Asn Cys Val Leu Lys Gly Ser Ser Cys Ala Leu Gly
Ser Thr Tyr Trp Tyr Phe Thr Lys Lys Gly Ala Thr Lys Lys Ala Ser
Leu Ser Thr Gly Gly Arg Tyr Ser Asp Thr Lys Asn Thr Ala Ser Lys
Ser Phe Ser Leu Arg Ile Ser Asp Leu Arg Val Glu Asp Ser Gly Thr
Tyr His Cys Glu Ala Cys Leu Gly Gly Pro Tyr Cys Asp Thr Leu Glu
Tyr Asp Tyr Tyr Glu Gly Gly Gly Thr Ile Leu Thr Val Lys
<210> 4
<211> 108
<212> PRT
<213>Upeneus bensari
<400> 4
Gln Arg Val Glu Gln Thr Pro Thr Thr Thr Thr Lys Glu Ala Gly Glu
Ser Leu Thr Ile Asn Cys Val Leu Lys Gly Ser Ser Cys Ala Leu Gly
Ser Thr Tyr Trp Tyr Phe Thr Lys Lys Gly Ala Thr Lys Lys Ala Ser
Leu Ser Thr Gly Gly Arg Tyr Ser Asp Thr Lys Asn Thr Ala Ser Lys
Ser Phe Ser Leu Arg Ile Ser Asp Leu Arg Val Glu Asp Ser Gly Thr
Tyr His Cys Glu Ala Ser Thr Val Gly Asp Cys Thr Ala Glu Tyr Asp
Tyr Tyr Glu Gly Gly Gly Thr Ile Leu Thr Val Lys
<210> 5
<211> 108
<212> PRT
<213>Upeneus bensari
<400> 5
Gln Arg Val Glu Gln Thr Pro Thr Thr Thr Thr Lys Glu Ala Gly Glu
Ser Leu Thr Ile Asn Cys Val Leu Lys Gly Ser Thr Cys Ala Leu Gly
Ser Thr Tyr Trp Tyr Phe Thr Lys Lys Gly Ala Thr Lys Lys Ala Ser
Leu Ser Thr Gly Gly Arg Tyr Ser Asp Thr Lys Asn Thr Ala Ser Lys
Ser Phe Ser Leu Arg Ile Ser Asp Leu Arg Val Glu Asp Ser Gly Thr
Tyr His Cys Glu Ala Tyr Ser Pro Val Gly Trp Asn Cys Ala Tyr Asn
Tyr Ile Glu Gly Gly Gly Thr Ile Leu Thr Val Lys
<210> 6
<211> 112
<212> PRT
<213>Upeneus bensari
<400> 6
Gln Arg Val Glu Gln Thr Pro Thr Thr Thr Thr Lys Glu Ala Gly Glu
Ser Leu Thr Ile Asn Cys Val Leu Lys Gly Ser Ser Cys Ala Leu Ser
Ser Thr Tyr Trp Tyr Phe Thr Lys Lys Gly Ala Thr Lys Lys Ala Ser
Leu Ser Thr Gly Gly Arg Tyr Ser Asp Thr Lys Asn Thr Ala Ser Lys
Ser Phe Ser Leu Arg Ile Ser Asp Leu Arg Val Glu Asp Ser Gly Thr
Tyr His Cys Glu Ala Tyr Lys Thr Ala Gly Met Ser Tyr Cys Glu Leu
Glu Leu Asp Ser Tyr Ile Glu Gly Gly Gly Thr Ile Leu Thr Val Lys
<210> 7
<211> 112
<212> PRT
<213>Upeneus bensari
<400> 7
Gln Arg Val Glu Gln Thr Pro Thr Thr Thr Thr Lys Glu Ala Gly Glu
Ser Leu Thr Ile Asn Cys Val Leu Lys Gly Ser Ser Cys Ala Leu Gly
Ser Thr Tyr Trp Tyr Phe Thr Lys Lys Gly Ala Thr Lys Lys Ala Ser
Leu Ser Thr Gly Gly Arg Tyr Ser Asp Thr Lys Asn Thr Ala Ser Lys
Ser Phe Ser Leu Arg Ile Ser Asp Leu Arg Val Glu Asp Ser Gly Thr
Tyr His Cys Glu Ala Tyr Asn Gln Ala Gly Arg Cys Asp Thr Ala Ser
Arg Trp Ile Gly Ser His Glu Gly Gly Gly Thr Ile Leu Thr Val Lys
<210> 8
<211> 111
<212> PRT
<213>Upeneus bensari
<400> 8
Gln Arg Val Glu Gln Thr Pro Thr Thr Thr Thr Lys Glu Ala Gly Glu
Ser Leu Thr Ile Asn Cys Val Leu Lys Gly Ser Ser Cys Ala Leu Gly
Ser Thr Tyr Trp Tyr Phe Thr Lys Lys Gly Ala Thr Lys Lys Ala Ser
Leu Ser Thr Gly Gly Arg Tyr Ser Asp Thr Lys Asn Thr Ala Ser Lys
Ser Phe Ser Leu Arg Ile Ser Asp Leu Arg Val Glu Asp Ser Gly Thr
Tyr His Cys Glu Ala Tyr Ser Tyr Ser Trp Asp Asp Val Pro Val Ile
Arg Thr Ile Ala Val Glu Gly Gly Gly Thr Ile Leu Thr Val Lys
<210> 9
<211> 105
<212> PRT
<213>Upeneus bensari
<400> 9
Gln Arg Val Glu Gln Thr Pro Thr Thr Thr Thr Lys Glu Ala Gly Glu
Ser Leu Thr Ile Asn Cys Val Leu Lys Gly Ser Ser Cys Ala Leu Gly
Ser Thr Tyr Trp Tyr Phe Thr Lys Lys Gly Ala Thr Lys Lys Ala Ser
Leu Ser Thr Gly Gly Arg Tyr Ser Asp Thr Lys Asn Thr Ala Ser Lys
Ser Phe Ser Leu Arg Ile Ser Asp Leu Arg Val Glu Asp Ser Gly Thr
Tyr His Cys Glu Ala Tyr Pro Cys Tyr Ser Arg Thr His Asn Val Lys
Gly Gly Gly Thr Ile Leu Thr Val Lys
<210> 10
<211> 105
<212> PRT
<213>Upeneus bensari
<400> 10
Gln Arg Val Glu Gln Thr Pro Thr Thr Thr Thr Lys Glu Ala Gly Glu
Ser Leu Thr Ile Asn Cys Val Leu Lys Gly Ser Ser Cys Ala Leu Gly
Ser Thr Tyr Trp Tyr Phe Thr Lys Lys Gly Ala Thr Lys Lys Ala Ser
Leu Ser Thr Gly Gly Arg Tyr Ser Asp Thr Lys Asn Thr Ala Ser Lys
Ser Phe Ser Leu Arg Ile Ser Asp Leu Arg Val Glu Asp Ser Gly Thr
Tyr His Cys Glu Ala Tyr Ser Tyr Cys Leu Gly Gln Pro Pro Ile Glu
Gly Gly Gly Thr Ile Leu Thr Val Lys
<210> 11
<211> 111
<212> PRT
<213>Upeneus bensari
<400> 11
Gln Arg Val Glu Gln Thr Pro Thr Thr Thr Thr Lys Glu Ala Gly Glu
Ser Leu Thr Ile Asn Cys Val Leu Lys Gly Ser Ser Cys Ala Leu Ser
Ser Thr Tyr Trp Tyr Phe Thr Lys Lys Gly Ala Thr Lys Lys Ala Ser
Leu Ser Thr Gly Gly Arg Tyr Ser Asp Thr Lys Asn Thr Ala Ser Lys
Ser Phe Ser Leu Arg Ile Ser Asp Leu Arg Val Glu Asp Ser Gly Thr
Tyr His Cys Glu Ala Tyr Arg Asp Trp Asp Val Gly Asn Tyr Cys Tyr
Thr Asp Ser Tyr Ile Glu Gly Gly Gly Thr Ile Leu Thr Val Lys
<210> 12
<211> 105
<212> PRT
<213>Upeneus bensari
<400> 12
Gln Arg Val Glu Gln Thr Pro Thr Thr Thr Thr Lys Glu Ala Gly Glu
Ser Leu Thr Ile Asn Cys Val Leu Lys Gly Ser Arg Cys Ala Leu Phe
Asn Thr His Trp Tyr Phe Thr Lys Lys Gly Ala Thr Lys Lys Ala Ser
Leu Ser Thr Gly Gly Arg Tyr Ser Asp Thr Lys Asn Thr Ala Ser Lys
Ser Phe Ser Leu Arg Ile Ser Asp Leu Arg Val Glu Asp Ser Gly Thr
Tyr His Cys Glu Ala Tyr Arg Val Asp Cys Ser Pro His Tyr Ile Glu
Gly Gly Gly Thr Ile Leu Thr Val Lys
<210> 13
<211> 109
<212> PRT
<213>Upeneus bensari
<400> 13
Gln Arg Val Glu Gln Thr Pro Thr Thr Thr Thr Lys Glu Ala Gly Glu
Ser Leu Thr Ile Asn Cys Val Leu Lys Gly Ser Ser Cys Ala Leu Asp
Ser Thr Tyr Trp Tyr Phe Thr Lys Lys Gly Ala Thr Lys Lys Ala Ser
Leu Ser Thr Gly Gly Arg Tyr Ser Asp Thr Lys Asn Thr Ala Ser Lys
Ser Phe Ser Leu Arg Ile Ser Asp Leu Arg Val Glu Asp Ser Gly Thr
Tyr His Cys Glu Ala Tyr Gly Cys Ser Thr Val Ile Trp Gly Ser Thr
Pro Tyr Tyr Glu Gly Gly Gly Thr Ile Leu Thr Val Lys
<210> 14
<211> 109
<212> PRT
<213>Upeneus bensari
<400> 14
Gln Arg Val Glu Gln Thr Pro Thr Thr Thr Thr Lys Glu Ala Gly Glu
Ser Leu Thr Ile Asn Cys Val Leu Lys Gly Ser Ser Cys Ala Leu Asp
Ser Thr Tyr Trp Tyr Phe Thr Lys Lys Gly Ala Thr Lys Lys Ala Ser
Leu Ser Thr Gly Gly Arg Tyr Ser Asp Thr Lys Asn Thr Ala Ser Lys
Ser Phe Ser Leu Arg Ile Ser Asp Leu Arg Val Glu Asp Ser Gly Thr
Tyr His Cys Lys Ala Tyr Thr Ala Gly Arg Gly Tyr Cys Ser Ser Trp
Asp Tyr Tyr Glu Gly Gly Gly Thr Ile Leu Thr Val Lys
<210> 15
<211> 103
<212> PRT
<213>Upeneus bensari
<400> 15
Gln Arg Val Glu Gln Thr Pro Thr Thr Thr Thr Lys Glu Ala Gly Glu
Ser Leu Thr Ile Asn Cys Val Leu Lys Gly Ser Ser Cys Ala Leu Gly
Ser Thr Tyr Trp Tyr Phe Thr Lys Lys Gly Ala Thr Lys Lys Ala Ser
Leu Ser Thr Gly Gly Arg Tyr Ser Asp Thr Lys Asn Thr Ala Ser Lys
Ser Phe Ser Leu Arg Ile Ser Asp Leu Arg Val Glu Asp Ser Gly Thr
Tyr His Cys Glu Ala Tyr Cys Trp Gly Ser Ser Tyr Tyr Glu Gly Gly
Gly Thr Ile Leu Thr Val Lys
<210> 16
<211> 114
<212> PRT
<213>Upeneus bensari
<400> 16
Gln Arg Val Glu Gln Thr Pro Thr Thr Thr Thr Lys Glu Ala Gly Glu
Ser Leu Thr Ile Asn Cys Val Leu Lys Gly Ser Asp Cys Ala Leu Gly
Arg Thr Tyr Trp Tyr Phe Thr Lys Lys Gly Ala Thr Lys Lys Ala Ser
Leu Ser Thr Gly Gly Arg Tyr Ser Asp Thr Lys Asn Thr Ala Ser Lys
Ser Phe Ser Leu Arg Ile Ser Asp Leu Arg Val Glu Asp Ser Gly Thr
Tyr His Cys Glu Ala Tyr Pro Arg Pro Gly Ile Gly Val Phe Cys Ala
Leu Thr Ser Gly Ile Ser Asn Tyr Glu Gly Gly Gly Thr Ile Leu Thr
Val Lys
<210> 17
<211> 109
<212> PRT
<213>Upeneus bensari
<400> 17
Gln Arg Val Glu Gln Thr Pro Thr Thr Thr Thr Lys Glu Ala Gly Glu
Ser Leu Thr Ile Asn Cys Val Leu Lys Gly Ser Ser Cys Pro Met Gly
Ser Thr Tyr Trp Tyr Phe Thr Lys Lys Gly Ala Thr Lys Lys Ala Ser
Leu Ser Thr Gly Gly Arg Tyr Ser Asp Thr Lys Asn Thr Ala Ser Lys
Ser Phe Ser Leu Arg Ile Ser Asp Leu Arg Val Glu Asp Ser Gly Thr
Tyr His Cys Glu Ala Leu Gln Leu Gly Cys Ile Gly Ala Ala Gly Ile
Gly Arg Tyr Glu Gly Gly Gly Thr Ile Leu Thr Val Lys
<210> 18
<211> 43
<212> DNA
<213>It is artificial synthesized
<400> 18
tcgctaccgt ggcccaggcg gcccaacggg ttgaacaaac acc 43
<210> 19
<211> 43
<212> DNA
<213>It is artificial synthesized
<400> 19
tgatggtgct ggccggcctg gcctttcaca gtcagaatgg tgc 43
<210> 20
<211> 115
<212> PRT
<213>It is artificial synthesized
<400> 20
Gln Arg Val Glu Gln Thr Pro Thr Thr Thr Thr Lys Glu Ala Gly Glu
Ser Leu Thr Ile Asn Cys Val Leu Lys Gly Ser Ser Cys Pro Met Ser
Asn Thr Tyr Trp Tyr Phe Thr Lys Lys Gly Ala Thr Lys Lys Glu Ser
Leu Thr Asn Gly Gly Arg Tyr Ser Val Thr Met Asn Lys Ala Ser Lys
Ser Phe Ser Leu Arg Ile Ser Asp Leu Arg Val Glu Asp Ser Gly Thr
Tyr His Cys Glu Ala Tyr Ser Ser Ser Thr Ala Gly Ser Tyr Cys Tyr
Glu Ala Gly Ile Pro His Asn Tyr Ile Glu Gly Gly Gly Thr Ile Leu
Thr Val Lys
115
<210> 21
<211> 106
<212> PRT
<213>It is artificial synthesized
<400> 21
Gln Arg Val Glu Gln Thr Pro Thr Thr Thr Thr Lys Glu Ala Gly Glu
Ser Leu Thr Ile Asn Cys Val Leu Lys Gly Ser Ser Cys Ala Leu Gly
Ser Thr Tyr Trp Tyr Phe Thr Lys Lys Gly Ala Thr Lys Lys Ala Ser
Leu Ser Thr Gly Gly Arg Tyr Ser Asp Thr Lys Asn Thr Ala Ser Lys
Ser Phe Ser Leu Arg Ile Ser Asp Leu Arg Val Glu Asp Ser Gly Thr
Tyr His Cys Glu Ala Cys Lys Ala Gly Ala Thr Ser Gly Tyr Thr Pro
Glu Gly Gly Gly Thr Ile Leu Thr Val Lys
<210> 22
<211> 27
<212> PRT
<213>It is artificial synthesized
<400> 22
Gln Arg Val Glu Gln Thr Pro Thr Thr Thr Thr Lys Glu Ala Gly Glu
Ser Leu Thr Ile Asn Cys Val Leu Lys Gly Ser
<210> 23
<211> 50
<212> PRT
<213>It is artificial synthesized
<400> 23
Trp Tyr Phe Thr Lys Lys Gly Ala Thr Lys Lys Ala Ser Leu Ser Thr
Gly Gly Arg Tyr Ser Asp Thr Lys Asn Thr Ala Ser Lys Ser Phe Ser
Leu Arg Ile Ser Asp Leu Arg Val Glu Asp Ser Gly Thr Tyr His Cys
Glu Ala
<210> 24
<211> 9
<212> PRT
<213>It is artificial synthesized
<400> 24
Gly Gly Gly Thr Ile Leu Thr Val Lys
<210> 25
<211> 8
<212> PRT
<213>Upeneus bensari
<400> 25
Ser Cys Ala Leu Gly Ser Thr Tyr
<210> 26
<211> 8
<212> PRT
<213>Upeneus bensari
<400> 26
Thr Cys Ala Leu Gly Ser Thr Tyr
<210> 27
<211> 8
<212> PRT
<213>Upeneus bensari
<400> 27
Ser Cys Ala Leu Ser Ser Thr Tyr
<210> 28
<211> 8
<212> PRT
<213>Upeneus bensari
<400> 28
Arg Cys Ala Leu Phe Asn Thr His
<210> 29
<211> 8
<212> PRT
<213>Upeneus bensari
<400> 29
Ser Cys Ala Leu Asp Ser Thr Tyr
<210> 30
<211> 8
<212> PRT
<213>Upeneus bensari
<400> 30
Asp Cys Ala Leu Gly Arg Thr Tyr
<210> 31
<211> 8
<212> PRT
<213>Upeneus bensari
<400> 31
Ser Cys Pro Met Gly Ser Thr Tyr
<210> 32
<211> 16
<212> PRT
<213>Upeneus bensari
<400> 32
Cys Leu Gly Gly Pro Tyr Cys Asp Thr Leu Glu Tyr Asp Tyr Tyr Glu
<210> 33
<211> 14
<212> PRT
<213>Upeneus bensari
<400> 33
Ser Thr Val Gly Asp Cys Thr Ala Glu Tyr Asp Tyr Tyr Glu
<210> 34
<211> 14
<212> PRT
<213>Upeneus bensari
<400> 34
Tyr Ser Pro Val Gly Trp Asn Cys Ala Tyr Asn Tyr Ile Glu
<210> 35
<211> 18
<212> PRT
<213>Upeneus bensari
<400> 35
Tyr Lys Thr Ala Gly Met Ser Tyr Cys Glu Leu Glu Leu Asp Ser Tyr
Ile Glu
<210> 36
<211> 18
<212> PRT
<213>Upeneus bensari
<400> 36
Tyr Asn Gln Ala Gly Arg Cys Asp Thr Ala Ser Arg Trp Ile Gly Ser
His Glu
<210> 37
<211> 17
<212> PRT
<213>Upeneus bensari
<400> 37
Tyr Ser Tyr Ser Trp Asp Asp Val Pro Val Ile Arg Thr Ile Ala Val
Glu
<210> 38
<211> 11
<212> PRT
<213>Upeneus bensari
<400> 38
Tyr Pro Cys Tyr Ser Arg Thr His Asn Val Lys
<210> 39
<211> 11
<212> PRT
<213>Upeneus bensari
<400> 39
Tyr Ser Tyr Cys Leu Gly Gln Pro Pro Ile Glu
<210> 40
<211> 17
<212> PRT
<213>Upeneus bensari
<400> 40
Tyr Arg Asp Trp Asp Val Gly Asn Tyr Cys Tyr Thr Asp Ser Tyr Ile
Glu
<210> 41
<211> 11
<212> PRT
<213>Upeneus bensari
<400> 41
Tyr Arg Val Asp Cys Ser Pro His Tyr Ile Glu
<210> 42
<211> 15
<212> PRT
<213>Upeneus bensari
<400> 42
Tyr Gly Cys Ser Thr Val Ile Trp Gly Ser Thr Pro Tyr Tyr Glu
<210> 43
<211> 15
<212> PRT
<213>Upeneus bensari
<400> 43
Tyr Thr Ala Gly Arg Gly Tyr Cys Ser Ser Trp Asp Tyr Tyr Glu
<210> 44
<211> 9
<212> PRT
<213>Upeneus bensari
<400> 44
Tyr Cys Trp Gly Ser Ser Tyr Tyr Glu
<210> 45
<211> 20
<212> PRT
<213>Upeneus bensari
<400> 45
Tyr Pro Arg Pro Gly Ile Gly Val Phe Cys Ala Leu Thr Ser Gly Ile
Ser Asn Tyr Glu
<210> 46
<211> 15
<212> PRT
<213>Upeneus bensari
<400> 46
Leu Gln Leu Gly Cys Ile Gly Ala Ala Gly Ile Gly Arg Tyr Glu
<210> 47
<211> 8
<212> PRT
<213>Upeneus bensari
<400> 47
Ser Cys Pro Met Ser Asn Thr Tyr
<210> 48
<211> 21
<212> PRT
<213>Upeneus bensari
<400> 48
Tyr Ser Ser Ser Thr Ala Gly Ser Tyr Cys Tyr Glu Ala Gly Ile Pro
His Asn Tyr Ile Glu
<210> 49
<211> 12
<212> PRT
<213>Upeneus bensari
<400> 49
Cys Lys Ala Gly Ala Thr Ser Gly Tyr Thr Pro Glu
Claims (1)
1. a kind of single domain antibody peptide backbone, which is characterized in that its sequence composition is followed successively by FR1, CDR1, FR2, CDR3 and FR3
Area, wherein FR1, FR2, FR3 are that fixed amino acid sequence, CDR1 and CDR3 determine area for complementary antibody, and amino acid sequence is variable,
CDR1 and CDR3 is determined and different antigen bindings；The amino acid sequence in the FR1 areas such as SEQ ID NO:Shown in 22, FR2 areas
Amino acid sequence such as SEQ ID NO:Shown in 23, the amino acid sequence such as SEQ ID NO in FR3 areas:Shown in 24；The single domain
For antibody sources in Upeneus bensari, the amino acid sequence of the CDR1 is SEQ ID NO:25, the amino acid sequence of the CDR3
For SEQ ID NO：49；Or the amino acid sequence of the CDR1 is SEQ ID NO:47, the amino acid sequence of the CDR3 is
SEQ ID NO:48。
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| CN111197074B (en) * | 2018-11-20 | 2023-08-15 | 深圳华大生命科学研究院 | Stripe bamboo shark immunoglobulin new antigen receptor variable region library primer and application |
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