CN106928366A - A kind of FAP nano antibodies Nb67 - Google Patents
A kind of FAP nano antibodies Nb67 Download PDFInfo
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- CN106928366A CN106928366A CN201511021663.2A CN201511021663A CN106928366A CN 106928366 A CN106928366 A CN 106928366A CN 201511021663 A CN201511021663 A CN 201511021663A CN 106928366 A CN106928366 A CN 106928366A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
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- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/24—Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
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- 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®
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Abstract
The invention discloses a kind of FAP nano antibodies for being directed to FAP peptide molecule epitopes, while also disclosed the gene order for encoding the FAP nano antibodies and the expression vector and host cell of the FAP nano antibodies can be expressed, and disclose the purposes of the FAP nano antibodies.By FAP nano antibodies disclosed by the invention, gene order and host cell etc., high efficient expression can go out FAP nano antibodies in Escherichia coli, its, affinity good with FAP immune responses specificity is high, can be applied to prepare FAP detection reagents or antineoplastic etc..
Description
Technical field
The invention belongs to biomedical or biological pharmacy technical field, it is directed to more specifically to one kind
The nano antibody (Nb67) of FAP peptide molecule epitope molecules, its coded sequence and purposes.
Background technology
Fibroblast activation protein (fibroblast activation protein, FAP) is tumour related into fibre
Tie up a kind of surface antigen of cell specific expression, with dipeptidyl peptidase and collagenase activity, its genome
Stabilization, with growth of tumour cell is promoted, attacks and immunosuppressant effect.FAP is by 761 amino
Acid composition, the level of glycosylation in different mammalian cells is different, and monomer relative molecular mass has 8.8
×104、9.5×104、9.7×104Etc. various reports, dimer relative molecular mass is 1.7 × 105。FAP
Structure be divided into cytoplasmic domain, transmembrane region and the part of extracellular region three, wherein cytoplasmic domain is by starting 6 amino acid
The short peptide chain of composition, transmembrane region is made up of 19 amino acid, plays the hydrophobic transmembrane fragment of fixation,
But the overwhelming majority of amino acid sequence 20~761 is all exposed in extracellular microenvironment in FAP molecules,
This part is exactly extracellular region, is crucial enzymatic area.FAP have simultaneously dipeptidylpeptidase activity and
Collagenase activity, dipeptides and NTx that can be in matrix degradation.FAP with belong to dipeptidyl peptidase man
The DPPIV (dipeptidyl peptidase IV/CD26) of race has 50% homology, is respectively provided with peptide chain
5 prime excision enzyme activity, is commonly formed compound.Thus it is speculated that FAP can be by adjusting some peptides in tumor stroma
Class growth factor, modification activities peptide simultaneously changes its function, so as to promote tumour to develop.
In malignant tumour, FAP positive fibroblast only accounts for the 2% of tumour cell sum, but when it
After being removed, cancer cell and stroma cell can all occur rapid necrosis.It can thus be seen that FAP is to disliking
Property tumour occurrence and development there is very important effect, and then have in terms of oncotherapy it is wide should
Use prospect.Target tumor microenvironment, the therapeutic strategy for especially targetting FAP plays in oncotherapy
Very important role.During tumorigenesis, tumour cell does not escape siberian crabapple only passively
The attack of system, while also suppress the normal function of the immunocyte in its growing environment on one's own initiative, it and its institute
The microenvironment at place interacts, and forms inhibition tumor microenvironment and promotes the malignant progression of tumour, these because
Element is existed for inquiring into elaboration of tumour mechanism and opens up new immunotherapy of tumors scheme there is provided thinking.
Several FAP monoclonal antibody specifics have been developed at present, and starts in clinical diagnosis and treatment
In be applied, its affinity to tumor tissues is improved.But there is antibody in this conventional method
The shortcomings of stability is poor, sensitivity is low, production cost is high.
Nano antibody technology, be biomedical science man on the basis of conventional antibodies, with molecular biology
The antibody engineering revolution that the concept of technology combination nano-particle science is carried out, so as to the newest and minimum developed
Antibody molecule, initially found in camel blood by Belgian scientist Lai Mande Hamars.Common
Antibody protein is made up of two heavy chains and two light chains, and the novel antibodies found from camel blood only have two
Bar heavy chain, does not have light chain, and these " heavy chain antibodies " can tightly be tied as normal antibody with the target such as antigen
Close, but it is blocking that aggregation is mutually adhered unlike single-chain antibody.Nanometer based on " heavy chain antibody " resists
Not only molecular weight is the 1/10 of common antibody to body, and chemical property is also more flexible, and good stability can
Dissolubility is high, and expression is easy and is readily available, and is easily coupled other molecules.But, in the prior art not
Have and develop applicable nano antibody for FAP.
The content of the invention
The technical problem to be solved in the present invention is to be directed to receiving for FAP epitopes for lacking in the prior art
The defect of meter Kang Ti, there is provided a kind of FAP nano antibodies, while providing the DNA for encoding the FAP nano antibodies
Purposes of molecule and the nano antibody etc..
The technical solution adopted for the present invention to solve the technical problems is：The first aspect of the present invention, there is provided
A kind of FAP nano antibodies, the FAP nano antibodies are the nano antibody for FAP epitopes, including two
Such as SEQ ID NO：The VHH chains of amino acid sequence shown in 9.The FAP nano antibodies can be with the present invention
Referred to as Nb67.
A kind of second aspect present invention, there is provided VHH chains for FAP nano antibodies, including framework region
FR and complementary determining region CDR, the framework region FR include the amino acid sequence of the FR of the following group：SEQ ID
NO：FR1 shown in 1, SEQ ID NO：FR2 shown in 2, SEQ ID NO：FR3 shown in 3,
SEQ ID NO：FR4 shown in 4；The complementary determining region CDR includes the amino acid of the CDR of the following group
Sequence：SEQ ID NO：CDR1 shown in 5, SEQ ID NO：CDR2 shown in 6, SEQ ID NO：
CDR3 shown in 7.Framework plot structure is guarded relatively, mainly plays a part of Protein requirement structure；Mutually
Mend and determine that plot structure is relatively diversified, the identification of main responsible antibody.
Preferably, the VHH chains for FAP nano antibodies, it has SEQ ID NO：Shown in 9
Amino acid sequence.
A kind of third aspect present invention, there is provided DNA molecular, it is used to encode the protein being selected from the group：
FAP nano antibodies Nb67 of the present invention, or it is of the present invention for FAP nano antibodies
VHH chains.
Preferably, described DNA molecular, it has SEQ ID NO：Nucleotide sequence shown in 8.
The side that the FAP nano antibodies that the present invention is provided can be recombinantly expressed by Phage amplification or genetic engineering
Formula is largely prepared.Phage amplification refers to the bacteriophage that will show and have FAP nano antibodies Nb67, is led to
The mode of biological amplification is crossed, amount reproduction production displaying there are the bacteriophage particles of FAP nano antibodies Nb67.
The mode of genetic engineering recombination expression refers to the gene that will encode FAP nano antibodies Nb67, by being cloned into
Expression vector, carries out a large amount of preparations of nano antibody in the form of protein expression.
A kind of the fourth aspect of the present invention, there is provided expression vector, its NO of ID containing SEQ：Core shown in 8
Nucleotide sequence.
A kind of the fifth aspect of the present invention, there is provided host cell, it can express FAP of the invention and receive
Meter Kang Ti Nb67.
The sixth aspect of the present invention, there is provided FAP nano antibodies Nb67 of the present invention is preparing FAP
Purposes in terms of detection reagent.FAP nano antibodies Nb67 of the invention can substitute traditional FAP and resist
Body, prepares FAP detection reagents, for detecting FAP peptide molecules.Present invention also offers FAP nanometers
Purposes of the antibody Nb67 in terms of antineoplastic is prepared.The present invention is also accordingly detected there is provided a kind of FAP
Reagent or antineoplastic, including foregoing FAP nano antibodies Nb67.
The seventh aspect of the present invention, there is provided FAP nano antibodies Nb67 of the present invention is in non-treatment mesh
Immunology detection in purposes.The type of the immunology detection includes MBP enzyme linked immuno-adsorbent assay, collaurum
The immune analysis detection types based on antigen and antibody specific reaction such as immunochromatography, immunodotting hybridization.
FAP nano antibodies Nb67 of the present invention is in use, the displaying that can be obtained by Phage amplification has FAP
The bacteriophage particles of nano antibody Nb67 are directly used in analysis detection and pass through FAP nano antibodies Nb67
Immunology detection analysis is carried out in the form of albumen after crossing prokaryotes or eucaryote expression.
The eighth aspect of the present invention, there is provided FAP nano antibodies Nb67 of the present invention is preparing combination
Application in absorption FAP reagents.For example, FAP nano antibodies of the present invention can be made immune affinity column,
Absorption crawl FAP, for further research etc. after enrichment.
Implement the present invention, have the advantages that：The present invention synthesizes FAP polypeptides first, and has it
There is immunogenicity, then by FAP molecule coupling labeleds on ELISA Plate, show the correct space structure of the albumen,
Antigen in this format screens immune nano Antibody geometric mean titer (camel heavy chain antibody using display technique of bacteriophage
Phage display gene pool), so as to obtain the specific nano antibody genes of FAP, this gene is gone to
In Escherichia coli, can be in the strain of the nano antibody of E. coli so as to establish；The inventive method
The FAP nano antibodies that obtain of screening have with FAP immune response characteristics, and specific good, affinity is high,
Can be applied to prepare FAP detection reagents or antineoplastic etc..
Brief description of the drawings
Below in conjunction with drawings and Examples, the invention will be further described, in accompanying drawing：
Fig. 1 is the amino acid number and structural representation of FAP nano antibodies；
Fig. 2 is the DNA electrophoretograms of FAP nano antibodies；
Fig. 3 is electricity of the FAP nano antibodies through the SDS-PAGE after nickel post resin gel affinitive layer purification
Swimming figure.
Specific embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, below in conjunction with accompanying drawing and reality
Example is applied, the present invention will be described in further detail.
The present invention utilizes display technique of bacteriophage, and energy and target are screened in the single domain heavy chain antibody storehouse being immunized from hunchbacked source
Single domain heavy chain antibody (VHH) phage clone that molecule FAP antigentic specificities are combined, and obtain energy
In the nano antibody strain of E. coli, i.e. FAP nano antibodies Nb67.
With reference to specific embodiment, the present invention is expanded on further.
Embodiment 1：It is directed to the structure in the nano antibody library of FAP：
(1) synthesize FAP polypeptides first, 1mg FAP are mixed in equal volume with Freund's adjuvant, be immunized one
Xinjiang one-humped camel, once in a week, is immunized 7 times altogether, stimulates B cell to express the nanometer of antigentic specificity
Antibody；
After (2) 7 immune end, extract 100mL camels PBLC and simultaneously extract total serum IgE；
(3) synthesize cDNA and expand VHH using sleeve type PCR；
(4) restriction enzyme PstI and NotI digestion 20ug pComb3 Vector for Phage Display is utilized
(Biovector China plasmid vector strain cell gene collection is supplied) and 10ug VHH are simultaneously connected
Two fragments；(5) connection product is converted in turning competent cell TG1 to electricity, builds FAP nanometers and resist
Body library simultaneously determines storage capacity, and storage capacity size is 1.85 × 108。
Embodiment 2：For the nano antibody screening process of FAP：
(1) 100mM NaHCO are dissolved in3, the 20ug FAP in pH 8.2 be coupled at NUNC
On ELISA Plate, 4 DEG C stand overnight；
Add within (2) second days the caseins of 100uL 0.1%, room temperature closing 1-3h；
(3) after 1-3h, 100uL bacteriophages (5 × 10 are added11The immune camel nano antibody phagocytosis exhibitions of tfu
Show gene pool), room temperature effect 1-2h；
(4) washed 4-6 times with 0.05%PBS+Tween-20, to wash uncombined bacteriophage off；
(5) under the bacteriophage specifically bound with FAP is dissociated with 100mM TEA (triethylamine),
And the e. coli tg1 in logarithmic phase growth is infected, 37 DEG C of culture 1h, are produced and purified phage is used
In the screening of next round, identical screening process repeats 3-5 wheels, is progressively enriched with.
Embodiment 3：Specific single positive colony is screened with the enzyme-linked immunoassay method (ELISA) of bacteriophage：
(1) from the Tissue Culture Dish containing bacteriophage after above-mentioned 3-5 wheel screenings, 96 single bacterium are selected
Fall and be inoculated in TB culture mediums (1 liter of TB culture medium of the ampicillin containing 100 micrograms per millilitres
In contain 2.3 grams of potassium dihydrogen phosphates, 12.52 grams of dipotassium hydrogen phosphates, 12 grams of peptones, 24 grams of yeast are extracted
Thing, 4 milliliters of glycerine) in, after growing to logarithmic phase, plus 1 mM of final concentration isopropylthio gala
Glucosides (IPTG), 30-35 DEG C of overnight incubation.
(2) obtained using osmosis and slightly carry antibody, and antibody is transferred to through the elisa plate of antigen coat
In, place 1-1.5 hours at room temperature.
(3) uncombined antibody is washed away with PBST, adds anti-HA antibody (the mouse anti-HA of primary antibody mouse
Tag antibody, purchased from Beijing CoWin Bioscience Co., Ltd.), place 1-1.5 hours at room temperature.
(4) uncombined antibody is washed away with PBST, adds anti-mouse alkaline phosphatase
Conjugate (goat-anti-mouse alkaline phosphatase enzyme mark antibody, purchased from Amy victory Science and Technology Ltd.),
Place 1-1.5 hours at room temperature.
(5) uncombined antibody is washed away with PBST, alkaline phosphatase nitrite ion is added, in ELISA instrument
On, in 405nm wavelength, read absorption value.
(6) when sample well OD values are more than more than 3 times of control wells OD values, it is judged to positive colony hole.
(7) bacterium in positive colony hole is turned to shake in the LB liquid containing 100 micrograms per millilitres to carry
Take plasmid and be sequenced.
By above-mentioned experiment, the present invention has obtained the knot that 6 positive colony holes go out more than 3 times with antigen expression
With joint efforts, it is considered as primary dcreening operation positive clone strain.Each primary dcreening operation is analyzed according to sequence alignment program Vector NTI positive
The gene order of clone strain, CDR1, the strain of CDR2, CDR3 sequence identical is considered as same clone strain,
And the different strain of its sequence is considered as different clone strains, one group of amino acid sequence of the VHH chains of antibody can be obtained
Row such as SEQ ID NO：Shown in 9.The FAP nano antibodies are numbered as Nb67, its amino acid number and
Structural representation is as shown in Figure 1.
Fig. 2 is referred to, is the DNA electrophoretograms of FAP nano antibodies.Marked as first of M in figure
The DNA bands of gel pore are：The DNA molecular marker of 1000bp, behind from left to right marked as 1-24
The DNA bands of gel pore be FAP nano antibody DNA electrophoretic bands.These 1-24 gel pores
DNA is the PCR primer of FAP nano antibodies DNA in foregoing positive clone strain, the PCR primer
Band about 500bp.
Embodiment 4：Nano antibody is in Host Strains expression in escherichia coli, purifying：
(1) the amino acid sequence such as SEQ ID NO for obtaining above sequencing analysis：Clone strain shown in 9
Plasmid electricity be transformed into Escherichia coli WK6, and it is i.e. blue or green containing ammonia benzyl to be coated on LA+glucose
On the culture plate of mycin and glucose, 37 DEG C of overnight incubations；
(2) select single bacterium colony to be seeded in the LB nutrient solutions that 5mL contains ampicillin, 37 DEG C
Shaking table culture is overnight；
(3) in overnight strain to the 330mL TB nutrient solutions of inoculation 1mL, 37 DEG C of shaking table cultures, training
When supporting OD values and reaching 0.6~1, IPTG is added, 30-35 DEG C of shaking table culture is overnight；
(4) it is centrifuged, receives bacterium；
(5) osmosis is utilized, antibody crude extract is obtained；
(6) albumen that purity is up to more than 90% can be prepared through nickel post ion affinity chromatography.
Fig. 3 is referred to, is FAP nano antibodies after nickel post resin gel affinitive layer purification
The electrophoretogram of SDS-PAGE.Label M represents protein molecular mark, and label Nb67 represents present invention implementation
The SDS-PAGE bands of the FAP nano antibodies Nb67 that example 4 is obtained, unit is KDa in figure.As a result
It has been shown that, the size of FAP nano antibodies Nb67 is about 15KDa, and FAP nano antibodies Nb67 is by upper
Purge process is stated, its purity can reach more than 95%.
It is affine that FAP nano antibody Nb67 of the present invention also to above-described embodiment 4 after purification has carried out dynamics
Power is analyzed.It is 4.21 × 10 that experiment measures association rate constant4(unit M-1S-1), dissociation rate constant is
2.87×10-3(unit S-1), it is 6.82 × 10 to be thus calculated equilibrium dissociation constant-8(unit M).
Compared with the affinity data of other clone strains selected from phage library, the FAP nano antibodies
The equilibrium dissociation constant of Nb67 is relatively low, is difficult to dissociate between representing antibody antigen, and its affinity is stronger.
The present invention is described according to specific embodiment, but it will be understood by those skilled in the art that is not taking off
During from the scope of the invention, various change and equivalent can be carried out.Additionally, the spy to adapt to the technology of the present invention
Determine occasion or material, many modifications can be carried out to the present invention without deviating from its protection domain.Therefore, the present invention
Specific embodiment disclosed herein is not limited to, and including all implementations for dropping into claims
Example.
Claims (10)
1. a kind of FAP nano antibodies, it is characterised in that the FAP nano antibodies are for FAP tables
The nano antibody of position, including two such as SEQ ID NO：The VHH chains of amino acid sequence shown in 9.
2. a kind of VHH chains for FAP nano antibodies, including framework region FR and complementary determining region CDR,
Characterized in that, the framework region FR includes the amino acid sequence of the FR of the following group：SEQ ID NO：1
Shown FR1, SEQ ID NO：FR2 shown in 2, SEQ ID NO：FR3 shown in 3, SEQ ID
NO：FR4 shown in 4；The complementary determining region CDR includes the amino acid sequence of the CDR of the following group：
SEQ ID NO：CDR1 shown in 5, SEQ ID NO：CDR2 shown in 6, SEQ ID NO：7
Shown CDR3.
3. VHH chains for FAP nano antibodies according to claim 2, it is characterised in that
It has SEQ ID NO：Amino acid sequence shown in 9.
4. a kind of DNA molecular, it is characterised in that it is used to encode the protein being selected from the group：Right
It is required that the described FAP nano antibodies described in 1, or being received for FAP described in Claims 2 or 3
The VHH chains of meter Kang Ti.
5. DNA molecular according to claim 4, it is characterised in that it has SEQ ID NO：
Nucleotide sequence shown in 8.
6. a kind of expression vector, it is characterised in that its NO of ID containing SEQ：Nucleotide sequence shown in 8.
7. a kind of host cell, it is characterised in that it can express FAP nanometers described in claim 1
Antibody.
8. the FAP nano antibodies described in claim 1 are preparing FAP detection reagents or antineoplastic object space
The purposes in face.
9. a kind of FAP detection reagents or antineoplastic, it is characterised in that including described in claim 1
FAP nano antibodies.
10. the FAP nano antibodies described in claim 1 are preparing the application in combining absorption FAP reagents.
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| CN201511021663.2A CN106928366B (en) | 2015-12-30 | 2015-12-30 | FAP nano antibody Nb67 |
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| CN201511021663.2A CN106928366B (en) | 2015-12-30 | 2015-12-30 | FAP nano antibody Nb67 |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104447988A (en) * | 2014-12-11 | 2015-03-25 | 东南大学 | Bactrian camel-derived ApoE nano antibody as well as coding sequence and application thereof |
| CN105037544A (en) * | 2015-07-21 | 2015-11-11 | 南昌大佳科技有限公司 | AFP nanometer antibody A83 based on AFP antigen |
| CN105037547A (en) * | 2015-07-21 | 2015-11-11 | 南昌大佳科技有限公司 | AFP nanometer antibody A18 based on AFP antigen |
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2015
- 2015-12-30 CN CN201511021663.2A patent/CN106928366B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104447988A (en) * | 2014-12-11 | 2015-03-25 | 东南大学 | Bactrian camel-derived ApoE nano antibody as well as coding sequence and application thereof |
| CN105037544A (en) * | 2015-07-21 | 2015-11-11 | 南昌大佳科技有限公司 | AFP nanometer antibody A83 based on AFP antigen |
| CN105037547A (en) * | 2015-07-21 | 2015-11-11 | 南昌大佳科技有限公司 | AFP nanometer antibody A18 based on AFP antigen |
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