CN106928360A - A kind of CD105 nano antibodies Nb68 - Google Patents
A kind of CD105 nano antibodies Nb68 Download PDFInfo
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- CN106928360A CN106928360A CN201511021748.0A CN201511021748A CN106928360A CN 106928360 A CN106928360 A CN 106928360A CN 201511021748 A CN201511021748 A CN 201511021748A CN 106928360 A CN106928360 A CN 106928360A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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- B01D15/08—Selective adsorption, e.g. chromatography
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- 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
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- 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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- C07—ORGANIC CHEMISTRY
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- 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 CD105 nano antibodies for being directed to CD105 peptide molecule epitopes, while also disclosed the gene order for encoding the CD105 nano antibodies and the expression vector and host cell of the CD105 nano antibodies can be expressed, and disclose the purposes of the CD105 nano antibodies.By CD105 nano antibodies disclosed by the invention, gene order and host cell etc., high efficient expression can go out CD105 nano antibodies in Escherichia coli, its, affinity good with CD105 immune responses specificity is high, can be applied to prepare CD105 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 (Nb68) of CD105 peptide molecule epitope molecules, its coded sequence and purposes.
Background technology
CD105 also known as endoglin, is the glycoprotein of endothelial cell endoglin expression, is TGF
One of composition of β (transform growthfactor, TGF-β) receptor complex, but cell can be independently present in
Surface.CD105 contains 633 amino acid, and relative molecular mass is about 68051, is a kind of and propagation
Related and can be by the albumen of hypoxia inducible, its after birth outer portion contains 561 amino acid, and trans-membrane region contains
There is part in 25 amino acid, after birth to contain 47 amino acid, constitute the afterbody of CD105.In CD105
Extracellular part, have 4 potential glycosylation sites, be respectively the 63rd, 96,109 and
282 amino acids residues.These glycosylation sites can be modified by N- glycosidases or interior glycosidase F, because
This proves that these sites possess function.
CD105 as a kind of glycoprotein, in the endothelial cell and the new green blood of tumor tissues of propagation
It is in overexpression in endothelial cell, and is not expressed or weak expression in the vascular endothelial cell of normal mature.It
It is to suppress Tumor Angiongesis, the important target molecule for the treatment of tumour, is infiltrated with tumor tissues, shifts relevant.
Accordingly, as a kind of reliable tumor neogenetic blood vessels mark by sentencing for clinical diagnosis, treatment and prognosis
It is disconnected that important reference information is provided.
However, CD105 detection reagents currently used in the market are mainly being marked by FITC fluoresceins
Anti- CD105 monoclonal antibodies realize that this conventional method has that Antibody stability is poor, sensitivity
It is low, the shortcomings of 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 CD105.
The content of the invention
The technical problem to be solved in the present invention is to be directed to CD105 epitopes for lacking in the prior art
The defect of nano antibody, there is provided a kind of CD105 nano antibodies, while provide the CD105 nanometers of coding resisting
Purposes of the DNA molecular of body 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 CD105 nano antibodies, the CD105 nano antibodies are the nano antibody for CD105 epitopes,
Including two such as SEQ ID NO：The VHH chains of amino acid sequence shown in 9.The CD105 receives in the present invention
Meter Kang Ti can be referred to as Nb68.
A kind of second aspect present invention, there is provided VHH chains for CD105 nano antibodies, including framework
Area 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 CD105 nano antibodies, it has SEQ ID NO：9
Shown 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：
CD105 nano antibodies Nb68 of the present invention, or it is of the present invention anti-for CD105 nanometers
The VHH chains of body.
Preferably, described DNA molecular, it has SEQ ID NO：Nucleotide sequence shown in 8.
What the CD105 nano antibodies that the present invention is provided can be recombinantly expressed by Phage amplification or genetic engineering
Mode is largely prepared.Phage amplification refers to the phagocytosis that will show and have CD105 nano antibodies Nb68
Body, by way of biology amplification, amount reproduction production displaying has biting for CD105 nano antibodies Nb68
Phage particle.The mode of genetic engineering recombination expression refers to the base that will encode CD105 nano antibodies Nb68
Cause, 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 CD105 of the invention
Nano antibody Nb68.
The sixth aspect of the present invention, there is provided prepared by CD105 nano antibodies Nb68 of the present invention
Purposes in terms of CD105 detection reagents.CD105 nano antibodies Nb68 of the invention can substitute tradition
CD105 antibody, prepare CD105 detection reagents, for detecting CD105 peptide molecules.
The seventh aspect of the present invention, there is provided CD105 nano antibodies Nb68 of the present invention is controlled non-
Treat the purposes in purpose immunology detection.The type of the immunology detection includes MBP enzyme linked immuno-adsorbent assay, glue
The immune analysis based on antigen and antibody specific reaction such as body gold immunochromatography, immunodotting hybridization detect class
Type.CD105 nano antibodies Nb68 of the present invention is in use, the exhibition that can be obtained by Phage amplification
The bacteriophage particles for being shown with CD105 nano antibodies Nb68 are directly used in analysis detection and receive CD105
Meter Kang Ti Nb68 in the form of albumen after prokaryotes or eucaryote expression by carrying out immunology detection point
Analysis.
The eighth aspect of the present invention, there is provided CD105 nano antibodies Nb68 of the present invention is preparing knot
Close the application in absorption CD105 reagents.For example, CD105 nano antibodies of the present invention can be made immune
Affinity column, absorption crawl CD105, for further research etc. after enrichment.Present invention also offers CD105
Purposes of the nano antibody Nb68 in terms of antineoplastic is prepared.The present invention is also corresponding to provide one kind
CD105 detection reagents or antineoplastic, including foregoing CD105 nano antibodies Nb68.
Implement the present invention, have the advantages that：The present invention synthesizes CD105 polypeptides first, and makes it
With immunogenicity, then by CD105 molecule coupling labeleds on ELISA Plate, show the correct space of the albumen
Structure, antigen in this format is using display technique of bacteriophage screening immune nano Antibody geometric mean titer (camel weight
Chain antibody phage display gene pool), so as to obtain the specific nano antibody genes of CD105, by this
Gene is gone in Escherichia coli, can be in the strain of the nano antibody of E. coli so as to establish；This
The CD105 nano antibodies that inventive method screening is obtained have and CD105 immune response characteristics, and specificity
Good, affinity is high, can be applied to prepare CD105 immunology detection reagents 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 CD105 nano antibodies of the present invention；
Fig. 2 is the DNA electrophoretograms of CD105 nano antibodies of the present invention；
Fig. 3 is CD105 nano antibodies of the present invention after nickel post resin gel affinitive layer purification
The electrophoretogram of SDS-PAGE.
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 CD105 antigentic specificities are combined, and obtain
Can be in the strain of the nano antibody of E. coli, i.e. CD105 nano antibodies Nb68.
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 CD105：
(1) synthesize CD105 polypeptides first, 1mg CD105 are mixed in equal volume with Freund's adjuvant, exempt from
One Xinjiang one-humped camel of epidemic disease, once in a week, is immunized 7 times altogether, stimulates B cell to express antigentic specificity
Nano 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 CD105 nanometers
Antibody library simultaneously determines storage capacity, and storage capacity size is 1.85 × 108。
Embodiment 2：For the nano antibody screening process of CD105：
(1) 100mM NaHCO are dissolved in3, the 20ug CD105 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 CD105 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 CD105 nano antibodies are numbered as Nb68, its amino acid number
It is as shown in Figure 1 with structural representation.
Fig. 2 is referred to, is the DNA electrophoretograms of CD105 nano antibodies.Marked as the first of M in figure
The DNA bands of individual 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 CD105 nano antibody DNA electrophoretic bands.These 1-24 gel pores
DNA be the PCR primer of CD105 nano antibodies DNA, the PCR primer band is 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 CD105 nano antibodies after nickel post resin gel affinitive layer purification
The electrophoretogram of SDS-PAGE.Label M represents protein molecular mark, and label Nb68 represents present invention implementation
The SDS-PAGE bands of the CD105 nano antibodies Nb68 that example 4 is obtained, unit is KDa in figure.Knot
Fruit shows that the size of CD105 nano antibodies Nb68 is about 15KDa, CD105 nano antibodies Nb68
By above-mentioned purge process, its purity can reach more than 95%.
CD105 nano antibody Nb68 of the present invention also to above-described embodiment 4 after purification has carried out dynamics
Affinity analysis.It is 5.92 × 10 that experiment measures association rate constant4(unit M-1S-1), dissociation rate is normal
Number is 1.71 × 10-3(unit S-1), it is 2.89 × 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 CD105 nano antibodies
Nb68 equilibrium dissociation constants are relatively low, are 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 CD105 nano antibodies, it is characterised in that the CD105 nano antibodies are for CD105
The nano antibody of epitope, including two such as SEQ ID NO：The VHH chains of amino acid sequence shown in 9.
2. a kind of VHH chains for CD105 nano antibodies, including framework region FR and complementary determining region
CDR, it is characterised in that the framework region FR includes 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 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 CD105 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 CD105 nano antibodies described in 1, or described in Claims 2 or 3 for CD105
The VHH chains of nano antibody.
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 the CD105 that it can express described in claim 1 receives
Meter Kang Ti.
8. the CD105 nano antibodies described in claim 1 are preparing CD105 detection reagents or antitumor
Purposes in terms of medicine.
9. a kind of CD105 detection reagents or antineoplastic, it is characterised in that including claim 1 institute
The CD105 nano antibodies stated.
10. the CD105 nano antibodies described in claim 1 are in combination absorption CD105 reagents are prepared
Using.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114231559A (en) * | 2020-09-09 | 2022-03-25 | 广西医科大学 | Expression plasmid containing CAR nucleic acid fragment, CD 105-targeted CAR-T cell containing expression plasmid, and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103421115A (en) * | 2013-09-02 | 2013-12-04 | 东南大学 | CD38 nanometer antibody and application |
| 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 |
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2015
- 2015-12-30 CN CN201511021748.0A patent/CN106928360B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103421115A (en) * | 2013-09-02 | 2013-12-04 | 东南大学 | CD38 nanometer antibody and application |
| 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 |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114231559A (en) * | 2020-09-09 | 2022-03-25 | 广西医科大学 | Expression plasmid containing CAR nucleic acid fragment, CD 105-targeted CAR-T cell containing expression plasmid, and preparation method and application thereof |
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