CN106928360B - CD105 nano antibody Nb68 - Google Patents
CD105 nano antibody Nb68 Download PDFInfo
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- CN106928360B CN106928360B CN201511021748.0A CN201511021748A CN106928360B CN 106928360 B CN106928360 B CN 106928360B CN 201511021748 A CN201511021748 A CN 201511021748A CN 106928360 B CN106928360 B CN 106928360B
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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
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- 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®
Abstract
The invention discloses a CD105 nano antibody aiming at CD105 polypeptide molecule epitope, simultaneously discloses a gene sequence for coding the CD105 nano antibody, an expression vector and a host cell capable of expressing the CD105 nano antibody, and discloses application of the CD105 nano antibody. The CD105 nano antibody, the gene sequence, the host cell and the like disclosed by the invention can be used for efficiently expressing the CD105 nano antibody in escherichia coli, has good specificity and high affinity with CD105 immunoreaction, and can be applied to preparation of CD105 detection reagents or antitumor drugs and the like.
Description
Technical Field
The invention belongs to the technical field of biomedicine or biopharmaceutical, and particularly relates to a nano antibody (Nb68) aiming at an epitope molecule of a CD105 polypeptide molecule, a coding sequence and application thereof.
Background
CD105, also known as endoglin, is a glycoprotein expressed by endothelial cell membranes and is one of the components of the TGF- β receptor complex, but can be independently present on the cell surface. CD105 contains 633 amino acids, has a relative molecular mass of about 68051, is a proliferation-associated hypoxia-inducible protein, and has 561 amino acids in the extracellular domain, 25 amino acids in the transmembrane domain, and 47 amino acids in the intracellular domain, constituting the tail of CD 105. In the extracellular portion of CD105, there are 4 potential glycosylation sites, amino acid residues 63, 96, 109 and 282, respectively. These glycosylation sites can be modified by N-glycosidases or endoglycosidases F, thus demonstrating that these sites are functional.
CD105, a cell membrane glycoprotein, is overexpressed in proliferating endothelial cells and in neovascular endothelial cells of tumor tissues, but not or weakly expressed in normal mature vascular endothelial cells. It is an important target molecule for inhibiting tumor angiogenesis and treating tumors, and is related to tumor tissue infiltration and metastasis. Therefore, the tumor angiogenesis marker as a reliable tumor angiogenesis marker can provide important reference information for clinical diagnosis, treatment and prognosis judgment.
However, the CD105 detection reagent used in the current market is mainly realized by an FITC fluorescein labeled anti-CD 105 monoclonal antibody, and the traditional method has the defects of poor antibody stability, low sensitivity, high production cost and the like.
The nano antibody technology is an antibody engineering revolution carried out by biomedical scientists on the basis of traditional antibodies by applying molecular biology technology and combining the concept of nano particle science, so as to develop the latest and smallest antibody molecules, which are originally discovered in camel blood by leiman. While the common antibody proteins consist of two heavy chains and two light chains, the novel antibodies found in camelid blood have only two heavy chains and no light chains, these "heavy chain antibodies" bind tightly to targets such as antigens as normal antibodies, but do not aggregate together into clumps as single chain antibodies do. The nano antibody based on the 'heavy chain antibody' has the advantages of molecular weight of only 1/10 of common antibodies, more flexible chemical property, good stability, high solubility, easy expression and easy acquisition, and is easy to couple with other molecules. However, no suitable nanobody has been developed against CD105 in the prior art.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a CD105 nano antibody, a DNA molecule for coding the CD105 nano antibody, application of the nano antibody and the like aiming at the defect that the prior art lacks of the nano antibody aiming at the CD105 epitope.
The technical scheme adopted by the invention for solving the technical problems is as follows: in a first aspect of the present invention, a CD105 nanobody is provided, wherein the CD105 nanobody is a nanobody directed to a CD105 epitope, and comprises two sequences as shown in SEQ ID NO: 9, or a VHH chain of the amino acid sequence set forth in 9. The CD105 nanobody of the present invention may be abbreviated as Nb 68.
In a second aspect of the invention, there is provided a VHH chain directed against a CD105 nanobody, comprising a framework region FR and a complementarity determining region CDR, the framework region FR comprising the amino acid sequences of FRs of the group: SEQ ID NO: FR1 shown in SEQ ID NO: FR2 as shown in 2, SEQ ID NO: FR3 as shown in SEQ ID NO: FR4 shown in FIG. 4; the complementarity determining region CDR includes the amino acid sequences of CDRs of the group: SEQ ID NO: 5, CDR1 shown in SEQ ID NO: 6, CDR2 shown in SEQ ID NO: CDR3 shown in FIG. 7. The structure of the framework region is relatively conserved, and the framework region mainly plays a role in maintaining the structure of the protein; the CDR structure is relatively diverse and is primarily responsible for antibody recognition.
Preferably, the VHH chain of the CD105 nanobody has the amino acid sequence of SEQ ID NO: 9, or a pharmaceutically acceptable salt thereof.
In a third aspect of the invention, there is provided a DNA molecule for encoding a protein selected from the group consisting of: the CD105 nanobody Nb68 of the present invention or the VHH chain of the CD105 nanobody of the present invention.
Preferably, said DNA molecule has the sequence of SEQ ID NO: 8.
The CD105 nano antibody provided by the invention can be prepared in a large scale by a phage amplification or genetic engineering recombinant expression mode. Phage amplification refers to the mass propagation and production of phage particles displaying CD105 nano antibody Nb68 by means of biological amplification of phage displaying CD105 nano antibody Nb 68. The gene engineering recombination expression mode refers to that the gene of the CD105 nano antibody Nb68 is cloned to an expression vector to carry out mass preparation of the nano antibody in a protein expression mode.
In a fourth aspect of the invention, there is provided an expression vector comprising SEQ ID NO: 8.
In a fifth aspect of the invention, a host cell is provided that can express the CD105 nanobody Nb68 of the invention.
In a sixth aspect of the invention, the invention provides an application of the CD105 nano antibody Nb68 in preparing a CD105 detection reagent. The CD105 nano antibody Nb68 can replace the traditional CD105 antibody to prepare a CD105 detection reagent for detecting CD105 polypeptide molecules.
In a seventh aspect of the invention, the invention provides the use of the CD105 nanobody Nb68 in the immunological detection for non-therapeutic purposes. The immunological detection types comprise immunological analysis detection types based on antigen-antibody specific reaction, such as enzyme-linked immunosorbent assay, colloidal gold immunochromatography, immunodot hybridization and the like. When the CD105 nano antibody Nb68 is applied, phage particles which are obtained by phage amplification and display CD105 nano antibody Nb68 are directly used for analysis and detection, and CD105 nano antibody Nb68 is subjected to prokaryotic organism or eukaryotic organism expression and then is subjected to immunological detection and analysis in the form of protein.
In an eighth aspect of the invention, the invention provides an application of the CD105 nanobody Nb68 in preparing a binding and adsorbing CD105 reagent. For example, the CD105 nanobody of the present invention may be prepared into an immunoaffinity column, which adsorbs and captures CD105 for further study after enrichment, etc. The invention also provides application of the CD105 nano antibody Nb68 in preparation of antitumor drugs. The invention also correspondingly provides a CD105 detection reagent or an anti-tumor drug, which comprises the CD105 nano antibody Nb 68.
The implementation of the invention has the following beneficial effects: firstly, synthesizing CD105 polypeptide, enabling the polypeptide to have immunogenicity, then coupling CD105 molecules on an enzyme label plate, displaying the correct spatial structure of the protein, screening an immune nano antibody gene library (camel heavy chain antibody phage display gene library) by using the antigen in the form through a phage display technology, thereby obtaining CD105 specific nano antibody genes, transferring the genes into escherichia coli, and establishing a nano antibody strain capable of being efficiently expressed in the escherichia coli; the CD105 nano antibody obtained by screening by the method has the characteristics of immunoreaction with CD105, good specificity and high affinity, and can be applied to the preparation of CD105 immunological detection reagents and the like.
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The invention will be further described with reference to the accompanying drawings and examples, in which:
FIG. 1 is a schematic diagram of the amino acid numbering and structure of the CD105 nanobody of the present invention;
FIG. 2 is a DNA electrophoresis of the CD105 nanobody of the present invention;
FIG. 3 is an electrophoresis diagram of SDS-PAGE of the CD105 nanobody of the present invention after purification by nickel column resin gel affinity chromatography.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments.
The invention utilizes phage display technology to screen single-domain heavy-chain antibody (VHH) phage clones which can be specifically combined with target molecule CD105 antigen from camel source immunized single-domain heavy-chain antibody library, thereby obtaining a nano antibody strain which can be efficiently expressed in escherichia coli, namely CD105 nano antibody Nb 68.
The invention will be further illustrated with reference to the following specific examples.
Example 1: construction of nanobody library against CD 105:
(1) firstly, synthesizing CD105 polypeptide, mixing 1mg of CD105 with Freund's adjuvant in equal volume, immunizing a Xinjiang dromedary, immunizing once a week for 7 times in total, and stimulating B cells to express antigen-specific nano antibodies;
(2) after 7 times of immunization, extracting 100mL camel peripheral blood lymphocytes and extracting total RNA;
(3) synthesizing cDNA and amplifying VHH by using nested PCR;
(4) utilizing restriction enzymes PstI and NotI to cut 20ug pComb3 phage display vector (supplied by Biovector Chinese plasmid vector strain cell gene collection center) and 10ug VHH and connect the two fragments, (5) transforming the connection product into electrotransformation competent cell TG1 to construct CD105 nano antibody library and determine the library capacity, wherein the size of the library capacity is 1.85 × 108。
Example 2: nanobody screening process for CD 105:
(1) the solution was dissolved in 100mM NaHCO3、pH 820ug of CD105 from 2 was coupled to NUNC plate and left overnight at 4 ℃;
(2) adding 100uL of 0.1% casein in the next day, and sealing for 1-3h at room temperature;
(3) after 1-3h, 100uL phage (5 × 10) was added11tfu immune camel nanometer antibody phage display gene bank), acting for 1-2h at room temperature;
(4) washing 4-6 times with 0.05% PBS + Tween-20 to wash away unbound phage;
(5) the phage specifically binding to CD105 were dissociated with 100mM TEA (triethylamine) and infected with E.coli TG1 in log phase growth, cultured at 37 ℃ for 1h, phage generated and purified for the next round of screening, and the same screening process was repeated for 3-5 rounds to gradually enrich.
Example 3: screening of specific single positive clones by phage enzyme-linked immunosorbent assay (ELISA):
(1) from the phage-containing cell culture dishes after the 3-5 rounds of selection described above, 96 individual colonies were picked and inoculated into TB medium containing 100. mu.g per ml of ampicillin (2.3 g of potassium dihydrogen phosphate, 12.52 g of dipotassium hydrogen phosphate, 12 g of peptone, 24 g of yeast extract, 4 ml of glycerol in 1L of TB medium), grown to a logarithmic phase, followed by addition of Isopropylthiogalactoside (IPTG) at a final concentration of 1 mmol and overnight incubation at 30-35 ℃.
(2) Crude antibody is obtained by using a permeation method, and the antibody is transferred to an ELISA plate coated by the antigen and is placed for 1-1.5 hours at room temperature.
(3) Unbound antibody was washed away with PBST, and a primary anti-mouse anti-HA antibody (mouse anti-HA tagganty, available from Beijing kang, century Biotechnology Co., Ltd.) was added and allowed to stand at room temperature for 1-1.5 hours.
(4) Unbound antibody was washed away with PBST, and anti-mouse alkali line phosphataseconjugate (goat anti-mouse alkaline phosphatase-labeled antibody, available from Eimei technologies, Ltd.) was added and allowed to stand at room temperature for 1 to 1.5 hours.
(5) Unbound antibody was washed away with PBST, and absorbance was read on an ELISA instrument at 405nm by adding an alkaline phosphatase developing solution.
(6) And when the OD value of the sample well is more than 3 times larger than that of the control well, judging the sample well to be a positive clone well.
(7) The bacteria of the positive cloning wells were shaken in LB liquid containing 100. mu.g per ml to extract the plasmid and sequenced.
Through the experiment, the invention obtains 6 positive cloning holes which show more than 3 times of binding force with antigen, and the positive cloning holes are regarded as primary screening positive cloning strains. Analyzing the gene sequences of the primary screening positive clones according to the Vector NTI of the sequence alignment software, regarding the strains with the same CDR1, CDR2 and CDR3 sequences as the same clones, and regarding the strains with different sequences as different clones, so as to obtain the amino acid sequences of VHH chains of a group of antibodies, such as SEQ ID NO: shown at 9. The CD105 nanobody is numbered as Nb68, and the amino acid numbering and structural diagram are shown in FIG. 1.
FIG. 2 shows the DNA electrophoresis of the CD105 nanobody. The DNA bands of the first gel well labeled M in the figure are: the DNA molecular markers of 1000bp, the DNA bands of the gel holes marked from left to right with the labels of 1-24 are all CD105 nano antibody DNA electrophoresis bands. The DNA of each 1-24 gel hole is the PCR product of CD105 nanometer antibody DNA, and the PCR product is about 500 bp.
Example 4: the nano antibody is expressed and purified in host bacterium escherichia coli:
(1) the amino acid sequence obtained by the previous sequencing analysis is shown as SEQ ID NO: the plasmid of the clone shown in 9 is electrically transformed into escherichia coli WK6, and is coated on a culture plate containing LA + glucose, namely ampicillin and glucose, and is cultured overnight at 37 ℃;
(2) selecting a single colony, inoculating the single colony in 5mL LB culture solution containing ampicillin, and carrying out shake culture at 37 ℃ overnight;
(3) inoculating 1mL of overnight strain into 330mL of TB culture solution, carrying out shake culture at 37 ℃, adding IPTG (isopropyl-beta-thiogalactoside) when the OD value reaches 0.6-1, and carrying out shake culture at 30-35 ℃ overnight;
(4) centrifuging and collecting bacteria;
(5) obtaining antibody crude extract by using an osmosis method;
(6) the protein with purity of more than 90 percent can be prepared by nickel column ion affinity chromatography.
FIG. 3 shows an electrophoresis chart of SDS-PAGE of CD105 nanobody purified by nickel column resin gel affinity chromatography. The reference numeral M represents a protein molecular marker, and the reference numeral Nb68 represents an SDS-PAGE band of the CD105 nano antibody Nb68 obtained in example 4 of the invention, and the unit in the figure is KDa. The result shows that the size of the CD105 nano antibody Nb68 is about 15KDa, and the purity of the CD105 nano antibody Nb68 can reach more than 95% after the purification process.
The invention also carries out kinetic affinity analysis on the CD105 nano antibody Nb68 purified in the example 4, and the experiment shows that the binding rate constant is 5.92 × 104(Unit M)-1S-1) Dissociation rate constant of 1.71 × 10-3(unit S)-1) From this, an equilibrium dissociation constant of 2.89 × 10 was calculated-8(unit M). Compared with the affinity data of other clones selected from a phage library, the CD105 nano antibody Nb68 has a low equilibrium dissociation constant, represents that the antibody and the antigen are difficult to dissociate, and has strong affinity.
While the invention has been described with reference to specific embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed, but that the invention will include all embodiments falling within the scope of the appended claims.
Claims (9)
1. A CD105 nanobody, which is a nanobody against a CD105 epitope, and the amino acid sequence of the VHH chain thereof is shown in SEQ ID NO: shown at 9.
2. A VHH chain directed against a CD105 nanobody comprising a framework region FR and a complementarity determining region CDR, wherein the framework region FR comprises the amino acid sequence of the FR of the group: SEQ ID NO: FR1 as shown in 1, SEQ ID NO: FR2 as shown in 2, SEQ ID NO: FR3 as shown in SEQ ID NO: FR4 shown in FIG. 4; the complementarity determining region CDR includes the amino acid sequences of CDRs of the group: SEQ ID NO: 5, CDR1 shown in SEQ ID NO: 6, CDR2 shown in SEQ ID NO: CDR3 shown in FIG. 7.
3. A DNA molecule for encoding a protein selected from the group consisting of: the CD105 nanobody of claim 1, or the VHH chain of claim 2 directed against a CD105 nanobody.
4. The DNA molecule of claim 3, having a nucleotide sequence as set forth in SEQ ID NO: shown in fig. 8.
5. An expression vector comprising the nucleotide sequence of SEQ ID NO: 8.
6. A host cell capable of expressing the CD105 nanobody of claim 1.
7. The use of the CD105 nanobody of claim 1 in the preparation of CD105 detection reagents or antitumor drugs.
8. A CD105 detection reagent or an antitumor agent comprising the CD105 nanobody of claim 1.
9. Use of the CD105 nanobody of claim 1 for the preparation of a binding-adsorbing CD105 reagent.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| 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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| 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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