CN109897108B - Alpaca single domain antibody of anti-human endothelial protein C receptor and application thereof - Google Patents
Alpaca single domain antibody of anti-human endothelial protein C receptor and application thereof Download PDFInfo
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Abstract
The invention discloses an alpaca single domain antibody of an anti-human endothelial protein C receptor and application thereof. The method utilizes a phage display technology to carry out multi-round screening on an alpaca phage antibody library, and enriches phage capable of specifically binding to a human protein C receptor; through the culture of phage, antibody is prepared and identified to obtain positive clone, and its corresponding coding sequence is obtained through sequencing. The antibody of the invention maintains high binding capacity to EPCR and can be used for expression detection of EPCR; the antibody of the invention can be competitively bound with protein C to EPCR, thereby preventing the activation of the signal path of protein C-EPCR and being used for treating diseases taking the signal path of protein C-EPCR as a target.
Description
Technical Field
The invention relates to an antibody of an anti-human endothelial protein C receptor, in particular to a novel specific single domain antibody of the anti-human endothelial protein C receptor, a DNA coding sequence of a variable region thereof, and application of the single domain antibody.
Background
The endothelin C receptor (EPCR) is a newly discovered member of the anticoagulant pathway of protein C, and is another anticoagulant-related protein that is found on the surface of tumor cells following thrombomodulin. Research shows that the protein C receptor can activate protease activated receptor (PAR-1) by combining with Activated Protein C (APC), promote cell proliferation and inhibit apoptosis, and EPCR plays a key mediating role in the process; recent research shows that activated protein C can improve the mobility and chemotaxis of breast cancer cell lines by combining with EPCR and PAR-1, reveals that the over-expression of EPCR in cells and tissues is favorable for the proliferation of the cells and tissues, and inhibits apoptosis, thereby promoting the progression and metastasis of tumors. The current EPCR functions include participation in anticoagulation, regulation of inflammatory response, inhibition of apoptosis, and participation in tumor resistance and metastasis.
The open reading frame of human EPCR contains 714 nucleotides, encodes 238 amino acids, and 17 amino acid signal peptides are removed during the processing process, so that the mature protein consisting of 221 amino acids is formed. EPCR consists of an extracellular domain, a transmembrane domain and a short, highly conserved cytoplasmic domain. EPCR on cell membranes belongs to a combined receptor, and researches prove that EPCR has expression in various tumor cells such as lung cancer, breast cancer, ovarian cancer, renal cancer, colon cancer, malignant glioma, melanoma and the like and leukemia cells, particularly malignant solid tumors. The high expression of EPCR gene in various tumor cells suggests that EPCR gene can be used as a tumor marker for cancer diagnosis.
One antibody that lacks a light chain, consists only of heavy chains, but has full function, is found in camelids such as bactrian camels, dromedary, alpacas, and llamas, called heavy chain antibodies (hcAb). Like conventional antibodies, the heavy chain antibody variable region has good antigen recognition and binding ability, and can normally function as an antibody. The variable region of the heavy chain antibody (VHH) has a molecular weight of about 15kDa, is 1/10, which is only the size of a conventional antibody, and is the smallest molecular fragment with complete antibody function that is currently available, and is called single domain antibody (sdAb). The antigen-binding region of camelid single domain antibodies consists of only three hypervariable regions of VHH (H1-H3), forming in space an antigen-binding domain that differs from the typical structure of conventional antibodies. Wherein the average length of H3 is longer than that of conventional antibodies, and the single domain antibody has a finger-shaped structure protruding in space, so that the single domain antibody can bind to an epitope which can not be accessed by some conventional antibodies. In addition, high expression of recombinant VHH in E.coli could be obtained due to the substitution of hydrophilic amino acids at four positions in the framework region FR 2.
The method for obtaining the gene sequence of the single domain antibody is to screen through a phage antibody library. The phage antibody library is a population which is formed by cloning the alpaca complete set of sdAb variable region genes by using a gene cloning technology, assembling the cloned genes into an expression vector and expressing the expression vector on the surface of a phage. Screening of phage antibody libraries involves two main steps of panning and identification: the panning is to incubate phage antibody library with the target antigen together, elute several times, collect the combined phage, infect the bacterium with the obtained phage and amplify, and then pan for the next round, can enrich to the polyclonal bacterial strain infected with phage specifically combined with target antigen after several rounds of panning; the identification process is to select a monoclonal bacterial strain from the phage-infected polyclonal bacterial strains, i.e. the screened phage-infected bacteria are plated and selected to obtain a high-specificity monoclonal bacterial strain, and the variable region gene sequence and the encoding protein of the related antibody can be obtained after gene sequencing, analysis and comparison. The identified single-domain antibody is further cloned and constructed, and a proper vector is selected for expression to obtain the single-domain antibody. Compared with IgG and scFv antibodies, the single domain antibody has the advantages of small molecule, low immunogenicity, strong penetrating power and the like, so that the single domain antibody has wide application prospects in the fields of basic research, drug development and the like.
At present, there has been no antibody screening for an anti-human endothelin C receptor using the full length of human EPCR as a target antigen using an alpaca phage antibody library.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a specific single-domain antibody for resisting a human endothelial protein C receptor. Through screening of an alpaca phage antibody library, two single-domain antibodies capable of tightly binding with the EPCR are found, the binding can generate competitive influence on the binding of the protein C and the EPCR, and the binding can inhibit a signal path of the protein C-EPCR, so that the protein C-EPCR has potential treatment effect on diseases depending on the signal path.
In order to achieve the purpose, the invention adopts the following technical scheme
The invention provides a single domain antibody of an anti-human endothelin C receptor, wherein the amino acid sequence of the single domain antibody is any one of SEQ ID NO.1, SEQ ID NO.2, an amino acid sequence with homology of more than 95% with the sequence shown in SEQ ID NO.1 or an amino acid sequence with homology of more than 95% with the sequence shown in SEQ ID NO.2,
SEQ ID NO:1
AVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATSLGGAWVVAGNCPALDFNSWGQGTQVTVSS；
SEQ ID NO:2
QVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATGLGGAWTVASNCPALDFGSWGQGTQVTVSS。
in a second aspect of the invention, there is provided a nucleotide sequence encoding the single domain antibody described above, wherein the nucleotide sequence encoding the single domain antibody shown in SEQ ID NO.1 is shown in SEQ ID NO. 3; the nucleotide sequence of the single domain antibody shown in the SEQ ID NO.2 is shown in SEQ ID NO. 4.
In a third aspect of the invention, there is provided a recombinant expression vector comprising the nucleotide sequence as described above.
Further, the vector is selected from prokaryotic or eukaryotic expression vectors, and is preferably selected from pcDNA3.1.
In a fourth aspect of the invention, there is provided a host cell comprising a nucleotide sequence as described above.
Further, the host cell contains the recombinant expression vector described above.
Further, the above-mentioned host cell can express the single domain antibody against human endothelin C receptor.
Further, the host cell is preferably a HEK293 cell.
The fourth aspect of the invention provides the application of the single domain antibody of the anti-human endothelial protein C receptor in the preparation of a reagent for detecting or removing the human endothelial protein C receptor.
Furthermore, the invention also provides application of the amino acid sequence or the nucleotide sequence in preparing a reagent for detecting or removing the human endothelial protein C receptor.
In the fifth aspect of the invention, the invention provides the application of the single domain antibody of the anti-human endothelin C receptor in the preparation of drugs for treating diseases taking a protein C-EPCR signal pathway as a target.
Further, the disease targeting the protein C-EPCR signaling pathway is a tumor.
The invention has the beneficial effects that:
the method utilizes a phage display technology to carry out multi-round screening on an alpaca phage antibody library, and enriches phage capable of specifically binding to a human protein C receptor; through the culture of phage, antibody is prepared and identified to obtain positive clone, and its corresponding coding sequence is obtained through sequencing. The single domain antibody of the anti-human endothelin C receptor maintains high binding capacity to EPCR and can be used for expression detection of EPCR; the antibody of the invention can be competitively combined with protein C to EPCR, thereby preventing the activation of the signal path of protein C-EPCR, and can be used for treating diseases taking the signal path of protein C-EPCR as a target.
Drawings
FIG. 1 is a graph showing the determination of the optimal antigen-antibody reaction concentration in the screening of candidate single-domain antibodies against human endothelin C receptor in enzyme-linked immunosorbent assay (ELISA);
FIG. 2 shows the result of detecting the affinity of a candidate anti-human endothelin C receptor single domain antibody to an antigen by ELISA;
FIG. 3 shows the results of detecting competitive binding ability of candidate monoclonal antibodies against human endothelin C receptor by ELISA;
FIG. 4 shows SDS-PAGE electrophoresis detection of expression of single domain antibodies against human endothelin C receptor;
FIG. 5 is a graph of the time course of tumor volume in breast cancer model mice injected with anti-EPVR single domain antibody No. 6 and anti-EPVR single domain antibody No. 8;
FIG. 6 is a graph showing the effect of injection of anti-EPVR single domain antibody No. 6 and anti-EPVR single domain antibody No. 8 on tumor size in breast cancer model mice.
Detailed Description
The features and advantages of the present invention will be further understood from the following detailed description taken in conjunction with the accompanying drawings. The examples provided are merely illustrative of the method of the present invention and do not limit the remainder of the disclosure in any way.
It will be understood by those skilled in the art that, based on the disclosure of the present application in combination with common general knowledge in the art, the DNA sequences and amino acid sequences disclosed in the present application can be synthesized by other methods commonly used in the art, for example, by chemical synthesis to obtain the sequences disclosed in the present application. Furthermore, the skilled person can construct the novel DNA sequences obtained in the present application into any suitable vector, host cell. The following is merely an exemplary illustration of the scope of the claims of the present application and various changes and modifications of the invention of the present application may be made by those skilled in the art based on the disclosure, which should also fall within the scope of the claims of the present application.
The various chemical agents used in the examples of the present invention are commercially available in a conventional manner unless otherwise specified.
Example 1 obtaining human Endoglin C receptor protein
1. Preparation of immunogens
The amino acid sequence of the human EPCR holoprotein is as follows: MLTTLLPILLLSGWAFCSQDASDGLQRLHMLQISY FRDPYHVWYQGNASLGGHLTHVLEGPDTNTTIIQLQPLQEPESWARTQSG LQSYLLQFHGLVRLVHQERTLAFPLTIRCFLGCELPPEGSRAHVFFEVAVNG SSFVSFRPERALWQADTQVTSGVVTFTLQQLNAYNRTRYELREFLEDTCV QYVQKHISAENTKGSQTSRSYTSLVLGVLVGGFIIAGVAVGIFLCTGGRRC (SEQ ID NO: 5).
2. Whole protein construction
pcDNA3.1 vector was constructed, purified after expression:
synthesizing EPCR target gene, double enzyme digestion with NheI and HindIII, inserting into expression vector pcDNA3.1, constructing pcDNA3.1-EPCR recombinant plasmid, transfecting into HEK293 cell for expression, and purifying EPCR protein by affinity chromatography.
Example 2 screening of Single Domain antibodies against human endothelin C receptor
First, mRNA of human EPCR in immune B cells in unimmunized camels is reversely transcribed into cDNA, then the variable region is cloned to Phagemid (Phagemid, phage plasmid) M13K07 (purchased from Stratagene, USA), transformed into Escherichia coli strain XL1-Blue, and finally EPCR antibody protein fragments are displayed on the surface of phage M13K07 under the condition of amplification and packaging of helper phage M13K07 system, so as to become the alpaca natural single-domain heavy chain antibody library.
The natural single-domain heavy-chain antibody library of the alpaca used in the invention is obtained from Nanjing Kinsrui Biotech, Inc.
Screening and enrichment of EPCR antibodies
1) M13K07 Titer assay
1. A single colony, XL1-Blue, was inoculated into 2 XTY/Tet (15. mu.g/ml) medium overnight at 37 ℃.
2. The cells were re-inoculated in fresh 2 × TY medium at a ratio of 1:100 and 37 ℃ to an OD600 of 1.0.
3. Phage stock solutions were diluted in a series of 10-fold gradients, dissolved in 0.1ml aliquots of 2 × TY medium, in duplicate, to give one 10 per ml3-105The concentration of the phage.
4.2 XTY Top agar was thawed, at least 3ml of each phage dilution, and cooled to 45 ℃.
Mu.l of phage solution was added to 500. mu.l of fresh XL1-Blue cells in a 10ml culture tube 10 minutes later with 3ml of top agar, mixed well quickly, the top agar mixture was immediately poured onto LB agar plates, the plates were shaken to allow the top agar to spread out, preventing the top agar from clumping, and left to stand overnight at 37 ℃.
6. The number of plaques multiplied by 100 times the corresponding dilution fold equals the titer of phage in the original stock.
2) Amplification of M13K07
1. A single plaque was picked up by pipette tip from an overnight-cultured plate, flushed in 1ml of fresh XL1-Blue cells, and incubated at 37 ℃ for 1 hour with shaking.
2. Aspirate 100. mu.l of culture in 2 XTY 50ml of 50. mu.g/ml tetracycline and shake culture at 37 ℃ for 24 hours.
3. The culture was transferred to a sterile tube at 14000rpm for 10 minutes at 4 ℃ and the supernatant was transferred to a new tube, 1/5 volume percent 40% PEG4000 &2.5M NaCl solution, shaken vigorously and ice-cooled for 15 minutes.
4.14000rpm, 4 ℃ for 15 minutes, the supernatant was removed as much as possible, the pellet was resuspended in 1/20 volumes of 1 XTE buffer, 15000rpm4 ℃ for 5 minutes, the supernatant was transferred to a sterile tube, OD268 ═ 1.0 for 5X 10ml12And (4) phage.
3) Mass amplification of the original EPCR antibody library
1. A single colony, XL1-BLUE, was inoculated in 2 XTY/Tet (15. mu.g/ml) medium overnight at 37 ℃.
2. The culture medium was re-inoculated in a ratio of 1:100 in a fresh 2 XTY medium at 37 ℃ until OD600 became 0.4-0.5.
3. By 1010-1011An order of magnitude of phage infested 50ml of XL1-Blue and left to stand for 40 minutes at 37 ℃.
4. 100 μ l of the cell suspension was diluted in 2 XTY medium in a 10-fold gradient (10)-1-10-12) Mu.l of each diluted cell solution was spread on an LB-ampicillin plate and left overnight at 30 ℃.
5. The remaining culture was incubated at 37 ℃ for 1 hour, then 500ml of 2 XTY medium was added and incubation at 37 ℃ was continued for 1 hour.
6. Using 20 times of M13K07 (10)13)100 μ L of the mixture was subjected to infection and left standing at 37 ℃ for 40 minutes.
7.3000g, 15 min at RT, and the pellet resuspended in 1000ml2 XTY + AMP + KAN + GLU, incubated overnight at 30 ℃ with shaking at 150 rpm.
8. The supernatant was transferred to a sterile tube at 14000rpm for 10 minutes at 4 ℃ and transferred to a new tube, and 100. mu.l of 40% PEG4000 &2.5M NaCl solution was added to each 400. mu.l of the supernatant, and the tube was shaken vigorously and iced for 15 minutes.
9.14000rpm, 4 ℃ for 15 minutes, the supernatant was removed and dissolved in 4ml of 1 XTE buffer to a titer of 1011pfu/μl。
4) Screening and enrichment of EPCR antibodies, first round
1. Mu.g of the prepared immunogen human EPCR holoprotein was weighed, dissolved in 3ml of sodium carbonate-sodium bicarbonate coating solution (pH 9.5), and added with 4ml Nunc-ImmunoTM MaxisorpTMIn an immune tube, 4 ℃ overnight.
Tris-HCl blocks unsaturated protein coupling sites of the immuno tubes. The immunocontubes were first washed 3 times with the coupling solution, the supernatant was removed as much as possible, 4ml of 0.1M Tris-HCl buffer (pH 8.0) was added, and the mixture was allowed to stand at room temperature for 2 hours to block the active sites.
BSA blocks potential protein binding sites. 4ml of 5% BSA was placed in an immune tube, shaken slowly at room temperature for 2 hours, washed 3 times with PBS and spun clean.
4.1ml phage antibody library solution, 10 in total14Adding the phage into an immune test tube, slowly shaking a mixer for 30 minutes at room temperature, standing for 90 minutes at room temperature, washing the immune test tube 10 times by PBST, washing the immune test tube 10 times by PBS, and spin-drying.
5. And (5) eluting the phage. Mu.l of 10M MHCl was added to the cuvette and slowly shaken for 30 minutes at room temperature, then 100. mu.l of 0.1M Tris-HCl was added to adjust the pH to 7.5, 2ml of fresh XL1-Blue suspension with OD 7.0 was added, the mixture was left to stand at 37 ℃ for 50 minutes, transferred to a 50ml centrifuge tube, and 5ml of 2 XTY was added and shaken for 30 minutes at 37 ℃.
6. Add 1. mu.l of 1011M13K07 phage was left to stand at 37 ℃ for 50 minutes at 3000g, centrifuged for 5 minutes, the supernatant was removed, and the pellet was dissolved in 50ml of 2 XTY + AMP + KAN + GLU medium at 30 ℃ overnight.
Phage in PEG4000 pellet Medium supernatant, centrifuged at 14000rpm, and 2ml of 1 XTE solubilized phage to a titer of about 1011。
5) Screening and enrichment of EPCR antibodies
The second, third, fourth and fifth round, the steps are the same as the first round.
6) ELISA for detecting antibody enrichment degree
1. Antigen coating: the ELISA plates were coated with protein C at a concentration of 1. mu.g/ml.
Blocking BSA;
3. add 10 to each ELISA well9Antibody phagemids with different enrichment degrees are diluted into PBST, 100 mu l/hole and kept stand for 1 hour.
Anti-phage antibody (rabbit derived), dilution 1:5000, 100. mu.l/well, 37 ℃, 1 hour.
5. Goat anti-rabbit IgG antibody (HRP) at a dilution of 1:10000, 100. mu.l/well, 37 ℃ for 1 hour.
As one round of screening is performed, antibodies that specifically recognize binding protein C are gradually enriched.
(II) monoclonal antibody colony selection
The antibody enrichment degree of cystatin C is detected by an ELISA method, 60 antibody single colonies are randomly selected from an antibody library obtained by a third round of screening in a platform stage, 30 antibody single colonies are randomly selected from an antibody library in a fourth round of the platform stage, M13K07 is used for auxiliary amplification respectively, centrifuged supernatant is directly used as an antibody solution for ELISA, the single colony with the highest photometric value is selected as a monoclonal antibody, 21 positive colonies are selected for sequencing in the fourth round and the fifth round of screening according to the result of monoclonal antibody phagemid (Phage-VHH) ELISA, and the result shows that the coding gene sequence of the nano antibody has a repeated phenomenon and only 9 specific sequences.
(III) screening by enzyme-linked immunosorbent assay (ELISA)
Enzyme-linked immunosorbent assays (ELISAs) can be used for: the method for detecting the macromolecular antigen, the specific antibody and the like has the advantages of rapidness, sensitivity, simplicity, convenience, easy standardization of a carrier and the like. The basic principle is that the antigen or antibody is immobilized and the antigen or antibody is labeled by enzyme, the antigen or antibody combined on the surface of the solid phase carrier still keeps the immunological activity, after a substrate solution is dripped, a color reaction appears, and the antigen or antibody labeled by the enzyme not only keeps the immunological activity, but also keeps the activity of the enzyme.
1) ELISA adsorption experiment procedure
1. Coating antigen: the antigen EPCR was dissolved in 50mM carbonate coating buffer (pH 9.6) to give an antigen concentration of 5. mu.g/ml, and 100. mu.l/well was applied to a 96-well microplate and left overnight at 4 ℃.
2. The next day, the coating solution was discarded, and washed 3 times with PBST, and 150. mu.l of 1% BSA was added to each well and blocked at 37 ℃ for 1 hour.
After 3 PBST washes, 100. mu.l of the antibody to be detected (1. mu.g/mL) was added to each well, and a control sample was added and incubated at 37 ℃ for 2 hours.
After 3 PBST washes, 100. mu.l of diluted secondary HRP-labeled antibody was added and incubated at 37 ℃ for 1 hour.
5. And (3) color development, namely washing the PBST for 5 times, gently patting the PBST to dry, adding 100 mu l of color development agent into each hole, and reacting for 5-10 min at room temperature.
6. Mu.l of stop solution was added to each well and mixed well to stop the color reaction.
7. The absorbance was measured at 450nm with a microplate reader.
2) Chessboard method for determining optimal reaction concentration of antigen and antibody
Antigen protein C is respectively coated with 4 concentration gradients of 5.00 mu g/ml, 2.50 mu g/ml, 1.25 mu g/ml and 0.625 mu g/ml, a negative control group is coated with BSA with corresponding concentration, after the coating is closed, the No.1 clone antibody is diluted by 5 percent skim milk powder in a gradient way, the antibody concentration is sequentially diluted by 10 times, and the concentration is respectively 1 multiplied by 109PFU/μl、1×108PFU/μl、1×107PFU/μl、1×106PFU/mul, adding the antibody diluent of each gradient into corresponding holes respectively, standing for 1h at 37 ℃ in each hole of 100 mul, and selecting No.1 clone to perform ELISA detection to determine the optimal reaction concentration of the antigen antibody. As shown in fig. 1.
With increasing antibody concentration, the OD450 also increased gradually, beginning at a concentration of 5 × 1010PFU per well into the plateau phase. In terms of antigen coating concentration, the binding capacity of the antibody and the antigen is not obviously increased along with the increase of the antigen concentration, and particularly under the condition of 5.0 mu g/ml and 2.5 mu g/ml, the increase range is small and is almost close to the plateau phase. Therefore, from the viewpoint of both the sensitivity and economy of the test, the optimum antigen coating concentration of the test was confirmed to be 2.5. mu.g/ml and the antibody incubation concentration was 5X 10 per well8PFU。
Example 3 obtaining Single Domain antibodies that bind EPCR with high affinity and Competition
1. Antibody affinity detection:
the affinity assay was performed using a conventional ELISA method as follows: fixing the candidate antibody on a carrier, then coating the antibody to react with the antigen, washing, then reacting with a detection antibody with a mark, washing, and finally carrying out chemiluminescence or enzyme-linked chromogenic reaction to detect a signal. The results are shown in FIG. 2.
2. Antibody competitive binding capacity assay:
the detection method by competitive binding ELISA was as follows: fixing the antigen EPCR on a carrier, then reacting the candidate antibody with the antigen (a control group), detecting the competitive binding capacity by adopting the steps of mixing the candidate antibody with the purified protein C, then reacting with the antigen (a detection group), washing, reacting with a labeled detection antibody, washing, and finally carrying out chemiluminescence or enzyme-linked chromogenic reaction to detect signals. The results are shown in FIG. 3.
Through affinity detection and competitive binding capacity detection, two antibody variable regions are obtained through screening, namely clone 6(clone 6) and clone 8(clone 8), and the two antibodies have stronger binding reaction with EPCR, but the binding capacity with EPCR is obviously reduced after the antibodies are mixed and incubated with protein C, which indicates that the binding with EPCR and the binding between protein C and EPCR are competitive. The variable region sequences of clones 6 and 8 were sequenced as follows:
clone 6:
GCGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACTTTGGATTATTATGCCATAGGCTGGTTCCGCCAGGCCCCAGGGAAGGAGCGCGAGGGGGTCTCATGTATTAGTAGTAGTGATGGTAGCACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCGACAAGTCTAGGTGGGGCATGGGTAGTAGCTGGTAACTGTCCCGCTCTTGACTTTAATTCCTGGGGCCAGGGGACCCAGGTC ACCGTCTCCTCG(SEQ ID NO:3)。
clone 8:
CAGGTACAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACTTTGGATTATTATGCCATAGGCTGGTTCCGCCAGGCCCCAGGGAAGGAGCGCGAGGGGGTCTCATGTATTAGTAGTAGTGATGGTAGCACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCGACAGGTCTAGGTGGGGCATGGACAGTAGCTAGTAACTGTCCCGCTCTTGACTTTGGTTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCG(SEQ ID NO:4)。
example 4 construction of heavy chain Single Domain antibody expression plasmid and purification of antibody
1. The variable regions (V) of the antibodies of clone 6 and clone 8 selected in example 3HH) The human antibody IgG1Fc sequence is connected behind the sequence, and is connected into the pcDNA3.1 expression vector through Hind III and Nhe1 enzyme cutting sites.
2. Expression of single domain antibody in HEK293 cells, purification:
(1) transforming the recombinant plasmid obtained in the step 1 with correct sequencing into escherichia coli DH5 alpha, selecting a single clone, inoculating the single clone into an LB liquid culture medium containing ampicillin, culturing overnight at 37 ℃ by 200 revolutions per minute of a shaking table, and extracting the plasmid;
(2) transfecting the extracted recombinant plasmid into HEK293 cells by utilizing PEI (polyetherimide) for expression, and culturing in a large scale;
(3) the SDS-PAGE electrophoresis detection results of the specific heavy chain single domain antibodies 6 and 8 obtained by collecting the cells and the supernatant and purifying the antibody protein by using a conventional affinity chromatography method are shown in FIG. 4.
[ example 5 ] in vivo tumor inhibition experiments with specific heavy chain single domain antibodies 6 and 8
1. The preparation method comprises the following steps: when the MDA-MB-231 breast cancer cells are in the logarithmic growth phase, firstly removing the culture solution, washing the MDA-MB-231 breast cancer cells twice by PBS, adding 0.25% protease into a culture bottle for digestion for 1 minute, removing 0.25% protease, adding 3ml of serum-free culture medium, blowing and beating the cells to prepare suspension, centrifuging for 5 minutes at 1000rpm, removing the supernatant, adding a proper amount of cell culture solution, and preparing the tumor cell culture solution.
2. Breast cancer cell culture solution transplantation: the tumor cell culture fluid is stained by trypan blue and counted by a blood cell counting plate, when the living cell ratio is more than 90 percent, the cell concentration is 1 multiplied by 106～1×107When the cells are used per ml, 0.2ml of the cells can be inoculated into the axillary abdominal wall under the skin of each nude mouse. Selecting 6-8 week old mice, weighing 16-22g, inoculating MDA-MB-231 cells into each mouse subcutaneously by 1 × 107A breast cancer model was constructed at 200. mu.l.
3. Antibody therapy: selecting mice with transplanted tumors which have already grown into tumors and are similar in tumor volume, randomly dividing the mice into a control group, an anti-EPCR clone No. 6 group and an anti-EPCR clone No. 8 group, wherein six mice in each group are treated by antibody after being inoculated for two weeks, adopting a mode of tail vein injection of the mice, respectively injecting No. 6 antibody and No. 8 antibody into an experimental group according to 3 mu g/kg, injecting physiological saline into the control group, observing the growth condition of the tumors every day, measuring and recording the tumor volume every three days during the period, and taking the tumors and taking pictures after 40 days of transplanting tumor cells. The tumor size data measured in vivo from three groups of mice were plotted in a graph, fig. 5, which shows that the control mice had faster tumor growth with increasing time, and that the in vivo tumor growth was significantly inhibited in mice injected with antibodies No. 6 and No. 8. FIG. 6 shows that after 40 days of inoculation, the mice were treated, and the tumors were removed and measured, and it can be seen that the mean tumor volume in the mice of the experimental group was significantly smaller than that in the control group, indicating that the anti-EPCR antibodies prepared by us can effectively inhibit the growth of breast cancer tumors in the mice.
Sequence listing
<110> Ding Heng
Wuhan Hengsai Biotech Co Ltd
<120> alpaca single domain antibody of anti-human endothelin C receptor and application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 128
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Ser Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Ser Leu Gly Gly Ala Trp Val Val Ala Gly Asn Cys Pro Ala
100 105 110
Leu Asp Phe Asn Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 2
<211> 128
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Ser Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Gly Leu Gly Gly Ala Trp Thr Val Ala Ser Asn Cys Pro Ala
100 105 110
Leu Asp Phe Gly Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 3
<211> 384
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gcggtgcagc tggtggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60
tcctgtgcag cctctggatt cactttggat tattatgcca taggctggtt ccgccaggcc 120
ccagggaagg agcgcgaggg ggtctcatgt attagtagta gtgatggtag cacatactat 180
gcagactccg tgaagggccg attcaccatc tccagagaca atgccaagaa cacggtgtat 240
ctgcaaatga acagcctgaa acctgaggac acggccgttt attactgtgc gacaagtcta 300
ggtggggcat gggtagtagc tggtaactgt cccgctcttg actttaattc ctggggccag 360
gggacccagg tcaccgtctc ctcg 384
<210> 4
<211> 384
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
caggtacagc tggtggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60
tcctgtgcag cctctggatt cactttggat tattatgcca taggctggtt ccgccaggcc 120
ccagggaagg agcgcgaggg ggtctcatgt attagtagta gtgatggtag cacatactat 180
gcagactccg tgaagggccg attcaccatc tccagagaca atgccaagaa cacggtgtat 240
ctgcaaatga acagcctgaa acctgaggac acggccgttt attactgtgc gacaggtcta 300
ggtggggcat ggacagtagc tagtaactgt cccgctcttg actttggttc ctggggccag 360
gggacccagg tcaccgtctc ctcg 384
<210> 5
<211> 238
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Met Leu Thr Thr Leu Leu Pro Ile Leu Leu Leu Ser Gly Trp Ala Phe
1 5 10 15
Cys Ser Gln Asp Ala Ser Asp Gly Leu Gln Arg Leu His Met Leu Gln
20 25 30
Ile Ser Tyr Phe Arg Asp Pro Tyr His Val Trp Tyr Gln Gly Asn Ala
35 40 45
Ser Leu Gly Gly His Leu Thr His Val Leu Glu Gly Pro Asp Thr Asn
50 55 60
Thr Thr Ile Ile Gln Leu Gln Pro Leu Gln Glu Pro Glu Ser Trp Ala
65 70 75 80
Arg Thr Gln Ser Gly Leu Gln Ser Tyr Leu Leu Gln Phe His Gly Leu
85 90 95
Val Arg Leu Val His Gln Glu Arg Thr Leu Ala Phe Pro Leu Thr Ile
100 105 110
Arg Cys Phe Leu Gly Cys Glu Leu Pro Pro Glu Gly Ser Arg Ala His
115 120 125
Val Phe Phe Glu Val Ala Val Asn Gly Ser Ser Phe Val Ser Phe Arg
130 135 140
Pro Glu Arg Ala Leu Trp Gln Ala Asp Thr Gln Val Thr Ser Gly Val
145 150 155 160
Val Thr Phe Thr Leu Gln Gln Leu Asn Ala Tyr Asn Arg Thr Arg Tyr
165 170 175
Glu Leu Arg Glu Phe Leu Glu Asp Thr Cys Val Gln Tyr Val Gln Lys
180 185 190
His Ile Ser Ala Glu Asn Thr Lys Gly Ser Gln Thr Ser Arg Ser Tyr
195 200 205
Thr Ser Leu Val Leu Gly Val Leu Val Gly Gly Phe Ile Ile Ala Gly
210 215 220
Val Ala Val Gly Ile Phe Leu Cys Thr Gly Gly Arg Arg Cys
225 230 235
Claims (8)
1. A single domain antibody of an anti-human endothelin C receptor is characterized in that the amino acid sequence of the single domain antibody is shown as SEQ ID NO.1 or SEQ ID NO. 2.
2. The single domain antibody of claim 1, wherein the nucleotide sequence encoding the single domain antibody of SEQ ID No.1 is set forth in SEQ ID No. 3; the nucleotide sequence of the single domain antibody shown in the SEQ ID NO.2 is shown in SEQ ID NO. 4.
3. A recombinant expression vector comprising the nucleotide sequence of claim 2.
4. The recombinant expression vector of claim 3, wherein the vector is pcDNA3.1.
5. A host cell capable of expressing a single domain antibody against a human endothelin C receptor comprising the nucleotide sequence of claim 2.
6. The host cell of claim 5, wherein the host cell is a HEK293 cell.
7. Use of the single domain antibody against human endothelin C receptor according to claim 1 or 2 for the preparation of a reagent for detecting, inhibiting or reducing human endothelin C receptor.
8. Use of a single domain antibody against human endothelin C receptor as claimed in claim 1 or 2 for the manufacture of a medicament for the treatment of a disease targeting the protein C-EPCR signaling pathway, wherein the disease targeting the protein C-EPCR signaling pathway is breast cancer.
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Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE69732847T2 (en) * | 1996-11-08 | 2006-04-13 | Oklahoma Medical Research Foundation, Oklahoma | ENDOTHEL SPECIFIC EXPRESSION UNDER INFLUENCE OF EPCR CONTROL ELEMENTS |
| WO2005069003A1 (en) * | 2003-06-27 | 2005-07-28 | Oklahoma Medical Research Foundation | Methods for predicting susceptibility to cardiovascular disease |
| JP2005035943A (en) * | 2003-07-16 | 2005-02-10 | Chemo Sero Therapeut Res Inst | Proliferation inhibitor of malignant tumor cell |
| CA2479968A1 (en) * | 2004-09-01 | 2006-03-01 | Keith R. Walley | Protein c and endothelial protein c receptor polymorphisms as indicators of patient outcome |
| ES2268961B1 (en) * | 2005-03-17 | 2008-03-16 | Proyecto De Biomedicina Cima, S.L. | NEW EPCR SOLUBLE PROTEIN OF NON-PROTEOLITICAL ORIGIN AND ITS USE. |
| US8153766B2 (en) * | 2007-10-26 | 2012-04-10 | Oklahoma Medical Research Foundation | Monoclonal antibodies against activated protein C |
| BRPI1007917A2 (en) * | 2009-02-06 | 2019-09-24 | Astute Medical Inc | method for assessing renal status in an individual, and use of one or more markers of renal injury |
| CN107502658B (en) * | 2017-07-18 | 2021-07-27 | 深圳市亿立方生物技术有限公司 | Method for preparing cancer diagnosis and treatment products aiming at expression of EPCR gene or EPCR protein |
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Effective date of registration: 20210302 Address after: 4 / F, building 1, 356 ZHENGBO Road, Lingang New District, China (Shanghai) pilot Free Trade Zone, Fengxian District, Shanghai, 201413 Applicant after: Shanghai Hengsai Biotechnology Co.,Ltd. Address before: Room 1703, unit 1, building 2, Chutian City Yayuan, 230 Zhongbei Road, guoguohu street, Wuchang District, Wuhan City, Hubei Province, 430000 Applicant before: Ding Heng Applicant before: WUHAN HENGSAI BIOLOGICAL TECHNOLOGY Co.,Ltd. |
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