CN116178535B - Nanometer antibody targeting Yes related protein, and preparation method and application thereof - Google Patents
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Abstract
The application relates to the technical field of biology, in particular to a nano antibody targeting Yes related proteins, a preparation method and application thereof. The provided nano antibody for targeting the Yes-related protein comprises a nano antibody NbE7, wherein the amino acid sequence of the nano antibody NbE7 is shown as seq. ID No.1, the provided nano antibody NbE7 has higher affinity with the Yes-related protein YAP, can target the Yes-related protein YAP through the antibody, has high-strength binding capacity with the Yes-related protein YAP, is favorable for developing nano antibody-related drugs of the Yes-related protein YAP, and has wide application.
Description
Technical Field
The application relates to the technical field of biology, in particular to a nano antibody targeting Yes related proteins, a preparation method and application thereof.
Background
Yes-related protein (Yes-associated protein, YAP) is a transcriptional coactivator and is also a key effector in the Hippo signaling pathway (Hippo pathway), and up-regulation and activation of this protein promotes cell proliferation, growth and anti-apoptosis, and is closely related to the development and progression of many diseases. Structurally, YAP is a multi-domain protein with an N-terminal proline-rich domain, a TEAD binding domain (TAD), a WW domain, an SH3 binding motif, a transcriptional activator binding domain (TID), and a C-terminal PDZ binding motif. Wherein the TID and TAD domains are essential for YAP to exert transcriptional promotion. Since YAP lacks a DNA binding domain, it is involved in expression regulation of genes in the identity of co-activators by interacting with DNA binding transcription factors. The TID domain serves primarily to recruit transcriptional activators, while the TAD domain binds to TEA domain family members 1-4 (TEAD 1-4), which together promote transcription of downstream target genes.
The Hippo signaling pathway, also known as the Salvador/Warts/Hippo (SWH) pathway, is named Hippo (Hpo), a protein kinase mainly derived from Drosophila, and consists of a series of conserved kinases that control organ size, mainly by regulating cell proliferation and apoptosis. Its core components include upstream kinase cascade transcription and downstream effectors. The upstream kinase cascade of the Hippo signaling pathway in mammalian cells mainly includes mammalian STE 20-like kinase 1/2 (MST 1/2), large tumor suppressor gene 1/2 (LATS 1/2), sav family WW domain protein 1 (SAV 1), and MOB kinase activator 1 (MOB 1), the two major downstream effectors being YAP, TAZ. The Hippo signaling pathway promotes activation of the Hippo pathway by receiving externally generated growth inhibitory signals, such as Merlin, scribble, which are localized at cell adhesion attachment sites or cell membrane surfaces when cell densities are too high, by forming complexes with a range of kinases, further promoting phosphorylation at the Ser-127 (p-YAPSer 127) and Ser-89 (p-TAZSer 89) residues of YAP and TAZ after mediating phosphorylation of MST1/2, LATS1/2, allowing them to bind to 14-3-3 proteins, thus retaining them in the cytoplasm, or by phosphorylating Ser-127 (p-YAPSer 381) and Ser-89 (p-TAZSer 311) of TAZ and further degrading by the ubiquitinated proteasome system (USP). Thereby inhibiting YAP/TAZ from entering the nucleus, and down regulating the expression of downstream target genes, thereby producing the effect of inhibiting the growth and proliferation of cells.
Mutant inactivation of the Hippo signaling pathway reduces the activity of the upstream kinase cascade, ultimately leading to reduced YAP phosphorylation levels, causing nuclear accumulation, enhancing transcriptional expression of downstream target genes, and promoting tumor development. Research shows that the phenomenon of up-regulation of YAP activity exists in most tumors at present. In Primary Liver Cancer (PLC), YAP expression is associated with tumor progression, differentiation, and up-regulation of YAP appears to be positively correlated with alpha fetoprotein levels; overactive mutations in YAP are commonly observed in non-small cell lung cancer (NSCLC) patients, while down-regulation of LATS2 is observed in 60% of cases, and up-regulation of YAP is observed in tumors such as gastric cancer, colon cancer, ovarian cancer, and prostate cancer.
Given the importance of the Hippo-YAP pathway in tumor development, current therapies targeting this pathway are also emerging in the field of view, and inhibition of YAP and TEAD interactions has been shown in current preclinical studies to block this interaction to inhibit tumor growth and progression. Researchers have found that Verteporfin (VP) binds to YAP and alters its conformation to effectively block YAP-TEAD interactions, thereby alleviating tumor cell growth due to YAP overexpression, but there are still limitations in clinical use for treatment of YAP-dependent tumors due to its proteotoxicity and poor pharmacokinetics. In addition, prevention of YAP nuclear localization transfer is also a targeting strategy, and drugs such as statin lipid-lowering drugs and zoledronic acid have been found to block YAP nuclear localization by acting on kinases downstream of Rho GTPase; the tankyrase inhibitor XAV939 blocks AMOT degradation, localizes YAP to the cytoplasm, reducing YAP nuclear accumulation. In addition, YAP overexpression mediated tumorigenesis was also found to be associated with immune escape, and the overactivated YAP protein in tumor cells was able to induce the expression of both CCL2 and CSF1 chemokines, thereby strongly recruiting macrophages to the tumor initiating cell periphery. Further studies indicate that recruited macrophages are type 2 macrophages that allow tumor initiating cells to escape immune surveillance through T cell inhibition, avoiding clearance. Thus, inhibition of macrophage recruitment is probably a new therapeutic approach for YAP-highly expressed tumor cells.
By observing the series of therapeutic agents and the direction of treatment, it was found that tumors caused by inactivation of the Hippo signaling pathway could direct spearhead towards YAP overactivation, and thus how to eliminate YAP effects became a key step in the treatment of YAP-dependent tumors. Current treatment regimens for YAP hyperactivated tumors are essentially all by inhibiting their transcriptional promotion of downstream target genes, reducing their role in the nucleus. This type of treatment may have a long onset of action, incomplete onset of action, and off-target potential. Therefore, development of a novel material capable of directly targeting YAP protein is urgently needed, ubiquitin-proteasome system (UPS), autophagy and the like are utilized to promote the degradation of YAP protein from the source, eliminate a subsequent series of effects caused by the degradation, and inhibit the occurrence and development of YAP overactive tumor.
Disclosure of Invention
The application aims to provide a nano antibody targeting Yes-related proteins, a preparation method and application thereof, and aims to solve the problem that the nano antibody targeting Yes-related proteins is lack to perform corresponding actions in the prior art.
In a first aspect, the application provides a nanobody targeting a Yes-related protein, the nanobody comprising a nanobody NbE7, wherein the amino acid sequence of the nanobody NbE7 is shown as seq id No. 1.
In a second aspect, the application provides a method for preparing a nanobody targeting Yes-related proteins, comprising the steps of:
designing and synthesizing Yes related protein YAP-GST gene, and carrying out protein expression and purification to obtain Yes related protein YAP target protein;
coating the Yes related protein YAP target protein on an immune tube for enrichment screening to obtain a phage library;
selecting monoclonal molecules from the eluent of the phage library, performing ELISA (enzyme-linked immunosorbent assay) verification, performing second-generation sequencing, and synthesizing a gene sequence of the nano antibody according to a sequencing result;
cloning the gene sequence of the nano antibody into an expression vector to obtain a recombinant plasmid, transferring the recombinant plasmid into a host cell to induce expression and purifying to obtain the nano antibody targeting Yes related protein.
In a third aspect, the application provides the use of a nanobody targeting a Yes-related protein in the manufacture of a medicament for targeting inflammation or immunotherapy associated with YAP protein.
According to the nano antibody for targeting the Yes-related protein, which is provided by the first aspect of the application, the nano antibody comprises a nano antibody NbE7, wherein the amino acid sequence of the nano antibody NbE7 is shown as seq. ID No.1, the provided nano antibody NbE7 has higher affinity with the Yes-related protein YAP, the antibody can target the Yes-related protein YAP, the high-strength binding capacity of the antibody with the Yes-related protein YAP is favorable for developing nano antibody related drugs of the Yes-related protein YAP, and various small molecular compounds, PROTAC (protein-targeting chimeras) and other substances are connected to the nano antibody, so that the degradation of the YAP protein from the source is promoted by utilizing Ubiquitin-Proteasome System (UPS), autophagy and the like, a series of effects caused by the degradation of the protein are eliminated, and the development of YAP overactive tumor is inhibited, so that the nano antibody is very widely applied.
According to the preparation method of the nano antibody targeting the Yes-related protein, which is provided by the second aspect of the application, three rounds of screening proteins of the Yes-related protein YAP are completed based on a phage natural nano antibody library, ELISA verification shows that one nano antibody NbE7 has higher binding force with the Yes-related protein YAP, so that the nano antibody NbE7 has better application value, and the preparation method is quick, simple and convenient, is favorable for large-scale screening, and improves screening efficiency.
The third aspect of the present application provides an application of a nano antibody targeting a Yes-related protein in preparing a drug targeting inflammation or immunotherapy related to a YAP protein, because the obtained nano antibody targeting a Yes-related protein comprises a nano antibody NbE7, which has a high-strength binding capacity to a Yes-related protein YAP, the development of a nano antibody-related drug related to a Yes-related protein YAP can be facilitated, the nuclear invasion of a dephosphorylated YAP protein can be affected by binding a small molecular compound, related gene transcription induced in the nucleus by the dephosphorylated YAP protein can be eliminated, and the YAP protein can be degraded by ubiquitination or delivered to a lysosome by fusion with a nano antibody targeting HSP90/70, so that the YAP protein overactivated in tumor cells can be eliminated, and the broad application is facilitated.
Drawings
In order to more clearly illustrate the embodiments of the application or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the application, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
Wherein:
FIG. 1 is a schematic diagram of YAP-GST protein purification in one embodiment.
FIG. 2 is a schematic diagram of screening of phage nanobody libraries in one embodiment.
FIG. 3 is a schematic representation of nanobody purification in one embodiment.
FIG. 4 is a graph of ELISA binding validation analysis of nanobodies in one example.
FIG. 5 is a schematic diagram showing affinity analysis of NbE7 and YAP fusion proteins in one embodiment.
Detailed Description
The following description of the embodiments of the present application will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
According to a first aspect of the embodiment of the application, a nano-antibody targeting Yes-related proteins is provided, wherein the nano-antibody comprises a nano-antibody NbE7, and the amino acid sequence of the nano-antibody NbE7 is shown as seq. ID No. 1.
According to the nano antibody for targeting the Yes-related protein provided by the embodiment of the application, the nano antibody comprises a nano antibody NbE7, wherein the amino acid sequence of the nano antibody NbE7 is shown as seq. ID No.1, the provided nano antibody NbE7 has higher affinity with the Yes-related protein YAP, the antibody can target the Yes-related protein YAP, the high-strength binding capacity of the antibody with the Yes-related protein YAP is beneficial to developing nano antibody related medicines of the Yes-related protein YAP, and various small molecular compounds, PROTAC (protein-targeting chimeras) and other substances are connected to the nano antibody, so that the degradation of the YAP protein from the source is promoted by using Ubiquitin-Proteasome System (UPS), autophagy and the like, a series of effects caused by the degradation of the protein are eliminated, and the development of YAP overactive tumor is inhibited, so that the nano antibody is very widely applied.
In some embodiments, the nanobody NbE7 has an amino acid sequence as shown in seq.id No.1, wherein seq.id No.1 is specifically:
MAVQLVESGGGSVQPGGSLRLSCVASRNIFNRGGMGWYRQAPGKQRELVTAITTDGIIHYTDSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNTALLEGTTWFSISPFDYWGQGTQVTVSS。
in some embodiments, the nanobody comprises 4 framework regions FR1, FR2, FR3, FR4, and 3 complementarity determining regions CDR1, CDR2, CDR3.
In some embodiments, in the nanobody NbE7, the amino acid sequence of FR1 is shown in SEQ ID No.2, and SEQ ID No.2 specifically is: MAVQLVESGGGSVQPGGSLRLSCVASRNIF.
In some embodiments, in the nanobody NbE7, the amino acid sequence of FR2 is shown in SEQ ID No.3, and SEQ ID No.3 specifically is: WYRQAPGKQRELVTAI.
In some embodiments, in the nanobody NbE7, the amino acid sequence of FR3 is shown in SEQ ID No.4, and SEQ ID No.4 specifically is: RFTISRDNAKNTVYLQMNSLKPEDTAVYYCNT.
In some embodiments, in the nanobody NbE7, the amino acid sequence of FR4 is shown in SEQ ID No.5, and SEQ ID No.5 specifically is: WGQGTQVTVSS.
In some embodiments, in the nanobody NbE7, the amino acid sequence of CDR1 is shown in SEQ ID No.6, and SEQ ID No.6 is specifically: NRGGMG.
In some embodiments, in the nanobody NbE7, the amino acid sequence of CDR2 is shown in SEQ ID No.7, and SEQ ID No.7 specifically: TTDGIIHYTDSVRG.
In some embodiments, in the nanobody NbE7, the amino acid sequence of CDR3 is shown in SEQ ID No.8, and SEQ ID No.8 specifically is: ALLEGTTWFSISPFDY.
In some embodiments, the nanobody NbE7 has a base sequence as shown in SEQ ID No.9, wherein SEQ ID No.9 is specifically:
ATGGCGGTGCAGCTGGTGGAGTCTGGGGGAGGCTCGGTCCAGCCTGGGGGGTCTCTGAGGCTCTCCTGTGTAGCCTCTCGAAACATCTTCAATCGCGGTGGCATGGGCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGTTGGTCACAGCCATTACTACTGATGGTATCATACACTATACAGACTCCGTGAGGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTCTATTATTGTAATACAGCTCTTTTGGAGGGTACTACTTGGTTCTCGATTTCCCCGTTTGATTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA。
the second aspect of the embodiment of the application provides a preparation method of a nano antibody targeting Yes related proteins, which comprises the following steps:
s01, designing and synthesizing a Yes related protein YAP-GST gene, and carrying out protein expression and purification to obtain a Yes related protein YAP target protein;
s02, coating the Yes related protein YAP target protein on an immune tube for enrichment screening to obtain a phage library;
s03, selecting monoclonal molecules from eluent of the phage library, performing ELISA (enzyme-linked immunosorbent assay) verification, performing second-generation sequencing, and synthesizing a gene sequence of the nano antibody according to a sequencing result;
s04, cloning the gene sequence of the nano antibody into an expression vector to obtain a recombinant plasmid, transferring the recombinant plasmid into a host cell to induce expression, and purifying to obtain the nano antibody targeting the Yes related protein.
According to the preparation method of the nano antibody targeting the Yes related protein, which is provided by the embodiment of the application, three rounds of screening proteins of the Yes related protein YAP are completed based on a phage natural nano antibody library, ELISA verification shows that one nano antibody NbE7 has higher binding force with the Yes related protein YAP, so that the nano antibody NbE7 has better application value, and the preparation method is quick, simple and convenient, is beneficial to large-scale screening, and improves screening efficiency.
In the step S01, the Yes related protein YAP-GST gene is designed and synthesized, and protein expression and purification are carried out to obtain the Yes related protein YAP target protein.
In some embodiments, the steps of protein expression and purification are as follows: a) To prevent inclusion body formation and protein degradation, induction conditions were sought at 16 ℃ by different concentrations of IPTG; b) Performing a large amount of induction expression according to the pre-experiment induction conditions, and performing bacteria breaking under the working condition of 1000W of a high-pressure bacteria breaker; c) Centrifuging 17000g at 4deg.C for 30min, and incubating supernatant with Ni filler at 4deg.C for 1 hr; d) Gradient concentration imidazole elution target protein; e) After Ni column purification, molecular sieve separation was performed to remove the impurity proteins, AKTA parameters were set at 0.5mL flow rate/min, and collected every 1 mL. f) The purity of the target protein is determined according to the electrophoresis result, and the protein concentration is measured by the BCA method.
In step S02, the Yes related protein YAP target protein is coated on an immune tube for enrichment screening to obtain a phage library.
In some embodiments, the concentration of the protein of interest is 40 to 45 μg/mL.
In some embodiments, in the step of enriching the screening, 2-3 rounds of enriching screening are performed.
In some embodiments, the natural alpaca-derived phage display nanobody library is screened using an immune tube method, with a selected phage display library capacity of 2x10 9 . The screening steps are as follows: a) Coating target protein on an immune tube according to the concentration of 40 mug/mL, and carrying out enrichment screening for 3 rounds; obtaining a phage library.
In step S03, monoclonal molecules are selected from the eluent of the phage library, ELISA verification is carried out, second generation sequencing is carried out, and the gene sequence of the nanobody is synthesized according to the sequencing result.
In some embodiments, using a third round of phage eluate plating, 192 monoclonal antibodies were randomly picked for ELISA validation, the ELISA 96-well plates were simultaneously coated with GST, BSA as controls, and the ELISA reading was 3-fold greater than the corresponding GST, BSA reading and greater than 0.5 as positive standard; and sequencing the positive monoclonal identified by the phage ELISA for 2 times to determine sequence information, extracting the sequence to obtain a nanobody protein sequence, performing comparative analysis on the sequence to obtain the distribution frequency of the positive sequence, and synthesizing the gene sequence of the nanobody according to the sequencing result.
In the step S04, the gene sequence of the nano antibody is cloned into an expression vector to obtain a recombinant plasmid, and the recombinant plasmid is transferred into a host cell to induce expression and purify to obtain the nano antibody targeting Yes related protein.
In some embodiments, the expression vector is selected from pCold vectors.
In some embodiments, nanobody gene sequences are cloned into pCold vectors while fusion expressing hemagglutinin tags (hemagglutinin HA tag) for subsequent detection. The expression purification steps are as follows: a) To prevent inclusion body formation and protein degradation, induction was performed at 16 ℃ using IPTG at a concentration of 0.2 mM; b) Performing a large amount of induction expression according to the pre-experiment induction conditions, and performing bacteria breaking under the working condition of 1000W of a high-pressure bacteria breaker; c) Centrifuging 17000g at 4deg.C for 30min, and incubating supernatant with Ni filler at 4deg.C for 1 hr; g) After Ni column purification, molecular sieve separation was performed, AKATA parameters were set at 0.5mL flow rate/min, and collected every 1 mL.
Further, the method also comprises the step of carrying out an Elisa experiment on the obtained nano-antibody, wherein the experiment is used for verifying whether the nano-antibody expressed and purified in vitro can directly interact with antigen proteins purified in vitro. The method comprises the following steps: a) Diluting antigen protein to 5ug/ml with PBS, diluting GST protein to 2.5ug/ml, plating at 100 ul/well, coating the well plate, and standing at 4deg.C overnight; b) Blocking 2h at room temperature with 3% PBS/BSA, 200 ug/well; c) Different concentrations of nanobody are prepared by 1% BSA/PBST, 100 ug/hole is prepared, and the mixture is incubated for 1h at room temperature; d) Incubation of secondary antibody anti-HA HRP (1: 3000 1h at room temperature; e) TMB color development; e) Stopping the reaction by using a stopping solution; g) The absorbance was measured at 450nm using a microplate reader and a curve was prepared based on the absorbance.
Further, the method also comprises the step of performing a surface plasmon resonance (surface plasmon resonance, SPR) test on the obtained nano-antibody, wherein the surface plasmon resonance test is used for further verifying the combination of the antigen and the nano-antibody, and calculating the equilibrium constant of the antigen and the nano-antibody. Purified antigen proteins are immobilized on a chip, nano antibodies with different concentrations are sequentially added to analyze the affinity with the antigen proteins, reaction signals within 360 seconds are recorded, a kinetic curve is made, and relevant parameters are calculated.
In a third aspect, the embodiment of the application provides an application of a nano antibody targeting Yes related proteins in preparing a drug targeting YAP protein related inflammation or immunotherapy.
According to the application of the nano antibody targeting the Yes-related protein in preparing the medicine for targeting the relevant inflammation or immunotherapy of the YAP protein, the obtained nano antibody targeting the Yes-related protein comprises a nano antibody NbE7 which has high-strength binding capacity to the Yes-related protein YAP, so that development of the nano antibody-related medicine for the Yes-related protein YAP can be facilitated, the nuclear invasion of the dephosphorylated YAP protein is influenced by combining a small molecular compound, related gene transcription caused by the dephosphorylated YAP protein in the nucleus is eliminated, and the YAP protein can be degraded through ubiquitination or delivered to a lysosome through fusion with the nano antibody targeting HSP90/70, so that the YAP protein which is excessively activated in tumor cells is eliminated, and the wide application is facilitated.
In some embodiments, the nanobody targeting the Yes-related protein affects the nuclear entry of the dephosphorylated YAP protein by binding small molecule compounds, eliminating related gene transcription initiated within the nucleus by the dephosphorylated YAP protein.
In some embodiments, the nanobody targeting the Yes-related protein degrades or delivers the YAP protein to lysosomes through fusion with the nanobody targeting HSP90/70 to eliminate the YAP protein from overactivation within the tumor cell.
The following description is made with reference to specific embodiments.
Example 1
Nano antibody for targeting Yes related protein and preparation method thereof
(1) YAP nanobody expression purification
YAP-GST gene was designed and synthesized and purified protein was expressed for nanobody screening. The expression purification steps are as follows: a) To prevent inclusion body formation and protein degradation, induction conditions were sought at 16 ℃ by different concentrations of IPTG; b) Performing a large amount of induction expression according to the pre-experiment induction conditions, and performing bacteria breaking under the working condition of 1000W of a high-pressure bacteria breaker; c) Centrifuging 17000g at 4deg.C for 30min, and incubating supernatant with Ni filler at 4deg.C for 1 hr; d) Gradient concentration imidazole elution target protein; e) After Ni column purification, molecular sieve separation was performed to remove the impurity proteins, AKTA parameters were set at 0.5mL flow rate/min, and collected every 1 mL. f) The purity of the target protein is determined according to the electrophoresis result, and the protein concentration is measured by the BCA method.
(2) Nanobody screening and ELISA (enzyme-Linked immuno sorbent assay) primary verification of positive clones
Screening a natural alpaca-derived phage display nanobody library by adopting an immune tube method, wherein the selected phage display library capacity is 2x109. The screening steps are as follows: a) Coating target protein on an immune tube according to the concentration of 40 mug/mL, and carrying out enrichment screening for 3 rounds; b) Using third phage eluent to plate, randomly picking 192 monoclonal antibodies for ELISA verification, making an ELISA 96-well plate and coating GST and BSA as a control, and taking ELISA reading being 3 times greater than corresponding GST and BSA reading and reading being greater than 0.5 as a positive standard; c) Sequencing and determining sequence information of positive monoclonal sent company identified by phage ELISA for 2 times, extracting sequences to obtain nanometer antibody protein sequences, and comparing and analyzing the sequences to obtain distribution frequency of positive sequences.
(3) Purification expression of nanobodies
The nanobody gene sequence was cloned into pCold vector while fusion expressing hemagglutinin tag (hemagglutinin HA tag) for subsequent detection. The expression purification steps are as follows: a) To prevent inclusion body formation and protein degradation, induction was performed at 16 ℃ using IPTG at a concentration of 0.2 mM; b) Performing a large amount of induction expression according to the pre-experiment induction conditions, and performing bacteria breaking under the working condition of 1000W of a high-pressure bacteria breaker; c) Centrifuging 17000g at 4deg.C for 30min, and incubating supernatant with Ni filler at 4deg.C for 1 hr; g) Performing molecular sieve separation after Ni column purification, setting AKATA parameters at a flow rate of 0.5 mL/min, and collecting once every 1 mL; obtaining the nano antibody NbE7 targeting the Yes related protein.
Elisa test of (two) nanobody
This experiment was used to verify whether expression of purified nanobodies in vitro and in vitro purified antigen proteins could directly interact. The method comprises the following steps: a) Diluting antigen protein to 5ug/ml with PBS, diluting GST protein to 2.5ug/ml, plating at 100 ul/well, coating the well plate, and standing at 4deg.C overnight; b) Blocking 2h at room temperature with 3% PBS/BSA, 200 ug/well; c) Different concentrations of nanobody are prepared by 1% BSA/PBST, 100 ug/hole is prepared, and the mixture is incubated for 1h at room temperature; d) Incubation of secondary antibody anti-HA HRP (1: 3000 1h at room temperature; e) TMB color development; e) Stopping the reaction by using a stopping solution; g) The absorbance was measured at 450nm using a microplate reader and a curve was prepared based on the absorbance.
(III) surface plasmon resonance experiments (surface plasmon resonance, SPR)
This experiment was used to further verify the binding of antigen and nanobody and calculate the equilibrium constants of both. Purified antigen proteins are immobilized on a chip, nano antibodies with different concentrations are sequentially added to analyze the affinity with the antigen proteins, reaction signals within 360 seconds are recorded, a kinetic curve is made, and relevant parameters are calculated.
Analysis of results
YAP-GST protein expression purification
YAP protein has molecular mass of 65kDa, and the fusion protein formed by the truncated fragment of the marker sequence, GST and His is purified, and the protein has the size of about 66kDa and is used as a screening target of nano antibodies. The YAP fusion gene is synthesized after codon optimization to carry out escherichia coli induced expression purification, in order to avoid protein degradation and inclusion body generation, 0.4mM IPTG is used for induction at a low temperature of 16 ℃ overnight, and finally, after purification through a Ni column and a molecular sieve, the target protein with high purity is obtained for subsequent nano antibody screening as shown in figure 1.
Screening, identifying and purifying YAP nano antibody
First, YAP fusion proteins were screened for phage nanobody libraries, and after two or three rounds of screening, the library was enriched approximately 100 and 10 fold as shown in FIG. 2. About 192 clones from the library obtained in the third round were picked for preliminary verification by phage ELISA twice, 72 positive clones were initially identified for sequencing, and the sequencing result showed 72 normal. According to the sequence before and after the nano antibody, the nano antibody sequence can be obtained from the sequencing result, 72 sequences are translated into amino acids, then the sequences are sequenced and subjected to multi-sequence comparison, 14 different nano antibody sequences are obtained, the 14 nano antibodies are purified, as shown in figure 3, preliminary verification shows that a false positive and a weak binding force exist, subsequent experiments exclude the false positive and the weak binding force, and the nano antibody with higher purity is obtained after purification.
Affinity detection of yap nanobodies
The binding of YAP nanobodies and YAP fusion proteins is further detected by ELISA, and the result shows that compared with the GST proteins in a control group, as shown in A of fig. 4, the ELISA detects the binding condition of the 12 nanobodies and the YAP fusion proteins, and the result shows that the 12 nanobodies (E1-E11 and E13) participating in the experiment all show better binding capacity, which indicates that the binding capacity of the purified 12 YAP nanobodies to the YAP fusion proteins in the experimental group is higher, and the nano antibodies have higher binding capacity to the YAP. Further, as shown in FIG. 4B, ELISA examined the binding capacity of the 12 nanobodies to GST fragments in the fusion protein to exclude false positive results (as a control) generated by the fragments, and the results showed that the binding of the 12 nanobodies to GST fragments was weak.
Next, a nanobody NbE7 with the strongest binding force was selected from the Elisa results, and a biocare experiment was performed to further examine the affinity constant of the nanobody with YAP fusion protein, while using GST as a control, as shown in fig. 5, the affinity of the nanobody with YAP fusion protein was found to be at nanomolar level, specifically, the affinity of NbE7 with YAP fusion protein was found to be 17.27nM. Further shows that the obtained nano antibody NbE7 has good binding capacity with YAP.
In summary, the provided nano antibody for targeting the Yes-related protein comprises a nano antibody NbE7, wherein the amino acid sequence of the nano antibody NbE7 is shown as seq. ID No.1, the provided nano antibody NbE7 has higher affinity with the Yes-related protein YAP, the antibody can target the Yes-related protein YAP, the high-strength binding capacity of the antibody with the Yes-related protein YAP is beneficial to developing nano antibody related medicines of the Yes-related protein YAP, and various small molecular compounds, PROTAC (protein-targeting chimeras) and other substances are connected to the nano antibody, so that the Ubiquitin-proteasome system (Ubiquinin-Proteasome System, UPS), autophagy and the like are utilized to promote the degradation of the YAP protein from the source, the subsequent series of effects caused by the degradation of the YAP protein are eliminated, the development of YAP over-activated tumor is inhibited, and the application is quite wide.
The foregoing disclosure is illustrative of the present application and is not to be construed as limiting the scope of the application, which is defined by the appended claims.
Claims (4)
1. A nanobody targeting Yes related proteins, which is characterized in that the nanobody is a nanobody NbE7, wherein the amino acid sequence of the nanobody NbE7 is shown as seq. ID No. 1.
2. The Yes-related protein targeting nanobody of claim 1, wherein the nanobody comprises 4 framework regions FR1, FR2, FR3, FR4 and 3 complementarity determining regions CDR1, CDR2, CDR3;
in the nano antibody NbE7, the amino acid sequence of FR1 is shown as SEQ ID NO.2, the amino acid sequence of FR2 is shown as SEQ ID NO.3, the amino acid sequence of FR3 is shown as SEQ ID NO.4, the amino acid sequence of FR4 is shown as SEQ ID NO.5, the amino acid sequence of CDR1 is shown as SEQ ID NO.6, the amino acid sequence of CDR2 is shown as SEQ ID NO.7, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 8.
3. The nanobody targeting Yes-related protein according to claim 1, wherein the base sequence of the nanobody NbE7 is shown as seq.id No. 9.
4. The use of a nanobody targeting a Yes-related protein according to any one of claims 1-3 in the preparation of a medicament for targeting YAP protein.
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