CN116178538B - Nanoantibodies targeting heat shock protein 70 and preparation methods and applications thereof - Google Patents
Nanoantibodies targeting heat shock protein 70 and preparation methods and applications thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a heat shock protein 70-targeted nano antibody, and a preparation method and application thereof. A nanobody targeting heat shock protein 70 is disclosed, comprising nanobody Nb H5, wherein the amino acid sequence of nanobody Nb H5 is shown as seq id No. 1. The provided nanometer antibody NbH targeting the heat shock protein 70 has higher affinity with the heat shock protein 70, can target the heat shock protein 70 through the nanometer antibody NbH, realizes the effect of starting the degradation of ubiquitinated proteasome, is favorable for constructing PROTAC complex for application, and has great clinical application value. Meanwhile, the nano antibody has small molecular weight, is favorable for drug assembly, is favorable for positioning when PROTAC complex is formed, does not influence the effect of targeted drugs, and has wide application.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a heat shock protein 70 targeting nano antibody, a preparation method and application thereof.
Background
Heat shock proteins (Heat shock protein, HSPs), also known as Stress proteins (sps), are a group of polypeptides that are highly conserved in structure, widely found in prokaryotic, eukaryotic cells. Heat shock proteins are involved in a variety of vital processes within cells, such as: chaperone function (maintenance of intracellular protein homeostasis, promotion of correct folding and assembly of proteins), enhancement of cell heat resistance and thermal protection (enhancement of cell tolerance to high temperature, maintenance of intracellular environment homeostasis at high temperature), anti-apoptosis (prevention of apoptosis phenomenon of cells under stress), anti-oxidation (promotion of SOD production of cells to resist damage of oxygen radicals), inflammation protection (inhibition of cytokine action, reduction of its content in circulation).
This type of protein was first found in Drosophila, and after 30 minutes of exposure to Drosophila larvae to 32℃, researchers found that a new bulge appeared on the chromosome of its large salivary glands, indicating that this region was enhanced in transcriptional activity, and was also called a "heat shock response", which was further found in subsequent studies to be accompanied by a specific set of proteins produced, while similar results were also observed on animals, bacteria, from which heat shock proteins were brought into the field of view of humans. Heat shock proteins are divided into several families, HSP100, HSP90, HSP70, HSP60 and small molecule shock proteins, according to their protein size, each family having a number of different members. Wherein the most HSP70 family members, there are 21 proteins under the HSP70 family, which are a group of evolutionarily highly conserved stress proteins mainly including HSP68, 72, 73; HSC70, GRP75, bip, etc.
HSP70 is a highly conserved polypeptide containing 641 amino acids and mainly consists of an N-terminal nucleotide binding domain (NBD, 45 kDa/1-381 aa) and a substrate binding domain (SBD, 25 kDa/398-505 aa), and comprises a connector (382-397 aa), an SBD bottom domain (506-605 aa) and a C-terminal domain (606-641 aa) in addition to two main domains. The N-terminal binding domain (NBD) is a V-shaped structural framework and consists of two ATP binding subdomains, and mainly plays a role in binding ATP and ADP; the Substrate Binding Domain (SBD) is likewise composed of two subdomains, the β -sheet domain (sbdβ) and the α helix (sbdα), respectively, that bind predominantly to a variety of HSP70 substrate proteins. These two domains together mediate refolding of misfolded substrate proteins under the action of ATP and ADP, which is also the most clear functional feature of current HSP70 studies. HSP70 initially binds to ATP and then an mediator protein HSP40 forms a complex with misfolded proteins that is in close proximity to HSP70 to promote hydrolysis of ATP followed by release of HSP40, with hydrolysis of ATP accompanied by a conformational change in HSP70 and enhancing its binding capacity to misfolded proteins. After HSP70 has helped refolding of the client proteins, a Nucleotide Exchange Factor (NEF) is recruited to the NBD of HSP70, where ADP dissociates from the NBD, and the client proteins are also released, HSP70 reverts back to the original state, cycling through the chaperone functions described above after binding to the next ATP.
Therefore, the research of the nanobody targeting the heat shock protein 70 can be beneficial to the analysis of the diseases related to the heat shock protein 70 and the research of the corresponding medicines related to the heat shock protein 70.
Disclosure of Invention
The application aims to provide a heat shock protein 70 targeting nano antibody, a preparation method and application thereof, and aims to solve the problem that the prior art lacks a heat shock protein 70 targeting nano antibody, so that the heat shock protein 70 target cannot be better utilized for preparing related medicines.
In order to achieve the purposes of the application, the technical scheme adopted by the application is as follows:
In a first aspect, the application provides a nanobody targeting heat shock protein 70, comprising nanobody Nb H5, wherein the amino acid sequence of nanobody Nb H5 is as shown in seq id No. 1.
Further, the nanobody includes 4 framework regions FR1, FR2, FR3, FR4, and 3 complementarity determining regions CDR1, CDR2, CDR3;
In the nano antibody NbH, 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.
Further, the base sequence of the nanobody NbH is shown as seq. ID No. 9.
In a second aspect, the application provides the use of a nanobody targeting heat shock protein 70 for the preparation of an imaging agent or tracer for tumor tissue highly expressing HSP 70.
In a third aspect, the application provides an application of a nano antibody targeting heat shock protein 70 in preparing a drug for targeted treatment of HSP70 high-expression tumor.
In a fourth aspect, the application provides an application of a nanobody targeting heat shock protein 70 in preparing a protein degradation targeting complex drug.
Further, the nanobody of the targeted heat shock protein 70 is combined and connected with a target protein to form a protein degradation targeted combination drug, and under the action of the nanobody of the targeted heat shock protein 70, E3 ligase CHIP is induced to approach, so that ubiquitination degradation of the target protein is mediated.
In a fifth aspect, the present application provides a method for preparing a nanobody targeting heat shock protein 70, comprising the steps of:
Designing and synthesizing HSP70-NBD genes, and carrying out expression and purification to obtain HSP70-NBD proteins;
coating the HSP70-NBD protein on an immune tube for enrichment screening to obtain a phage library;
Performing PCR amplification and ELISA verification on the eluent of the phage library, 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 of the targeted heat shock protein 70.
Further, the concentration of the target protein is 25 to 27. Mu.g/mL.
Further, the expression vector is selected from the group consisting of pCold vectors.
According to the heat shock protein 70-targeted nanobody provided by the first aspect of the application, the nanobody comprises the nanobody NbH, wherein the amino acid sequence of the nanobody NbH is shown as seq. ID No.1, as HSP70 has the function of starting ubiquitination proteasome degradation, the provided heat shock protein 70-targeted nanobody NbH5 has higher affinity with the heat shock protein 70, and the heat shock protein 70 can be targeted through the nanobody NbH5, so that the effect of starting ubiquitination proteasome degradation is realized, the construction of PROTAC complex for application is facilitated, and the clinical application value is very high. Meanwhile, the nano antibody has small molecular weight, is favorable for drug assembly, is favorable for positioning when PROTAC complex is formed, does not influence the effect of targeted drugs, and has wide application.
The application provides an application of the heat shock protein 70-targeted nanobody in preparing an imaging agent or a tracer of tumor tissues with high expression of HSP 70. Because the provided heat shock protein 70-targeting nanobody has higher affinity with the heat shock protein 70, the heat shock protein 70 can be targeted through the nanobody NbH, so that the heat shock protein 70-targeting nanobody is beneficial to being applied to preparing an imaging agent or a tracer of tumor tissues with high expression of HSP70, and the development speed and the development efficiency can be improved.
The application provides an application of a nano antibody targeting heat shock protein 70 in preparing a tumor drug for targeted treatment of HSP70 high expression. Because the provided nano antibody targeting the heat shock protein 70 has higher affinity with the heat shock protein 70, the nano antibody NbH can target the heat shock protein 70, so that the nano antibody targeting the heat shock protein 70 is favorable for being applied to preparing tumor medicaments for targeting treatment of HSP70 high expression and is favorable for wide application.
The application of the nano antibody of the targeted heat shock protein 70 in the preparation of the protein degradation targeted combination drug provided by the fourth aspect of the application, because the HSP70 has the function of starting ubiquitination proteasome degradation, the provided nano antibody of the targeted heat shock protein 70 is used for preparing the protein degradation targeted combination drug, the HSP70 nano antibody serves as an E3 ligase conjugate, achieves the effect of eliminating pathogenic protein level by combining with CHIP and promoting ubiquitination degradation of the targeted protein, and simultaneously provides a brand-new tool for construction of PROTAC complex.
According to the preparation method of the heat shock protein 70-targeted nanobody provided by the fifth aspect of the application, the preparation method is based on a phage natural nanobody library, three rounds of enrichment screening and ELISA verification prove that the heat shock protein 70-targeted nanobody NbH has higher binding force with the heat shock protein 70, so that the nano antibody NbH has better application value, and the preparation method is quick, simple and convenient, is favorable for large-scale screening, and improves screening efficiency.
Drawings
In order to more clearly illustrate the embodiments of the invention 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 invention, 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 diagram showing the analysis of HSP70-NBD, a target protein of high purity provided in example 1.
FIG. 2 is a three rounds of enrichment analysis of phage nanobody library provided in example 1.
Fig. 3 is an analytical graph of nanobody purification provided in example 1.
Fig. 4 is an analytical graph of nanobody purification provided in example 1.
FIG. 5 is a graph showing the analysis of the high binding capacity of nanobodies provided in example 1 to HSP 70.
Fig. 6 is a graph showing that the nanobody provided in example 1 has a good binding capacity with HSP 70.
Detailed Description
The following description of the embodiments of the present invention 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 invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
An embodiment of the present application provides a nanobody targeting heat shock protein 70, where the nanobody includes nanobody Nb H5, and an amino acid sequence of the nanobody Nb H5 is shown in seq id No. 1.
According to the heat shock protein 70 targeting nanobody provided by the embodiment of the application, the nanobody comprises the nanobody NbH, wherein the amino acid sequence of the nanobody NbH is shown as seq. ID No.1, as HSP70 has the function of starting degradation of ubiquitin-like proteasome, the heat shock protein 70 targeting nanobody NbH5 provided by the embodiment of the application has higher affinity with the heat shock protein 70, and the heat shock protein 70 can be targeted through the nanobody NbH, so that the effect of starting degradation of ubiquitin-like proteasome is realized, the construction of PROTAC complex for application is facilitated, and the application value of the heat shock protein 70 is very high. Meanwhile, the nano antibody has small molecular weight, is favorable for drug assembly, is favorable for positioning when PROTAC complex is formed, does not influence the effect of targeted drugs, and has wide application.
In some embodiments, the nanobody Nb H5 has an amino acid sequence as shown in seq.id No.1, seq.id No.1 specifically:
MAVQLVESGGGLVQAGGSLRLSCAASGRTFSNYTMGWFRQAPGKEREFV AAISWSGKTTYYADSVKGRFTISRDNAKNTVYLQMNSLKHEDTAVYYCHVM TTGPNRYWGQGTQVTVSS.
in some embodiments, the nanobody comprises 4 framework regions FR1, FR2, FR3, FR4, and 3 complementarity determining regions CDR1, CDR2, CDR3.
In the nano antibody NbH, the amino acid sequence of FR1 is shown as SEQ ID NO.2, and the SEQ ID NO.2 specifically comprises: MAVQLVESGGGLVQAGGSLRLSCAASGRTF.
The amino acid sequence of FR2 is shown as SEQ ID NO.3, and SEQ ID NO.3 specifically comprises: WFRQAPGKEREFVAAI.
The amino acid sequence of FR3 is shown as SEQ ID NO.4, and SEQ ID NO.4 specifically comprises: RFTISRDNAKNTVYLQMNSLKHEDTAVYYCHV.
The amino acid sequence of FR4 is shown as SEQ ID NO.5, and SEQ ID NO.5 specifically comprises: WGQGTQVTVSS.
The amino acid sequence of CDR1 is shown as SEQ ID NO.6, and SEQ ID NO.6 specifically comprises: SNYTMG.
The amino acid sequence of CDR2 is shown as SEQ ID NO.7, and SEQ ID NO.7 specifically comprises: SWSGKTTYYADSVKG.
The amino acid sequence of CDR3 is shown as SEQ ID NO.8, and SEQ ID NO.8 specifically comprises: MTTGPNRY.
In some embodiments, the base sequence of nanobody NbH is as shown in SEQ. ID No.9, SEQ ID No.9 is specifically:
ATGGCGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTACAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCAGTAACTATACCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTTGCAGCGATCAGCTGGAGTGGTAAGACCACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACATGAGGACACGGCCGTTTATTACTGTCACGTCATGACGACTGGACCAAATCGCTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA.
the second aspect of the embodiment of the application provides application of the nano antibody targeting the heat shock protein 70 in preparing an imaging agent or a tracer of tumor tissues with high expression of HSP 70.
The application of the nano antibody of the targeting heat shock protein 70 provided by the second aspect of the embodiment of the application in preparing an imaging agent or a tracer of tumor tissues with high expression of HSP 70. Because the provided heat shock protein 70-targeting nanobody has higher affinity with the heat shock protein 70, the heat shock protein 70 can be targeted through the nanobody NbH, so that the heat shock protein 70-targeting nanobody is beneficial to being applied to preparing an imaging agent or a tracer of tumor tissues with high expression of HSP70, and the development speed and the development efficiency can be improved.
The third aspect of the embodiment of the application provides an application of a nano antibody targeting heat shock protein 70 in preparing a drug for targeted treatment of HSP70 high-expression tumor.
The third aspect of the embodiment of the application provides an application of the nano antibody targeting heat shock protein 70 in preparing a drug for targeted treatment of HSP70 high-expression tumor. Because the provided nano antibody targeting the heat shock protein 70 has higher affinity with the heat shock protein 70, the nano antibody NbH can target the heat shock protein 70, so that the nano antibody targeting the heat shock protein 70 is favorable for being applied to preparing tumor medicaments for targeting treatment of HSP70 high expression and is favorable for wide application.
In a fourth aspect, embodiments of the application provide for the use of nanobodies targeting heat shock protein 70 in the preparation of protein degradation targeting complexes.
The application of the nano antibody of the targeted heat shock protein 70 in the preparation of the protein degradation targeted combination drug provided by the fourth aspect of the embodiment of the application, because the HSP70 has the function of starting ubiquitinated proteasome degradation, the provided nano antibody of the targeted heat shock protein 70 is used for preparing the protein degradation targeted combination drug, and the HSP70 nano antibody serves as an E3 ligase conjugate, achieves the effect of eliminating pathogenic protein level by combining with CHIP and promoting ubiquitination degradation of the targeted protein, and simultaneously provides a brand-new tool for construction of PROTAC complex.
In some embodiments, the nanobody targeting heat shock protein 70 binds to and links with a target protein of interest to form a protein degradation targeting complex drug, and under the influence of the nanobody targeting heat shock protein 70, induces E3 ligase CHIP to approach, mediating ubiquitination degradation of the target protein of interest.
The fifth aspect of the embodiment of the application provides a preparation method of a nanobody targeting heat shock protein 70, comprising the following steps:
S01, designing and synthesizing an HSP70-NBD gene, and carrying out expression and purification to obtain an HSP70-NBD protein;
s02, coating the HSP70-NBD protein on an immune tube for enrichment screening to obtain a phage library;
s03, performing PCR amplification and ELISA verification on the eluent of the phage library, 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 of the targeted heat shock protein 70.
According to the preparation method of the heat shock protein 70-targeted nanobody provided by the fifth aspect of the embodiment of the application, the three rounds of enrichment screening and ELISA verification prove that the heat shock protein 70-targeted nanobody NbH has higher binding force with the heat shock protein 70, so that the heat shock protein 70-targeted nanobody NbH has better application value, and the preparation method is quick, simple and convenient, is favorable for large-scale screening and improves the screening efficiency.
In the step S01, the HSP70-NBD gene is designed and synthesized, and expression and purification are carried out to obtain the HSP70-NBD protein.
In some embodiments, the HSP70-NBD gene is designed and synthesized and purified protein is 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.
In step S02, the HSP70-NBD protein is coated on an immune tube for enrichment screening to obtain a phage library.
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 2x109. The screening steps are as follows: a) Coating target protein on an immune tube according to the concentration of 25 mug/mL, and carrying out enrichment screening for 3 rounds; b) Phage libraries were obtained using a third round of phage eluate plating.
In step S03, the eluent of the phage library is subjected to PCR amplification and ELISA verification, then second generation sequencing is carried out, and the gene sequence of the nanobody is synthesized according to the sequencing result.
In some embodiments, 192 monoclonal antibodies were randomly picked for ELISA validation, and ELISA was performed with ELISA 96-well plates coated with BSA as a control, with ELISA readings 3-fold greater than the corresponding BSA readings and readings greater than 0.5 as positive criteria; and sequencing and determining sequence information by 2 times of positive monoclonal companies identified by phage ELISA, extracting the sequence to obtain a nano antibody protein sequence, and carrying out comparative analysis on the sequence to obtain the distribution frequency of the positive sequence.
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 of the targeted heat shock protein 70.
In some embodiments, nanobody gene sequences are cloned into pcold vectors while fusion expressing the 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) And (3) carrying out molecular sieve separation after Ni column purification, wherein AKATA parameters are set at a flow rate of 0.5 mL/min, and collecting once every 1mL to obtain the nano antibody of the targeted heat shock protein 70.
The following description is made with reference to specific embodiments.
Example 1
Nanometer antibody targeting heat shock protein 70 and preparation method thereof
The test process comprises the following steps:
1) HSP70-NBD nano antibody expression purification
The HSP70-NBD gene is designed and synthesized and the purified protein is 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 25 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 BSA as a control, and taking ELISA reading 3 times greater than corresponding BSA reading and reading 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 is cloned into pcold vector, and simultaneously the hemagglutinin tag (hemagglutinin HA tag) is fusion expressed 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 purification on a Ni column, molecular sieve separation was performed, AKATA parameters set at 0.5mL flow rate/min, and collected every 1 mL.
4) Elisa test of 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 into 5ug/ml with PBS, plating at 100 ul/hole, coating with pore 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.
5) Surface plasmon resonance experiment (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:
(1) HSP70-NBD protein expression purification
The molecular mass of the HSP70 protein is about 70kDa, and a fusion protein formed by the marker sequence fragment NBD, GST and His is intercepted and purified, and the protein is about 43kDa and is used as a screening target of the nano antibody. After codon optimization, synthesizing HSP70-NBD fusion gene to perform escherichia coli induced expression purification, and in order to avoid protein degradation and inclusion body generation, inducing with 0.4mM IPTG at low temperature of 16 ℃ overnight, and finally purifying by a Ni column and a molecular sieve to obtain high-purity target protein for subsequent nano antibody screening (shown in figure 1).
(2) Screening, identifying and purifying HSP70-NBD nano antibody
Firstly, screening a phage nanobody library on HSP70-NBD, after two or three rounds of screening, enriching the library approximately by 200 and 60 times (as shown in figure 2), picking about 192 clones from the library obtained in the third round, performing phage ELISA twice for preliminary verification, preliminarily identifying 11 positive clones, sequencing, and displaying 9 normal sequencing results. According to the sequence before and after the nano antibody, the nano antibody sequence can be obtained from the sequencing result, 9 sequences are translated into amino acids and then sequenced, and multiple sequence comparison is carried out, so that 6 different nano antibody sequences are obtained. Subsequently we purified these 6 nanobodies and performed coomassie blue staining (as shown in fig. 3) and WB validation (as shown in fig. 4). In addition, in preliminary experiments we found that A9, F10 belongs to false positive nanobodies, which we exclude in subsequent further binding force verification experiments.
(3) Affinity detection of HSP70-NBD nanobodies
Binding of the HSP70-NBD nanobody to HSP70 was further detected by ELISA, and the results showed that 4 of the above-purified HSP70-NBD nanobodies had higher binding to HSP70 of the experimental group compared to the control BSA, indicating that the nanobodies had high binding capacity to HSP70 (as shown in fig. 5). Next, using Biacore, the affinity constants of the 4 nanobodies and HSP70 were further tested, so as to obtain 1 nanobody NbH (as shown in fig. 6) with an affinity at nanomolar level, further demonstrating that nanobody NbH has a good binding ability with HSP 70.
In summary, the nano antibody of the heat shock protein 70 provided by the application comprises the nano antibody NbH, wherein the amino acid sequence of the nano antibody NbH is shown as seq. ID No.1, as HSP70 has the function of starting degradation of ubiquitin-like proteasome, the provided nano antibody NbH5 of the heat shock protein 70 has higher affinity with the heat shock protein 70, and the nano antibody NbH5 can target the heat shock protein 70 to realize the effect of starting degradation of ubiquitin-like proteasome, thereby being beneficial to constructing PROTAC complex for application and having great clinical application value. Meanwhile, the nano antibody has small molecular weight, is favorable for drug assembly, is favorable for positioning when PROTAC complex is formed, does not influence the effect of targeted drugs, and has wide application.
The foregoing disclosure is illustrative of the present invention and is not to be construed as limiting the scope of the invention, which is defined by the appended claims.
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