A mutant of an ELBD-targeting peptide and its application

Abstract

This invention discloses a mutant of an ELBD targeting peptide and its applications. The amino acid sequence of the mutant ELBD targeting peptide is shown in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. The ELBD targeting peptide and its mutants of this invention can be applied to various scenarios such as disease diagnosis, therapeutic drugs, targeted delivery, and targeted degradation and elimination, and can be developed into targeted therapeutic drugs for the treatment of related diseases. Introducing the targeting peptide onto nanobodies can give the nanobodies multi-target binding properties, making them suitable for more clinical applications. The fusion of the targeting peptide and dsRBD can directionally deliver therapeutic RNA molecules to specific target cells, affecting cellular biological functions and treating various diseases. After the targeting peptide is coupled to an IgG binding domain, the antibody's specific binding characteristic to antigen molecules can be utilized to internalize target molecules originally outside the cell into the cell, achieving a targeted degradation and elimination effect similar to PROTAC technology through intracellular lysosomal digestion and degradation.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to mutants of ELBD-targeting peptides and their applications. Background Technology

[0002] Targeted peptides are short peptides that specifically guide and transport coupled-to-the-target molecules (usually proteins, nucleic acids, nanoparticles, etc.) to specific regions of a cell. These specific cellular regions include specific sites on the cell membrane, the nucleus, mitochondria, endoplasmic reticulum (ER), Golgi apparatus, lysosomes, and peroxisomes. In medicine, targeted peptides are frequently used to transport drug molecules to specific target sites (cells, tissues, organs), resulting in a more concentrated concentration of drug molecules at the site of action, reducing adverse drug reactions, and enhancing the pharmacological efficacy of the drug. In cancer treatment, the application of targeted peptides can reduce the dosage of chemotherapy drugs, decrease the concentration of chemotherapy drugs in normal tissues, and increase the concentration in tumor cells, thereby reducing drug-induced adverse reactions and improving the effectiveness of cancer treatment. Targeted peptides, when conjugated with detectable markers (fluorescent substances, isotopes, radioactive substances, etc.), are also used for cancer diagnosis and as an adjunct to surgical treatment.

[0003] Nanobodies are small antigen-binding fragments (~15kDa) found in the peripheral blood of alpacas. These antibodies contain only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions.

[0004] Nanobodies possess unique characteristics not found in conventional antibodies, including small size, high solubility, stability, and the ability to penetrate deep tissues. These properties allow for the formulation of ready-to-use solutions with long shelf lives. The single-domain nature of nanobodies enables rapid and convenient screening for nanobodies targeting specific recognition sites using phage display technology. Furthermore, nanobodies are relatively easy to produce in bacterial, yeast, or mammalian cells, allowing for large-scale production at a reasonable cost. Nanobodies can be used for gene encoding, labeling, and expression in cells, and for in vivo localization and functional studies of target proteins. Currently, nanobody-based therapies employ three different strategies: ① as antagonists / maskers of target receptor sites, inhibiting or affecting receptor function; ② as targeting domains targeting a specific antigen, carrying conjugated drug / functional domains for specific targeted delivery; ③ as targeting molecules on the surface of nanoparticles / liposomes, guiding nanoparticles / liposomes to the target site. Nanobodies have become a promising tool for disease diagnosis and treatment. Similarly, introducing other targeting peptide sequences onto nanobodies can enhance their multi-target binding capabilities, making them suitable for a wider range of clinical applications.

[0005] Many proteins that interact with highly structured RNA contain double-stranded RNA-binding domains (dsRBDs). These proteins include the nuclease RNase III, Dicer, the protein kinase PKR, the RNA deaminase (ADAR), and Staufen (a protein responsible for mRNA localization). Double-stranded RNA-binding proteins (DRBPs) share a common, evolutionarily conserved sequence that interacts with dsRNA. Proteins containing dsRNA-binding domains (dsRBDs) have been reported to bind to dsRNAs as small as 11 bp, and this binding is largely independent of specific nucleotide sequences. However, several dsRBDs do exhibit high substrate specificity, which may have significant biological implications. More than 20 DRBPs have been identified, and these proteins are involved in numerous cellular functions, from RNA interference to antiviral mechanisms and other types of posttranscriptional gene regulation, playing a variety of crucial roles in the cell.

[0006] By fusing a targeted transmembrane peptide sequence with dsRBD, therapeutic RNA molecules can be delivered to specific target cells, affecting cellular biological functions and treating various diseases.

[0007] Protein domains from Streptococcus or Staphylococcus aureus that can bind to antibody molecules have long been used extensively for the isolation, purification, and large-scale commercial production of monoclonal antibodies. Summary of the Invention

[0008] To address the aforementioned issues, this invention provides mutants of ELBD-targeting peptides and their applications. The ELBD-targeting peptides and their mutants of this invention can be applied to various scenarios such as disease diagnosis, therapeutic drugs, targeted delivery, and targeted degradation and elimination, and can be developed into targeted therapeutic drugs for the treatment of related diseases.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] The present invention discloses a mutant of an ELBD targeting peptide, the amino acid sequence of which is shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

[0011] The ELBD sequence in this invention is the ELBD sequence (SEQ ID NO:24) listed in a divisional application of U.S. Patent Application 15562722: R C SHYTGIR C SHGIYTGIR C QH.

[0012] In this sequence, there are still three Cys residues. Considering the formation of disulfide bonds through Cys oxidation, one Cys residue must be in a free state, potentially leading to intermolecular cross-linking and polymerization of the recombinant protein. Therefore, this sequence is further optimized. The optimization plan is as follows: 1. Retain the first and second Cys residues and remove the third Cys residue, allowing this sequence to form a ring through the disulfide bonds formed by oxidation; 2. Optimize the ring size while preserving as many important conserved amino acid residues as possible.

[0013] Therefore, three mutants as shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 were obtained.

[0014] Another aspect of the present invention discloses the application of the mutant of the above-mentioned ELBD targeting peptide, and the use of the mutant of the ELBD targeting peptide in the preparation of targeted therapeutic drugs.

[0015] As a preferred embodiment of the present invention, the expression vector for targeted delivery of the mutant ELBD targeting peptide is an expression vector bound to a nanobody.

[0016] As a preferred embodiment of the present invention, the expression vector for targeted delivery of the mutant ELBD targeting peptide is an expression vector that binds to RNA.

[0017] As a preferred embodiment of the present invention, the expression vector for targeted delivery of the mutant ELBD targeting peptide is an expression vector with an antibody-binding sequence.

[0018] As a preferred embodiment of the present invention, the method for constructing a series of expression vectors for mutant ELBD targeting peptides is as follows: starting with the EGFP-ELBD-H2-pET28a expression plasmid, the original EGFP-ELBD-H2-pET28a expression vector is digested with EcoRI and SalI, and ligated with the synthesized fragment using T4 DNA ligase; the ligation product is transformed into E. coli DH5α strain, cultured, the recombinant plasmid DNA is recovered, and sequenced for verification, thus completing the construction of a series of EGFP-ELBD-H2-pET28a expression vectors, including EGFP-ELBD-N1-pET28a, EGFP-ELBD-N2-H2-pET28a, and EGFP-ELBD-N3-H2-pET28a.

[0019] As a preferred embodiment of the present invention, the expression vector for combining the mutant of the ELBD targeting peptide with the nanobody is constructed as follows: the DNA sequence of the GFP nanobody is synthesized by a DNA artificial solid-phase synthesis method, and then primers 1 and 2 are designed according to the gene sequence of the GFP nanobody for PCR amplification using the synthesized DNA sequence as a template. Primers 1 and 2 introduce Nde I and Bam HI restriction sites, respectively. The PCR amplification reaction conditions are as follows: denaturation at 95℃ for 5 min, followed by denaturation at 95℃ for 45 s, annealing at 60℃ for 30 s, extension at 72℃ for 60 s, repeated 33 times, and finally at 72℃ for 10 min.

[0020] The amplified fragments were purified using a DNA fragment recovery kit and digested with Nde I and Bam HI to recover the fragments. Simultaneously, the EGFP-ELBD-N1-H2-pET28a vector plasmid was digested with Nde I and Bam HI to recover the vector fragments. The fragments were then ligated using T4 DNA ligase and transformed into E. coli DH5α strain. The mixture was incubated overnight at 37°C for 15 h, and the recombinant plasmid DNA was purified using a plasmid DNA recovery kit.

[0021] The sequence of primer 1 is shown in SEQ ID NO:4, and the sequence of primer 2 is shown in SEQ ID NO:5.

[0022] As a preferred embodiment of the present invention, the construction of the expression vector for the mutant of the ELBD targeting peptide bound to RNA is as follows: the DNA sequence of the double-stranded RNA-binding protein RBD is synthesized by a DNA artificial solid-phase synthesis method; then, using the synthesized sequence as a template, primers 3 and 4 are designed according to the gene sequence of RBD for PCR amplification, and primers 3 and 4 introduce Nde I and Bam HI restriction sites, respectively.

[0023] PCR amplification reaction conditions: The PCR amplification reaction conditions are as follows: first denature at 95℃ for 5 min, then denature at 95℃ for 45 s, anneal at 60℃ for 30 s, extend at 72℃ for 60 s, repeat 33 times, and finally at 72℃ for 10 min.

[0024] The amplified fragments were purified using a DNA fragment recovery kit and digested with Nde I and Bam HI to recover the fragments. Simultaneously, the EGFP-ELBD-N1-H2-pET28a vector plasmid was digested with Nde I and Bam HI to recover the vector fragments. The fragments were then ligated using T4 DNA ligase and transformed into E. coli DH5α strain. The mixture was incubated overnight at 37°C for 15 h, and the recombinant plasmid DNA was purified using a plasmid DNA recovery kit.

[0025] The sequence of primer 3 is shown in SEQ ID NO:6, and the sequence of primer 4 is shown in SEQ ID NO:7.

[0026] As a preferred embodiment of the present invention, the construction of the expression vector of the mutant of the ELBD targeting peptide and the antibody binding sequence is as follows: the DNA sequence of the IgG binding protein IgGBD is synthesized by DNA artificial solid phase synthesis method; then, using the synthesized sequence as a template, primers 5 and 6 are designed according to the gene sequence of IgGBD for PCR amplification, and primers 5 and 6 introduce Nde I and Bam HI restriction sites, respectively.

[0027] PCR amplification reaction conditions: The PCR amplification reaction conditions are as follows: first denature at 95℃ for 5 min, then denature at 95℃ for 45 s, anneal at 60℃ for 30 s, extend at 72℃ for 60 s, repeat 33 times, and finally at 72℃ for 10 min.

[0028] The amplified fragments were purified using a DNA fragment recovery kit and digested with Nco I and Bam HI to recover the fragments. Simultaneously, the EGFP-ELBD-N1-H2-pET28a vector plasmid was digested with Nde I and Bam HI to recover the vector fragments. The fragments were then ligated using T4 DNA ligase, transformed into E. coli DH5α strain, and cultured overnight at 37°C for 15 h. The recombinant plasmid DNA was purified using a plasmid DNA recovery kit.

[0029] The sequence of primer 5 is shown in SEQ ID NO:8, and the sequence of primer 6 is shown in SEQ ID NO:9.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] 1) The ELBD targeting peptide and its mutants of the present invention can be applied to various scenarios such as disease diagnosis, therapeutic drugs, targeted delivery, targeted degradation and elimination, and developed into targeted therapeutic drugs for the treatment of related diseases.

[0032] 2) The mutant of the ELBD targeting peptide provided by this invention, when introduced onto nanobodies, can enable the nanobodies to have multi-target binding properties, making them suitable for more clinical application scenarios.

[0033] 3) The mutant of the ELBD-targeting peptide provided by this invention, fused with dsRBD, can deliver therapeutic RNA molecules to specific target cells, affecting the biological function of the cells and treating various diseases.

[0034] 4) The mutant of the ELBD-targeting peptide provided by this invention, after being coupled with the IgG binding domain, can utilize the characteristic of antibody-specific binding to antigen molecules to internalize the target molecule that was originally outside the cell into the cell. Through the digestive degradation of intracellular lysosomes, it can achieve a targeted degradation and elimination effect similar to that of PROTAC technology. Attached Figure Description

[0035] Figure 1 This is a schematic diagram illustrating the selectivity of the recombinant protein for cells in Example 1.

[0036] Figure 2 This is a schematic diagram illustrating the efficiency of nanobody delivery in Example 2.

[0037] Figure 3 This is a schematic diagram illustrating the efficiency of antibody IgG delivery in Example 3.

[0038] Figure 4 This is a schematic diagram illustrating the efficiency of siRNA delivery in Example 4. Detailed Implementation

[0039] To facilitate understanding of the technical means, creative features, objectives, and effects of this invention, the invention is further described below with reference to specific embodiments. However, the following embodiments are merely preferred embodiments of this invention and not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the scope of protection of this invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0040] In this invention, the ELBD sequence (SEQ ID NO:24): R C SHYTGIR C SHGIYTGIR C QH is listed in a divisional application of U.S. patent application 15562722.

[0041] This sequence still contains three Cys residues. Considering the formation of disulfide bonds through Cys oxidation, one Cys residue must be in a free state, potentially leading to intermolecular cross-linking and the polymerization of the recombinant protein. Therefore, this sequence is further optimized. The optimization plan is: 1. Retain the first and second Cys residues and remove the third Cys residue, allowing this sequence to form a ring through the disulfide bonds formed by oxidation; 2. Optimize the ring size while preserving as many important conserved amino acid residues as possible. The following three mutant sequences are listed below for illustration.

[0042] Among them, the targeted membrane-penetrating peptide composed of ELBD-N1 and H2 (i.e., CPP derived from HB-EGF, see SEQ ID NO: 12 in PCT / CN2017 / 076656, and SEQ ID NO: 10 in this application) has better membrane-penetrating effect, the expressed fusion protein can exist in more soluble form, the purified fusion protein is more stable and less prone to aggregation and precipitation.

[0043] Construction of EGFP-ELBD-H2 series expression vectors

[0044] A series of mutant nucleotide sequences with EcoRI and SalI restriction sites at both ends were synthesized using a DNA artificial solid-phase synthesis method. The AA sequences are shown in Table 1.

[0045] Starting with the EGFP-ELBD-H2-pET28a expression plasmid, the original EGFP-ELBD-H2-pET28a expression vector was double-digested with EcoRI and SalI, and then ligated with the synthesized fragment using T4 DNA ligase.

[0046] The ligation product was transformed into E. coli DH5α strain, cultured, and the recombinant plasmid DNA was recovered and sequenced for verification, thus completing the construction of a series of EGFP-ELBD-H2-pET28a expression vectors.

[0047] Including EGFP-ELBD-N1-H2-pET28a, EGFP-ELBD-N2-H2-pET28a, EGFP-ELBD-N3-H2-pET28a.

[0048] Table 1. AA sequences

[0049] Optimized ELBD RCSHYTGIRCSHGIYTGIRCQH ELBD-N1 RCSHYTGIVCHSYVGA SEQ ID NO: 1 ELBD-N2 RCSHYTGIICHPYHGE SEQ ID NO: 2 ELBD-N3 VCHSYVGAVCHSYVGA SEQ ID NO: 3

[0050] Construction of GFP nanobody-ELBD-N1-H2 expression vector

[0051] The DNA sequence of GFP nanobodies was synthesized using a DNA artificial solid-phase synthesis method. Then, using the synthesized DNA sequence as a template, primers were designed based on the GFP nanobody gene sequence for PCR amplification. The forward and reverse primers introduced Nde I and Bam HI restriction sites, respectively.

[0052] SEQ ID NO: 4, primer 1: 5'-CGCCATATGGTTCAGCTGGTTGAAAGCGGTGG-3';

[0053] SEQ ID NO:5, Primer 2: 5'-CGCGGATCCTTTGCTACTAACGGTAACCTGGGTGC-3'.

[0054] PCR amplification reaction conditions: The PCR amplification reaction conditions are as follows: first denature at 95℃ for 5 min, then denature at 95℃ for 45 s, anneal at 60℃ for 30 s, extend at 72℃ for 60 s, repeat 33 times, and finally at 72℃ for 10 min.

[0055] The amplified fragments were purified using a DNA fragment recovery kit and digested with Nde I and Bam HI. Simultaneously, the EGFP-ELBD-N1-H2-pET28a vector plasmid was digested with Nde I and Bam HI, and the vector fragment was recovered. Ligation was performed using T4 DNA ligase. The mixture was transformed into E. coli DH5α strain and cultured overnight at 37°C for 15 h. The recombinant plasmid DNA was purified using a plasmid DNA recovery kit and sent to a DNA sequencing company for sequencing verification.

[0056] Construction of double-stranded RNA binding protein (dsRBD)-ELBD-N1-H2 expression vector

[0057] The DNA sequence of double-stranded RNA-binding protein (dsRBD) was synthesized using a DNA solid-phase synthesis method. Using the synthesized sequence as a template, primers were designed based on the dsRBD gene sequence for PCR amplification, with the forward and reverse primers introducing NdeI and BamHI restriction sites, respectively.

[0058] SEQ ID NO:6, Primer 3: 5'-CGCCATATGGAACGTGCGATTCAGGGCAA-3';

[0059] SEQ ID NO: 7, primer 4: 5'-CGCGGATCCTTCGCTTTCCTTCTTAAAGGTTTCAATACCTTCC-3'.

[0060] PCR amplification reaction conditions: The PCR amplification reaction conditions are as follows: first denature at 95℃ for 5 min, then denature at 95℃ for 45 s, anneal at 60℃ for 30 s, extend at 72℃ for 60 s, repeat 33 times, and finally at 72℃ for 10 min.

[0061] The amplified fragments were purified using a DNA fragment recovery kit and digested with Nde I and Bam HI. Simultaneously, the EGFP-ELBD-N1-H2-pET28a vector plasmid was digested with Nde I and Bam HI, and the vector fragment was recovered. Ligation was performed using T4 DNA ligase. The mixture was transformed into E. coli DH5α strain and cultured overnight at 37°C for 15 h. The recombinant plasmid DNA was purified using a plasmid DNA recovery kit and sent to a DNA sequencing company for sequencing verification.

[0062] Construction of IgG-binding protein (IgGBD)-ELBD-N1-H2 expression vector

[0063] The DNA sequence of IgG binding protein (IgGBD) was synthesized using a solid-phase DNA synthesis method. Using the synthesized sequence as a template, primers were designed based on the IgGBD gene sequence for PCR amplification, with the forward and reverse primers introducing NdeI and BamHI restriction sites, respectively.

[0064] SEQ ID NO:8, Primer 5:

[0065] 5'-CGCCATATGGTTGATAACAAATTTAACAAAGAACAGCAGAACGCAT-3';

[0066] SEQ ID NO:9, Primer 6: 5'-CGCGGATCCTTTTGGTGCCTGTGCATCATTCAGC-3'.

[0067] PCR amplification reaction conditions: The PCR amplification reaction conditions are as follows: first denature at 95℃ for 5 min, then denature at 95℃ for 45 s, anneal at 60℃ for 30 s, extend at 72℃ for 60 s, repeat 33 times, and finally at 72℃ for 10 min.

[0068] The amplified fragments were purified using a DNA fragment recovery kit and digested with Nco I and Bam HI. Simultaneously, the EGFP-ELBD-N1-H2-pET28a vector plasmid was digested with Nde I and Bam HI, and the vector fragments were recovered. Ligation was performed using T4 DNA ligase. The mixture was transformed into E. coli DH5α strain and cultured overnight at 37°C for 15 h. The recombinant plasmid DNA was purified using a plasmid DNA recovery kit and sent to a DNA sequencing company for sequencing verification.

[0069] ELBD-N1 series recombinant protein expression and purification

[0070] (I) Expression and purification of EGFP-ELBD-H2 series recombinant proteins

[0071] 1) Pick a single colony transformed with EGFP-Tn-HBD-pET28a plasmid from the solid LB medium plate of the preserved strain and incubate it in 30 ml of LB liquid medium containing kanamycin (10-50 mg / L) at 37°C with shaking until the OD600 is about 0.5-1.0.

[0072] 2) Take this bacterial culture and inoculate it into a certain volume of culture medium containing a certain concentration of kanamycin (10-50 mg / L) at a 1% inoculation rate and continue to expand the culture until the OD600 is about 0.5-1.0.

[0073] 3) Adjust the culture temperature to the range of 15℃ to 20℃, add an appropriate amount of IPTG (0.1-10 MM) to induce the expression of the target protein, continue to culture for 10-20h, and collect the bacterial cells by low-speed centrifugation at 500-1000 rpm.

[0074] 4) The collected bacterial cells were resuspended in 20mM Tris-HCl buffer (pH 8.0-8.5) and sonicated at a certain power (10-100 W). The supernatant containing the target protein was collected by high-speed centrifugation (8000-13400 rpm) at low temperature (4-10℃).

[0075] 5) The collected supernatant was subjected to affinity chromatography using a nickel chelate affinity chromatography column. Similar to most nickel chelate affinity chromatography methods for purifying target proteins, gradient elution was performed using imidazole buffers of different concentrations (20 mM imidazole elution, 200 mM imidazole elution), and the target protein fractions eluted with different concentrations of imidazole were collected.

[0076] 6) The collected proteins were dialyzed in Tris-HCl buffer at pH 7.2 for 6 h.

[0077] (II) Expression and purification of GFP nanobody-ELBD-N1-H2 recombinant protein

[0078] 1) Pick a single colony transformed with EGFP-Tn-HBD-pET28a plasmid from the solid LB medium plate of the preserved strain and incubate it in 30 ml of LB liquid medium containing kanamycin (10-50 mg / L) at 37°C with shaking until the OD600 is about 0.5-1.0.

[0079] 2) Take this bacterial culture and inoculate it into a certain volume of culture medium containing a certain concentration of kanamycin (10-50 mg / L) at a 1% inoculation rate and continue to expand the culture until the OD600 is about 0.5-1.0.

[0080] 3) Adjust the culture temperature to the range of 15℃ to 20℃, add an appropriate amount of IPTG (0.1-10 MM) to induce the expression of the target protein, continue to culture for 10-20h, and collect the bacterial cells by low-speed centrifugation at 500-1000 rpm.

[0081] 4) The collected bacterial cells were resuspended in 20mM Tris-HCl buffer (pH 8.0-8.5) and sonicated at a certain power (10-100 W). The supernatant containing the target protein was collected by high-speed centrifugation (8000-13400 rpm) at low temperature (4-10℃).

[0082] 5) The collected supernatant was subjected to affinity chromatography using a nickel chelate affinity chromatography column. Similar to most nickel chelate affinity chromatography methods for purifying target proteins, gradient elution was performed using imidazole buffers of different concentrations (20 mM imidazole elution, 200 mM imidazole elution), and the target protein fractions eluted with different concentrations of imidazole were collected.

[0083] 6) The collected proteins were dialyzed in Tris-HCl buffer at pH 7.2 for 6 h.

[0084] (III) Expression and purification of recombinant IgG binding protein (IgGBD)-ELBD-N1-H2 protein

[0085] 1) Pick a single colony transformed with EGFP-Tn-HBD-pET28a plasmid from the solid LB medium plate of the preserved strain and incubate it in 30 ml of LB liquid medium containing kanamycin (10-50 mg / L) at 37°C with shaking until the OD600 is about 0.5-1.0.

[0086] 2) Take this bacterial culture and inoculate it into a certain volume of culture medium containing a certain concentration of kanamycin (10-50 mg / L) at a 1% inoculation rate and continue to expand the culture until the OD600 is about 0.5-1.0.

[0087] 3) Adjust the culture temperature to the range of 15℃ to 20℃, add an appropriate amount of IPTG (0.1-10 MM) to induce the expression of the target protein, continue to culture for 10-20h, and collect the bacterial cells by low-speed centrifugation at 500-1000 rpm.

[0088] 4) The collected bacterial cells were resuspended in 20mM Tris-HCl buffer (pH 8.0-8.5) and sonicated at a certain power (10-100 W). The supernatant containing the target protein was collected by high-speed centrifugation (8000-13400 rpm) at low temperature (4-10℃).

[0089] 5) The collected supernatant was subjected to affinity chromatography using a nickel chelate affinity chromatography column. Similar to most nickel chelate affinity chromatography methods for purifying target proteins, gradient elution was performed using imidazole buffers of different concentrations (20 mM imidazole elution, 200 mM imidazole elution), and the target protein fractions eluted with different concentrations of imidazole were collected.

[0090] 6) The collected proteins were dialyzed in Tris-HCl buffer at pH 7.2 for 6 h.

[0091] (iv) Expression and purification of double-stranded RNA-binding protein (dsRBD)-ELBD-N1-H2 recombinant protein

[0092] 1) Pick a single colony transformed with EGFP-Tn-HBD-pET28a plasmid from the solid LB medium plate of the preserved strain and incubate it in 30 ml of LB liquid medium containing kanamycin (10-50 mg / L) at 37°C with shaking until the OD600 is about 0.5-1.0.

[0093] 2) Take this bacterial culture and inoculate it into a certain volume of culture medium containing a certain concentration of kanamycin (10-50 mg / L) at a 1% inoculation rate and continue to expand the culture until the OD600 is about 0.5-1.0.

[0094] 3) Adjust the culture temperature to the range of 15℃ to 20℃, add an appropriate amount of IPTG (0.1-10 MM) to induce the expression of the target protein, continue to culture for 10-20h, and collect the bacterial cells by low-speed centrifugation at 500-1000 rpm.

[0095] 4) The collected bacterial cells were resuspended in 20mM Tris-HCl buffer (pH 8.0-8.5) and sonicated at a certain power (10-100 W). The supernatant containing the target protein was collected by high-speed centrifugation (8000-13400 rpm) at low temperature (4-10℃).

[0096] 5) The collected supernatant was subjected to affinity chromatography using a nickel chelate affinity chromatography column. Similar to most nickel chelate affinity chromatography methods for purifying target proteins, gradient elution was performed using imidazole buffers of different concentrations (20 mM imidazole elution, 200 mM imidazole elution), and the target protein fractions eluted with different concentrations of imidazole were collected.

[0097] 6) The collected proteins were dialyzed in Tris-HCl buffer at pH 7.2 for 6 h.

[0098] Example 1

[0099] Cellular selectivity of EGFP-ELBD-N1 recombinant protein

[0100] Eleven cell types were selected to study the tumor-targeting characteristics of the EGFP-ELBD-N1-H2 recombinant protein. These included seven human tumor cell types: HeLa (human cervical cancer cells), MCF-7 (human breast cancer cells), A549 (human lung cancer cells), H460 (human lung cancer cells), 22RV1 (human prostate cancer cells), SMMC-7721 (human liver cancer cells), Lovo (human liver cancer cells), MDA-MB-231 (human triple-negative breast cancer cells), and SKBR-3 (human breast cancer cells), as well as one normal human cell type: MRC-5 (human embryonic lung fibroblasts).

[0101] Recombinant protein samples at concentrations of 0.25 μM, 0.5 μM, 1 μM, and 2 μM were incubated with in vitro cultured tumor cells for 12 h. The cell-penetrating effects of various EGFP-ELBD-H2 recombinant proteins on the cells were compared and observed using an inverted fluorescence microscope.

[0102] See Figure 1 The results showed that the EGFP-ELBD-N1-H2 protein had significantly higher cell membrane penetration rates in HeLa, 22RV1, A549, MCF-7, Lovo, SKBR-3, and MDA-MB-231 cells than in SMMC-7721, H460, and normal MRC-5 cells. This phenomenon is related to the different levels of specific receptor expression on the surface of different cancer cells.

[0103] Example 2

[0104] Observation of the efficiency of GFP nanobody-ELBD-N1-H2 delivery of nanobodies

[0105] HeLa cells were selected, and 1 μM EGFP protein, 0.5 μM, 1 μM, and 2 μM GFP nanobody-ELBD-N1-H2 protein were co-incubated with in vitro cultured cells for 12 h. After washing with PBS and trypan blue, the nanobody was observed under an inverted fluorescence microscope to see if it could be carried into the cell by the ELBD-N1-H2 targeting transmembrane peptide.

[0106] See Figure 2 The results show that EGFP itself cannot enter the cell and there is no fluorescent signal inside the cell.

[0107] In the presence of GFPnanobody-ELBD-N1-H2, there is a significant intracellular fluorescence signal, and the fluorescence signal increases significantly with the increase of GFPnanobody-ELBD-N1-H2 protein concentration.

[0108] This demonstrates that GFP nanobody-ELBD-N1-H2 can carry extracellular EGFP protein into cells, and also proves that ELBD-N1 can deliver nanobodies into cells. These experimental results also verify that ELBD-N1 can deliver exogenous protein molecules into cells through non-covalent interactions.

[0109] Example 3

[0110] Observation of the efficiency of IgG delivery of antibody IgG by IgGBD-ELBD-N1-H2

[0111] HeLa cells were used. Antibody IgG, fluorescently labeled with Alexa Fluor 488 at a final concentration of 100 nM, was incubated with 0.25 μM, 0.5 μM, and 1 μM IgGBD-ELBD-N1-H2 protein at room temperature for half an hour. The mixture was then co-incubated with in vitro cultured cells for 12 h. After washing with PBS and trypan blue, the antibody IgG was observed using an inverted fluorescence microscope to determine whether it could be carried into the cells by the ELBD-N1-targeting transmembrane peptide. See [link to relevant documentation]. Figure 3 The results showed that the IgG antibody labeled with Alexa Fluor 488 could not enter the cell efficiently and there was no significant fluorescence signal inside the cell; while in the presence of IgGBD-ELBD-N1-H2, there was a significant fluorescence signal inside the cell, indicating that IgGBD-ELBD-N1-H2 can efficiently deliver extracellular IgG antibody into the cell.

[0112] Example 4

[0113] Observation of the efficiency of RBD-ELBD-N1-H2 delivery of siRNA

[0114] HeLa cells were used. siRNA labeled with Cy3 at a final concentration of 50 nM was incubated with 0.5 μM, 1 μM, and 2 μM RBD-ELBD-N1-H2 protein at room temperature for half an hour. The mixture was then co-incubated with in vitro cultured cells for 12 h. After washing with PBS and trypan blue, the siRNA was observed under an inverted fluorescence microscope to determine whether it could be carried into the cells by the ELBD-N1-targeting transmembrane peptide. See [link to relevant documentation]. Figure 4 The results showed that Cy3-labeled siRNA had some weak red fluorescence signals in the cells, which may be due to the non-specific uptake of double-stranded nucleic acids by the cells. The RBD-ELBD-N1-H2 protein could significantly enhance the intracellular red fluorescence signal, indicating that the RBD-ELBD-N1-H2 protein can be used as a highly efficient delivery vector for siRNA.

[0115] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. An ELBD-targeting peptide mutant fusion protein, characterized in that, The fusion protein is formed by the direct fusion of an ELBD-targeting peptide mutant and an H2 membrane-penetrating peptide from the N-terminus to the C-terminus; the amino acid sequence of the ELBD-targeting peptide mutant is shown in SEQ ID NO:1, and the ELBD-targeting peptide mutant forms a ring by forming an intramolecular disulfide bond through two cysteine ​​residues; the H2 membrane-penetrating peptide is a cell-penetrating peptide derived from HB-EGF, and its sequence is shown in SEQ ID NO:

10.

2. The application of the fusion protein according to claim 1 in the preparation of tumor-targeting delivery vectors, characterized in that, The tumor-targeting delivery vector is a fusion expression vector constructed by fusing the fusion protein of claim 1 with nanobodies, double-stranded RNA-binding protein dsRBD, and IgG-binding protein IgGBD, respectively, for mediating the intracellular delivery of nanobodies, IgG antibodies, and siRNA to tumor cells; the tumor is a tumor derived from HeLa, 22RV1, A549, MCF-7, Lovo, SKBR-3, or MDA-MB-231 cells.

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