Nanobody-assisted protein or polypeptide expression system, and preparation and use

Abstract

The present invention provides a nanobody-assisted protein or polypeptide expression system, and preparation and use, and specifically relates to a nanobody fusion protein, an expression vector, a host cell and a nanobody-assisted protein expression system for promoting protein or polypeptide expression and secretion, and preparation and use thereof.

Description

Nanobody-assisted protein or polypeptide expression system and its preparation and application Technical Field

[0001] The present invention relates to the field of biomedicine and relates to a nanobody-assisted protein expression system, and in particular to a nanobody fusion protein, vector, host cell, expression system, preparation, and application thereof that promotes protein or polypeptide expression and secretion. Background Art

[0002] Nanobody (abbreviated as Nb) is a heavy chain antibody variable region derived from camelids or sharks that naturally lacks light chains. It has the characteristics of small molecular weight (1 / 10 of the molecular weight of conventional antibodies), high stability, good water solubility, and strong penetrating power.

[0003] A protein expression system is a system consisting of a host (or in vitro cultured cells), exogenous genes, vectors, and auxiliary components. This system can achieve the purpose of expressing exogenous genes in a host (or in vitro cultured cells).

[0004] Biologically active biomacromolecules such as proteins and peptides have a wide range of applications in biopharmaceuticals, including cytokines, ligands, receptors, enzymes, protein or peptide antigens, and bioactive peptides. However, many secreted proteins and peptides suffer from poor expression and secretion in vivo or in cells, limiting their application. Proteins and peptides have extensive applications in biopharmaceuticals, and enhancing their production, stability, and correct localization in vivo is crucial for their application. Currently, Fc-based fusion protein technology is a commonly used technology. Fc-based fusion protein technology refers to a type of fusion protein in which another polypeptide is linked via the Fc domain of an immunoglobulin. The fusion partner can be any biologically active protein or peptide, such as a ligand that activates and interacts with a protein surface receptor; a protein or peptide antigen from a pathogen; or a protein fragment or peptide that recognizes a protein's target site. In most cases, the fusion partner is a protein fragment or peptide with therapeutic potential that can enhance its biopharmaceutical properties by linking to the Fc domain of an immune protein.

[0005] The disadvantages of Fc-based fusion protein technology are: (1) large molecular weight (Fc is a dimer with a molecular weight of approximately 50kDa), which may affect the function of active proteins (or polypeptides) due to steric hindrance; (2) Fc fragments have receptor binding sites that can bind to Fc receptors on the cell surface, affecting immune responses and having the ability to cause inflammation, antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cell-mediated phagocytosis (ADCP). During the application of proteins (or polypeptides), these additional reactions may cause serious toxic side effects; (3) Fc fragments themselves do not have the ability to specifically target specific cells or specific proteins.

[0006] Summary of the Invention

[0007] To address the problems existing in the prior art, we have utilized the characteristics of Nanobodies, such as small molecular weight, easy folding, stable structure, and easy secretion, to link Nanobodies with proteins or polypeptides to form fusion proteins. We have found that the fusion of Nanobodies has the effect of promoting the expression and secretion of proteins or polypeptides. Specifically, the present invention proposes the following technical solutions:

[0008] In a first aspect, the present invention provides a nanobody fusion protein that promotes the expression and secretion of an exogenous protein or polypeptide, characterized in that it comprises a nanobody and an exogenous protein or polypeptide, and the nanobody can promote the expression and secretion of the exogenous protein or polypeptide fused thereto.

[0009] Preferably, the nanobody fusion protein is characterized in that the nanobody comprises one or more nanobodies, and the nanobodies are directly connected to each other; preferably, there is a connecting peptide between the nanobodies.

[0010] Preferably, the nanobody fusion protein is characterized in that the nanobody is directly linked to the exogenous protein or polypeptide; preferably, the nanobody is linked to the C-terminus or N-terminus of the exogenous protein or polypeptide.

[0011] Preferably, the nanobody fusion protein is characterized in that there is a connecting peptide between the nanobody and the exogenous protein or polypeptide.

[0012] Preferably, the nanobody fusion protein is characterized in that the exogenous protein or polypeptide includes a protein or polypeptide with biological activity; preferably, the exogenous protein or polypeptide includes one of a cytokine, a ligand, a receptor, an enzyme and a polypeptide antigen; more preferably, the exogenous protein or polypeptide includes a chain-like unstructured protein or polypeptide; further preferably, it includes a prion protein fragment PrP encoded by the nucleic acid sequence shown in SEQ ID NO.1 1-110, one of the following: , the Shadoo protein encoded by the nucleic acid sequence shown in SEQ ID NO.12, and the somatostatin polypeptide encoded by the nucleic acid sequence shown in SEQ ID NO.11.

[0013] Preferably, the nanobody fusion protein is characterized in that one or more of the GFP-Nb nanobody encoded by the nucleic acid sequence shown in SEQ ID NO.2, the ALFA-Nb nanobody encoded by the nucleic acid sequence shown in SEQ ID NO.8, and the mcherry-Nb nanobody encoded by the nucleic acid sequence shown in SEQ ID NO.9 are connected to an exogenous protein or polypeptide.

[0014] In a second aspect, the present invention provides an expression vector for promoting the expression and secretion of exogenous proteins or polypeptides, characterized in that it comprises a polynucleotide sequence for encoding the nanobody fusion protein and a vector backbone, preferably, further comprising an inserted promoter nucleotide sequence; further preferably, comprising a CAG promoter nucleotide sequence.

[0015] Preferably, the expression vector for promoting the expression and secretion of exogenous proteins or polypeptides is characterized in that the vector backbone comprises a recombinant adeno-associated virus (rAAV) vector.

[0016] In a third aspect, the present invention provides a host cell for promoting the expression and secretion of exogenous proteins or polypeptides, characterized in that the expression vector is expressed in the host cell.

[0017] In a fourth aspect, the present invention provides a method for preparing the nanobody fusion protein, comprising culturing the host cell and isolating the nanobody fusion protein.

[0018] In a fifth aspect, the present invention provides a nanobody-assisted protein expression system for promoting the expression and secretion of exogenous proteins or polypeptides, characterized in that it includes the expression vector described above.

[0019] Preferably, the nanobody-assisted protein expression system for promoting the expression and secretion of exogenous proteins or polypeptides is characterized in that it includes an in vitro expression system and / or an in vivo expression system.

[0020] In a sixth aspect, the present invention provides a method for constructing a nano-antibody auxiliary protein expression system that promotes the expression and secretion of exogenous proteins or polypeptides, which is constructed by cloning the nucleotide sequence including the fusion protein into a vector backbone and then co-transfecting the host cells.

[0021] Preferably, the method for constructing the protein expression system is characterized in that the nanobodies include one or more directly connected nanobodies; preferably, there are connecting peptides between the nanobodies.

[0022] Preferably, the method for constructing the protein expression system is characterized in that the nanobody is directly connected to the exogenous protein or polypeptide; preferably, the nanobody is connected to the C-terminus or N-terminus of the exogenous protein or polypeptide.

[0023] Preferably, the method for constructing the protein expression system is characterized in that a connecting peptide is present between the nanobody and the exogenous protein or polypeptide.

[0024] Preferably, the method for constructing the protein expression system is characterized in that the exogenous protein or polypeptide includes a protein or polypeptide with biological activity; preferably, the exogenous protein or polypeptide includes one of a cytokine, a ligand, a receptor, an enzyme and a polypeptide antigen; more preferably, the exogenous protein or polypeptide includes a chain-like unstructured protein or polypeptide; further preferably, the exogenous protein or polypeptide includes a prion protein fragment PrP encoded by the nucleic acid sequence shown in SEQ ID NO.1 1-110 , one of the following: , the Shadoo protein encoded by the nucleic acid sequence shown in SEQ ID NO.12, and the somatostatin polypeptide encoded by the nucleic acid sequence shown in SEQ ID NO.11.

[0025] Preferably, the method for constructing the protein expression system is characterized in that one or more of the GFP-Nb nanobody encoded by the nucleic acid sequence shown in SEQ ID NO.2, the ALFA-Nb nanobody encoded by the nucleic acid sequence shown in SEQ ID NO.8, and the mcherry-Nb nanobody encoded by the nucleic acid sequence shown in SEQ ID NO.9 are connected to an exogenous protein or polypeptide.

[0026] Preferably, in the method for constructing the nanobody-assisted protein expression system that promotes the expression and secretion of exogenous proteins or polypeptides, the vector backbone will include a recombinant adeno-associated virus (rAAV) vector.

[0027] In the seventh aspect, the present invention provides a method for promoting the expression and secretion of exogenous proteins or polypeptides by nanoantibodies, characterized in that the nucleotide sequence comprising the fusion protein is cloned into an expression vector, preferably, further comprising a nucleotide sequence for inserting a promoter; further preferably, comprising a nucleotide sequence for inserting a CAG promoter.

[0028] In an eighth aspect, the present invention provides an application of the fusion protein, the expression vector and the protein expression system, characterized in that the nanobody promotes the expression and / or secretion of the exogenous protein or polypeptide fused thereto.

[0029] Preferably, the application of the fusion protein, expression vector and protein expression system is characterized by including application in biomedicine; preferably, including application in gene therapy and preparation of macromolecular biological products. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 shows the expression of PrP in cells cultured in vitro in Example 1 1-110 The secretory expression of protein and its fusion protein in the culture medium. Figure A is the result of immunoblotting analysis (WB). The first lane is PrP without fusion of any protein or polypeptide. 1-110 Protein, the second to eighth lanes are PrP 1-110 Fusion proteins with different proteins or polypeptides, where the second lane is PrP 1-110 -Nb fusion protein. Expresses PrP 1-110 -Nb culture medium (the second lane) was diluted 375 times, and the other lanes were directly loaded with undiluted culture medium. 1-110 Statistical chart of protein secretion. Figure C shows the removal of PrP 1-110 -Nb fusion protein secretion statistics of other proteins, thereby better showing the secretion expression statistics of other proteins;

[0031] Figure 2 shows the PrP in Example 2 1-110 -Nb expression in different mouse tissues. Panel A shows the expression results in PrP knockout mice, and Panel B shows the expression results in wild-type mice. The control is the PrP protein expression results in brain homogenate of wild-type mice to detect endogenous PrP expression (indicated by square brackets);

[0032] Figure 3 shows the PrP in Example 2 1-110 -Nb and PrP 1-110 Comparison of expression levels in mouse brain;

[0033] Figure 4 shows the promotion of PrP by different nanobodies (Nb) or Fc in Example 3 1-110 expression;

[0034] Figure 5 shows that the nanobody (Nb) in Example 4 promotes the expression and secretion of different polypeptides or proteins; "+" is the Nb fusion protein, "-" is the polypeptide somatostatin or protein Shadoo without Nb fusion; "secretion" refers to the secretion of proteins in the cell culture medium; "intracellular soluble" refers to the soluble protein in the cell lysate, representing the correctly folded protein that is being secreted outside the cell, and "intracellular insoluble" refers to the protein that is misfolded in the cell and will be degraded; the sample amount of "intracellular soluble" and "intracellular insoluble" is 5 times the sample amount of "secretion". DETAILED DESCRIPTION

[0035] The present invention now utilizes the characteristics of nano antibodies, such as small molecular weight, easy folding, stable structure and easy secretion, to connect nano antibodies and proteins or polypeptides together to form fusion proteins, so as to promote the expression and secretion of proteins or polypeptides.

[0036] Specifically, the present invention provides a Nanobody fusion protein that promotes the expression and secretion of a foreign protein or polypeptide, characterized in that it comprises a Nanobody and a foreign protein or polypeptide, and the Nanobody can promote the expression and secretion of the foreign protein or polypeptide fused thereto. The present invention also provides a method for preparing the Nanobody fusion protein. The present invention also provides an expression vector that promotes the expression and secretion of a foreign protein or polypeptide, characterized in that it comprises a polynucleotide sequence for the Nanobody fusion protein and a vector backbone. The present invention also provides a host cell that promotes the expression and secretion of a foreign protein or polypeptide, characterized in that the expression vector is expressed in the host cell. The present invention also provides a Nanobody auxiliary protein expression system that promotes the expression and secretion of a foreign protein or polypeptide, characterized in that it comprises the expression vector. The present invention also provides a method for constructing the Nanobody auxiliary protein expression system that promotes the expression and secretion of a foreign protein or polypeptide, comprising cloning the nucleotide sequence comprising the fusion protein into a vector backbone and then co-transfecting the system into a host cell. The present invention also provides a method for promoting the expression and secretion of a foreign protein or polypeptide using Nanobodies.

[0037] Nanobodies are conventional nanobodies that can be obtained by those skilled in the art according to conventional means, including one or more nanobodies derived from camels, alpacas or sharks, such as GFP-Nb nanobodies, ALFA-Nb nanobodies, mcherry-Nb nanobodies, PrP-Nb nanobodies, alpha-syn-Nb nanobodies, and IgG-Nb nanobodies.

[0038] Exogenous proteins or polypeptides are conventional proteins or polypeptides that can be obtained by those skilled in the art according to conventional means, especially proteins or polypeptides with disordered internal sequences, including but not limited to prion protein fragments PrP 1-110 , Shadoo protein, somatostatin polypeptide, beta-amyloid protein, tau protein, alpha synuclein, somatostatin, neurotensin, ghrelin, neuropeptide Y, neurokinin A, neurokinin B, substance P, oxytocin, vasopressin, neuroactive intestinal peptide, glucagon, glucagon-like peptide, endopioid peptide, atrial peptide, cholecystokinin, gastrin-releasing peptide, neuromedin B, neuromedin C, angiotensin, endothelin, galanin, bradykinin, motilin, etc.

[0039] The expression vector backbone is an expression vector backbone that can be obtained by those skilled in the art according to conventional means, including recombinant adeno-associated virus (rAAV) vectors, adenovirus vectors, lentivirus vectors, retrovirus vectors, eukaryotic cell expression vectors, insect baculovirus, etc. The expression vector promoter is a promoter that can be obtained by those skilled in the art according to conventional means, which can initiate the expression of foreign proteins or polypeptides in host cells, including but not limited to CAG, CMV, H2B, etc.

[0040] Host cells are host cells that can be obtained by those skilled in the art according to conventional means, including cultured eukaryotic cells in vitro, such as HEK293 cells, 293T cells, Hela cells, CHO cells, COS cells, Sf9 cells, etc., in which Nanobody fusion proteins are expressed, and Nanobodies can promote the expression and secretion of exogenous proteins or polypeptides fused thereto. In vivo hosts include expression in mammals, such as mice and Drosophila.

[0041] The technical solution of the present invention is further described in detail below with reference to specific embodiments and drawings.

[0042] Sources of biological materials in the embodiments of the present invention

[0043] The primers and protein PrP used in the embodiment of the present invention 1-110 , nanobody GFP-Nb, protein ZIP, nanobody mcherry-Nb, nanobody ALFA-Nb, protein shadoo and polypeptide somatostatin fused Nb and V5 tag nucleotide sequence (somatostatin-Nb-V5) were all synthesized and sold by Suzhou Jinweizhi Biotechnology Co., Ltd.; recombinant adeno-associated virus (rAAV) vector and CAG promoter are conventional vectors and promoters that can be obtained by those skilled in the art according to conventional means, specifically in this embodiment they are derived from the Vector Center of Beijing Institute of Brain Science and Brain-Inspired Technology; 293T cells are conventional cells that can be obtained by those skilled in the art according to conventional means, specifically in this embodiment they are derived from the Cell Resource Center of the Institute of Basic Medicine, Chinese Academy of Medical Sciences; antibody 6D11 was purchased from BioLegend; secondary antibody HRP-conjugated Affinipure Goat Anti-Mouse IgG (H+L) was purchased from Proteintech; Trans5α competent cells were purchased from Beijing Quanshijin Biological Co., Ltd., and antibodies against V5 tag protein were purchased from ThermoFisher Scientific.

[0044] The sources of the instruments and reagents used in the examples of the present invention are shown in Tables 1 and 2 below.

[0045] Table 1 Instrument sources

[0046] Table 2 Reagent sources

[0047] Example 1 Expression of PrP in in vitro cell culture 1-110 -Nb fusion protein

[0048] The N-terminus of prion protein (PrP) has a secretory signal peptide (SP) that guides PrP to be secreted outside the cell. The C-terminus of PrP has a well-folded structure, while the N-terminus is a flexible region without structure, so the N-terminus of prion protein is difficult to be highly expressed in cells or in vivo and secreted outside the cell. The nucleic acid sequence encoding the N-terminus of PrP (amino acids 1-110) is linked to the nucleic acid sequence encoding the nanobody (Nb) that recognizes green fluorescent protein (GFP), and the nucleic acid sequence encoding the fusion protein is linked to the recombinant adeno-associated viruses (rAAV) expression plasmid driven by the CAG promoter. Alternatively, PrP 1-110 The nucleic acid sequence of WT-WW polypeptide nucleic acid sequence, Variant-ww polypeptide nucleic acid sequence, Vilin headpiece polypeptide nucleic acid sequence, trp-cage polypeptide nucleic acid sequence, NTL9 1-39 The polypeptide nucleic acid sequence and ZIP protein nucleic acid sequence were connected by PCR, and then the product of PCR sequence amplification was connected with the expression plasmid vector to observe PrP 1-110 expression of the situation.

[0049] 1. Construction of fusion protein expression plasmid vector

[0050] Construction of PrP 1-110 -Nb as an example, the specific operations are as follows:

[0051] The nucleic acid sequence encoding the N-terminus of PrP (amino acids 1-110) (SEQ ID NO. 1) was used as a template and 1-F1 / 1-R1 as primers (primer sequences refer to Table 5) at an annealing temperature of 60°C to amplify the nucleic acid sequence encoding the N-terminus of PrP (amino acids 1-110) to obtain PrP. 1-110The Nb gene fragment was amplified by PCR using the nucleic acid sequence (SEQ ID NO. 2) encoding a nanobody (abbreviated as Nb) that recognizes green fluorescent protein (GFP) as a template and primers 1-F2 / 1-R2 (primer sequences are shown in Table 5) at an annealing temperature of 60°C. The reaction system used to amplify the target gene was 50 μL, as shown in Table 3:

[0052] Table 3 PCR reaction system

[0053] PCR reaction conditions are shown in Table 4:

[0054] Table 4 PCR reaction conditions

[0055] The primer sequences are shown in Table 5:

[0056] Table 5 Primer sequences used

[0057] The bacterial solution of the recombinant adeno-associated virus expression vector skeleton was added to ampicillin LB liquid medium (tryptone 0.01 g / mL; yeast powder 0.0005 g / mL; sodium chloride 0.01 g / mL; ampicillin concentration 100 μg / mL) and cultured overnight in a shaker at 37°C 220 rpm. The bacteria were collected and the plasmid was extracted according to the operation steps of the plasmid extraction kit, and the concentration was measured on Nanodrop. The plasmid was digested with restriction endonucleases BamHI and EcoRI and incubated at 37°C for 1 hour. The enzyme digestion system is shown in Table 6:

[0058] Table 6 Enzyme digestion system

[0059] The amplified PrP 1-110 The gene fragment and GFP-Nb gene fragment were connected to the recombinant adeno-associated virus expression vector backbone using the pEASY-Basic Seamless Cloning and Assembly Kit to form a fusion expression vector expressing PrP 1-110-Nb fusion protein (nucleic acid sequence shown in SEQ ID NO.7). The ligated product was added to Trans-5α competent cells thawed on ice, gently mixed, and placed in an ice bath for 30 minutes. After a 30-second incubation at 42°C, the cells were immediately returned to an ice bath for 2 minutes. 500 μL of LB liquid medium was then added to the tube. After incubation at 37°C and 220 rpm for 1 hour, 200 μL of the culture was evenly spread on ampicillin-resistant solid LB medium (tryptone 0.01 g / mL; yeast powder 0.005 g / mL; sodium chloride 0.01 g / mL; agarose 0.015 g / mL; ampicillin concentration 100 μg / mL) and incubated in a 37°C incubator overnight. Single clones were picked and incubated in 200 μL of ampicillin-resistant LB liquid medium for 2 hours before being sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing. After the sequencing results were aligned correctly, the cells were maintained and plasmid extraction and adeno-associated virus packaging were performed.

[0060] For the construction of other vectors, the template, primer sequence and annealing temperature are adjusted according to the construction of each vector. The following other vectors are constructed:

[0061] PrP 1-110 The -WT-WW vector uses the nucleic acid sequence encoding the PrP N-terminus (amino acids 1-110) (SEQ ID NO. 1) as a template, and performs the first round of PCR using 2-F1 / 2-R1 (annealing temperature Tm: 55°C). The first round of PCR amplification is then performed using the first round PCR product as a template using 2-F1 / 2-R2 (Tm: 55°C). The second round of PCR amplification is then performed using the second round PCR product as a template using 2-F1 / 2-R3 (Tm: 55°C). The fourth round of PCR amplification is performed using the product after the third round of amplification as a template using 2-F1 / 2-R4 (Tm: 60°C). The amplified product is then ligated to the expression plasmid vector backbone. The primer sequences used are shown in Table 11.

[0062] PrP 1-110 -Variant-WW vector construction method and PrP 1-110 -WT-WW was the same as that of WT-WW. The nucleic acid sequence encoding the N-terminus of PrP (amino acids 1-110) (SEQ ID NO. 1) was used as a template. The first round of PCR was performed with 3-F1 / 3-R1 (Tm: 55°C), the second round of PCR was performed with 3-F1 / 3-R2 (Tm: 55°C), the third round of PCR was performed with 3-F1 / 3-R3 (Tm: 55°C), and the fourth round of PCR was performed with 3-F1 / 3-R4 (Tm: 60°C). The amplified products were ligated to the expression vector backbone. The primer sequences used are shown in Table 11.

[0063] PrP 1-110-Vilin headpiece with the above PrP 1-110 -WT-WW was constructed in the same manner. The primer sequences used are shown in Table 11. The first round of PCR used the nucleic acid sequence encoding the PrP N-terminus (amino acids 1-110) (SEQ ID NO. 1) as a template and primers 4-F1 / 4-R1 (Tm: 60°C). The second round of PCR used the first-round product as a template and primers 4-F1 / 4-R2 (Tm: 56°C). The third round of PCR used the second-round PCR product as a template and primers 4-F1 / 4-R3 (Tm: 60°C). The fourth round of PCR used the third-round product as a template and primers 4-F1 / 4-R4 (Tm: 56°C).

[0064] PrP 1-110 -trp-cage vector construction and PrP 1-110 -WT-WW were the same, and the primer sequences used are shown in Table 11. The first round of PCR was performed with 5-F1 / 5-R1 (Tm: 55°C), the second round with 5-F1 / 5-R2 (Tm: 60°C), and the third round with 5-F1 / 5-R3 (Tm: 60°C).

[0065] PrP 1-110 -NTL9 1-39 Vector construction and PrP 1-110 -WT-WW was constructed in the same manner. The primer sequences used are shown in Table 11. Four rounds of PCR were required: the first round used primers 6-F1 / 6-R1 (Tm: 55°C), the second round used primers 6-F1 / 6-R2 (Tm: 60°C), the third round used primers 6-F1 / 6-R3 (Tm: 60°C), and the fourth round used primers 6-F1 / 6-R4 (Tm: 60°C).

[0066] PrP 1-110 -ZIP vector construction and PrP 1-110 The construction method of -Nb is the same. The primer sequences used are shown in Table 11. Using the nucleic acid sequence encoding the N-terminus of PrP (amino acids 1-110) (SEQ ID NO. 1) as a template and 7-F1 / 7-R1 (Tm: 59°C) as primers (primer sequences are shown in Table 11), the nucleic acid sequence encoding the N-terminus of PrP (amino acids 1-110) was amplified by PCR to obtain PrP. 1-110 The ZIP gene fragment was amplified by PCR using the ZIP nucleic acid sequence as a template and 7-F2 / 7-R2 (Tm: 50°C) as primers (primer sequences refer to Table 11) to obtain the ZIP gene fragment. The two amplified products were ligated to the expression vector backbone.

[0067] Table 7 PrP 1-110Fusion protein

[0068] Table 11

[0069] 2. In vitro cell expression system

[0070] 293T cells were cultured in complete medium (DMEM + 10% FBS + 1% PS) and passaged when the cell density reached 90%. After rinsing with PBS, they were digested with 0.25% trypsin for 30 seconds and then digested with complete medium. After centrifugation at 600 rpm for 5 minutes, they were resuspended in complete medium and the cells were counted after resuspension. The cells were plated at 5x10 5 100 μL DMEM medium was added with 2 μg of plasmid and then 2 μL of transfection reagent Neofect was added. TM DNA transfection reagent was mixed and allowed to stand for 15-30 minutes before being added dropwise to the cells in the six-well plate. After 24 hours of culture, the complete medium was replaced with serum-free medium (DMEM + 1% PS). After 24 hours of culture, samples were collected and analyzed.

[0071] For sample collection, the cultured cell culture medium was taken as the "secretory fraction" and the corresponding volume of 5x SDS loading buffer (Tris-HCl pH 6.8 250 mM; SDS 12%; glycerol 50%; bromophenol blue 0.04%; β-ME 10%) was added and incubated at 95°C in a metal bath for 10 min. After cooling, immunoblot analysis was performed.

[0072] Among them, PrP 1-110 -Nb cell culture medium was diluted 375-fold, while other culture media were not diluted.

[0073] 3. Western Blot Analysis (WB Analysis)

[0074] Prepare 14% separating gel, mix well and add to the middle of the gel plate, cool and add 5% stacking gel. The formulas of 14% separating gel and 5% stacking gel are shown in Tables 8 and 9.

[0075] Table 8 Separation gel (14%)

[0076] Table 9 Concentrated gel (5%)

[0077] After the stacking gel has solidified, place it in an electrophoresis tank and add 1x SDS running buffer (Tris 3g / L, SDS 1g / L, glycine 14.4g / L). Remove the comb and apply samples sequentially. Connect the power supply and set the parameters: 80V for 30 minutes, then 120V for 60 minutes, until the samples reach the bottom of the gel. After electrophoresis, wet-transfer the protein gel to a methanol-activated PVDF membrane at 35V for 16 hours on ice.

[0078] After transfer, the PVDF membrane was blocked with 5% (w / v) skim milk powder in TBST buffer (Tris 2.4 g / L, NaCl 8.8 g / L, Tween 20 0.1%) at room temperature for 1.5 hours. The membrane was then incubated with 6D11 antibody (1:2000) diluted in antibody diluent overnight at 4°C. After primary antibody incubation, the membrane was washed three times with TBST buffer for 5 minutes each. The membrane was incubated with secondary antibody (1:5000) diluted in TBST buffer for 1 hour at room temperature, followed by three washes with TBST buffer for 5 minutes each. The PVDF membrane was developed using an ECL colorimetric kit and imaged using an immunoblot imager.

[0079] As can be seen from Figure 1A, the expression of PrP 1-110 After the culture medium of -Nb fusion protein was diluted 375 times, the band intensity was still significantly stronger than that of PrP alone. 1-110 or PrP 1-110 -WT-WW, PrP 1-110 -Variant-WW, PrP 1-110 -Vilin-headpiece, PrP 1-110 -trp cage、PrP 1-110 -NTL9 1-39 , PrP 1-110 -ZIP fusion protein bands, showing that Nb can significantly help PrP that is not easy to express and secrete 1-110 It is expressed and secreted in cultured cells. Figure B shows PrP in the culture medium. 1-110 The statistical graph of protein secretion shows that its expression level is much higher than that of other samples. In order to clearly see the expression of other groups, Figure C is the one that removes PrP 1-110 -Nb statistical chart, statistics are based on PrP 1-110 The relative expression level of PrP was calculated as the standard. 1-110 -Nb secretion expression in the culture medium is PrP 1-110 Nearly 1000 times.

[0080] The nucleic acid sequence encoding the PrP N-terminus (amino acids 1-110) is SEQ ID NO. 1:

[0081] The nucleic acid sequence of the nanobody (Nb) that recognizes green fluorescent protein (GFP) is SEQ ID NO.2:

[0082] PrP 1-110 The nucleic acid sequence of the -Nb fusion protein is SEQ ID NO.7:

[0083] PrP 1-110 -WT-WW nucleic acid sequence is SEQ ID NO.13:

[0084] PrP 1-110 -Variant-WW nucleic acid sequence is SEQ ID NO.14:

[0085] PrP 1-110 -Vilin headpiece nucleic acid sequence is SEQ ID NO.15:

[0086] PrP 1-110 -trp-cage nucleic acid sequence is SEQ ID NO.16:

[0087] PrP 1-110 -NTL9 1-39 The nucleic acid sequence is SEQ ID NO.17:

[0088] PrP 1-110 -ZIP nucleic acid sequence is SEQ ID NO.18:

[0089] Example 2 In vivo expression of PrP 1-110 -Nb fusion protein

[0090] PrP 1-110 The vector of the Nb fusion protein was packaged as rAAV virus particles and injected into the lateral ventricle of newborn mice on the day of birth (animal experiments were in accordance with the experimental animal ethics of the Beijing Institute of Brain Science and Brain Inspiration, approval number: CIBR-IACUC-039) to detect PrP 1-110 -Nb fusion protein expression.

[0091] 1. Intracerebral Ventricular Injection in Mice

[0092] The constructed fusion plasmid expression vector PrP 1-110-Nb and PrP 1-110 Packaged as rAAV viral particles (packaged by the Vector Center of the Beijing Institute of Brain Science and Brain Inspiration). Place the mouse on the day of birth on a clean glove. Place the mouse to be injected on ice wrapped in rubber gloves (or towels) for anesthesia. After the mouse is anesthetized, use a glass microelectrode to inject 3×10 10 After the injection, the pups were returned to the mother mouse cage.

[0093] 2. Mouse Collection

[0094] Mice were anesthetized by intraperitoneal injection of tribromoethanol (350-500 mg / kg) according to their body weight. After anesthesia, the mice were fixed on a dissecting board, the thorax was opened to expose the heart, and a syringe containing 20 mL of PBS buffer was inserted into the left ventricle of the mouse. The right atrial appendage was cut open and perfused until clear fluid flowed out of the right atrial appendage. The brain, spinal cord, heart, liver, spleen, lungs, kidneys, intestines, muscles, and stomach of the mice were removed, placed in clean centrifuge tubes, and stored at -80°C.

[0095] 3. Extraction of Mouse Protein

[0096] Mouse tissues frozen at -80°C were removed and added to sterile 1× PBS at a ratio of 100 mg to 1 mL (w / v). The tissue was triturated (amplitude 8, 30 s, 3 times) to prepare a 10% tissue homogenate. 270 μL of the 10% homogenate was added to 30 μL of 10× cell lysis buffer (5% Triton X100 & sodium deoxycholate), vortexed, and incubated on ice for 10 minutes. The cells were sonicated in ice water (amplitude 10, on 30 s, off 30 s, for 5 minutes). Protein concentration was determined using a BCA assay. A 50 μL volume of 250 μg total protein was prepared, and 12.5 μL of 5× SDS loading buffer was added. The cells were incubated at 95°C for 10 minutes in a metal bath before immunoblotting analysis.

[0097] 4. Western Blot Analysis (WB Analysis)

[0098] According to the immunoblotting analysis method in Example 1, WB detection was performed on different mouse tissue homogenates using anti-PrP 6D11 antibody, as shown in Figure 2, Figure A shows the results of expression in PrP knockout mice, Figure B shows the results in wild-type mice, and the control is the brain homogenate of wild-type mice to detect endogenous PrP (shown in brackets). From Figure 2 A and Figure B, it can be seen that PrP 1-110 -Nb fusion protein is highly expressed in the central nervous system of mice (brain and spinal cord). It can be seen that endogenous PrP is expressed at a high level in the brain and spinal cord, and is also expressed in other tissues, but at a lower level. 1-110The expression of -Nb fusion protein in the mouse brain and spinal cord is even higher than that of endogenous PrP which is highly expressed in the central nervous system.

[0099] PrP 1-110 and PrP 1-110 The difference in the expression of -Nb in mouse brain is shown in Figure 3. 1-110 The expression level of -Nb fusion protein was significantly higher than that of PrP 1-110 , especially in PrP knockout mice.

[0100] Figures 2 and 3 show that Nb can also effectively help PrP 1-110 The expression in mice was consistent with the experimental results of in vitro cell culture.

[0101] Example 3 Different Nanobodies (Nb) Promote the Expression and Secretion of Proteins or Peptides

[0102] The nanobody that recognizes green fluorescent protein GFP (GFP-Nb), the nanobody that resists polypeptide ALFA-tag (ALFA-Nb), the nanobody that resists fluorescent protein mCherry (mcherry-Nb) and the nucleic acid sequence encoding the Fc fragment of mouse immunoglobulin (IgG1) were respectively combined with PrP 1-110 The fusion proteins were connected together (as shown in Table 10) and cloned into the CAG promoter-driven expression vector. These plasmids were then transfected into 293T cells, and the serum-free cell culture fluid was collected. Western blotting was performed using the anti-PrP 6D11 antibody to demonstrate that different nanobodies (Nb) promoted the expression and secretion of proteins or polypeptides.

[0103] 1. Construction of fusion protein expression plasmid vector

[0104] According to Example 1, PrP was constructed 1-110 -Nb fusion expression plasmid vector method, construction of PrP 1-110 -Nb(ALFA), with PrP 1-110 PCR amplification was performed using 8-F1 / 8-R1 (Tm: 60°C) as a template and 8-F2 / 8-R2 (Tm: 60°C) as primers using the anti-polypeptide ALFA-tag nanoantibody ALFA-Nb shown in SEQ ID NO.8 as a template. The two PCR products were connected to the expression vector backbone. 1-110 -Nb(mcherry) with PrP 1-110As a template, 9-F1 / 9-R1 (Tm: 60°C) was used for PCR amplification, and the anti-fluorescent protein mCherry nanoantibody mcherry-Nb shown in SEQ ID NO.9 was used as a template and primers 9-F2 / 9-R2 (Tm: 60°C) were used for amplification. 1-110 -Fc with PrP 1-110 PCR amplification was performed using 10-F1 / 10-R1 (Tm: 59°C) as a template. The nucleic acid sequence encoding the Fc fragment of mouse immunoglobulin (IgG1) as shown in SEQ ID NO. 10 was also amplified using 10-F2 / 10-R2 (Tm: 59°C) as a template. The amplified product was ligated into the expression vector backbone. See Table 12 for the primers listed above.

[0105] Table 10 Different Nanobodies and PrP 1-110 Fusion protein

[0106] Table 12

[0107] 2. In vitro cell expression system

[0108] According to the method for constructing an in vitro cell expression system in Example 1, the obtained plasmids were transfected into 293T cells, and the serum-free cell culture fluid was collected.

[0109] 3. Immunoblotting Analysis

[0110] According to the immunoblotting analysis method in Example 1, immunoblotting analysis was performed. From Figure 4, we can see that three different nanoantibodies PrP 1-110 -Nb(GFP), PrP 1-110 -Nb(ALFA), PrP 1-110 -Nb (mcherry) can help protein expression and secretion, and the effect is similar to PrP 1-110 -Fc fragment is similar.

[0111] The nucleic acid sequence of the nanobody ALFA-Nb is SEQ ID NO.8:

[0112] The nucleic acid sequence of the nanobody mcherry-Nb is SEQ ID NO.9:

[0113] The nucleic acid sequence of the Fc fragment of mouse immunoglobulin (IgG1) is SEQ ID NO.10

[0114] Example 4 Nanobodies (Nb) Promote the Expression and Secretion of Different Proteins or Peptides

[0115] The nanobody (GFP-Nb) that recognizes green fluorescent protein GFP was connected to somatostatin (a 28-amino acid polypeptide) and the difficult-to-express protein Shadoo to form fusion proteins. A V5 tag (V5-tag) was added to each protein for easy detection and cloned into an expression vector driven by the CAG promoter. These plasmids were then transfected into 293T cells, and serum-free cell culture fluid, cell-soluble proteins and cell-insoluble proteins were collected for Western blotting to prove that the nanobody (Nb) promotes the expression and secretion of different proteins or polypeptides.

[0116] 1. Construction of fusion expression plasmid vector

[0117] Fusion proteins of the different proteins or polypeptides listed in Table 13 with Nb were constructed separately. A V5 tag was added to each protein to facilitate detection. Following the method for constructing a fusion expression plasmid vector in Example 1, the somatostatin polypeptide shown in SEQ ID NO. 11 was used as a template and primers 11-F1 / 11-R1 (Tm: 58°C) were used for the first round of PCR amplification. The first round of PCR product was then used as a template and primers 11-F1 / 11-R2 (Tm: 60°C) were used for the second round of PCR. The PCR product was then ligated to the expression vector backbone. The vector Somatostatin-Nb-V5 was PCR amplified using Somatostatin-Nb-V5 (Tm: 58°C) synthesized in the company as a template and primers 12-F1 / 12-R1 (Tm: 60°C). Shadoo-V5 was amplified in the first round of PCR using the nucleic acid sequence of the Shadoo protein shown in SEQ ID NO. 12 as a template using 13-F1 / 13-R1 (Tm: 60°C). The first round of PCR amplification was then performed using the product from the first round as a template using 13-F1 / 13-R2 (Tm: 60°C). Shadoo-Nb-V5 was amplified in the first round of PCR using the nucleic acid sequence of the Shadoo protein shown in SEQ ID NO. 12 as a template using 14-F1 / 14-R1 (Tm: 60°C). The first round of PCR amplification was then performed using the nucleic acid sequence encoding the nanobody (Nb) that recognizes green fluorescent protein (GFP) (SEQ ID NO. 2) as a template using 14-F2 / 14-R2 (Tm: 60°C). The second round of PCR amplification was then performed using the product from the first round as a template using 14-F2 / 14-R3 (Tm: 60°C). The corresponding amplified products were ligated into the expression vector backbone. Primer sequences refer to Table 14.

[0118] Table 13 Different proteins or polypeptides and Nb fusion proteins

[0119] Table 14

[0120] 2. In vitro cell expression system

[0121] According to the method for constructing an in vitro cell expression system in Example 1, the obtained plasmids were transfected into 293T cells, serum-free cell culture fluid was collected, the cells were lysed, and the intracellular soluble proteins (supernatant) and intracellular insoluble proteins (precipitate) were collected by centrifugation.

[0122] For sample collection, the culture medium was collected as the "secretory fraction." After rinsing the cells with PBS, 200 μL of cell lysis buffer (50 mM NaCl, 5 mM Tris-HCl, pH 8.0, 2 mM EDTA, 0.5% Triton X-100, and 0.5% sodium deoxycholate) was added to each well. Cells were scraped from the bottom of the dish using a cell scraper and transferred to a centrifuge tube. After sonication in a waterbath (amplitude 10, time 5 min, 30 s on, 30 s off), the cells were centrifuged at 15,600 rpm at 4°C for 30 min. The supernatant was transferred to a new centrifuge tube and 50 μL of 5x SDS loading buffer (250 mM Tris-HCl, pH 6.8; 12% SDS; 50% glycerol; 0.04% bromophenol blue; 10% β-ME) was added. The supernatant was incubated at 95°C in a metal bath for 10 min to obtain the "intracellular soluble fraction." After cooling, the supernatant was analyzed by immunoblotting. The precipitate after centrifugation was the “intracellular insoluble fraction”, to which 250 μL 1× SDS loading buffer was added, and after sonication (amplitude 40, time 5 min, on 30 s, off 30 s), it was placed in a 95°C metal bath for 10 min and cooled before immunoblotting analysis.

[0123] 3. Immunoblotting Analysis

[0124] According to the method of immunoblotting analysis in Example 1, immunoblotting analysis was performed using an anti-V5-tag antibody (1:2000 dilution), and the amount of intracellular soluble and intracellular insoluble samples was 5 times the amount of secretion sample. As shown in Figure 5, "+" is the Nb fusion protein, "-" is the polypeptide somatostatin or Shadoo protein without Nb fusion, "secretion" is the protein secreted from the cell in the cell culture medium, "intracellular soluble" is the soluble protein in the cell lysate, representing the correctly folded protein that is being secreted outside the cell, and "intracellular insoluble" is the protein that is misfolded in the cell and will be degraded. As can be seen from Figure 5, most of Shadoo-Nb and somatostatin-Nb have been secreted outside the cell, and most of the intracellular is also soluble. This shows that nano antibodies can help different proteins or polypeptides to be effectively expressed and secreted.

[0125] The nucleic acid sequence of somatostatin polypeptide is SEQ ID NO.11:

[0126] Nucleic acid sequence of Shadoo protein SEQ ID NO.12:

[0127] The nucleic acid sequence of the V5 tag is SEQ ID NO.19:

Claims

1. A nanobody fusion protein that promotes the expression and secretion of exogenous proteins or polypeptides, characterized in that: It comprises nano antibodies and foreign proteins or polypeptides, and the nano antibodies can promote the expression and secretion of foreign proteins or polypeptides fused therewith.

2. The nanobody fusion protein according to claim 1, characterized in that The nanobody comprises one or more nanobodies, and the nanobodies are directly connected to each other; preferably, there is a connecting peptide between the nanobodies.

3. The nanobody fusion protein according to claim 1 or 2, characterized in that The nanobody is directly linked to the foreign protein or polypeptide; preferably, the nanobody is linked to the C-terminus or N-terminus of the foreign protein or polypeptide.

4. The nanobody fusion protein according to any one of claims 1 to 3, characterized in that There is a connecting peptide between the nanobody and the foreign protein or polypeptide.

5. The nanobody fusion protein according to any one of claims 1 to 4, characterized in that The exogenous protein or polypeptide includes a protein or polypeptide with biological activity; preferably, the exogenous protein or polypeptide includes one of a cytokine, a ligand, a receptor, an enzyme and a polypeptide antigen; more preferably, the exogenous protein or polypeptide includes a chain-like unstructured protein or polypeptide; further preferably, the exogenous protein or polypeptide includes a prion protein fragment PrP encoded by the nucleic acid sequence shown in SEQ ID NO.1 1-110 , one of the following: a Shadoo protein encoded by the nucleic acid sequence shown in SEQ ID NO.12, and a somatostatin polypeptide encoded by the nucleic acid sequence shown in SEQ ID NO.

11.

6. The nanobody fusion protein according to any one of claims 1 to 5, characterized in that One or more of the GFP-Nb nanobody encoded by the nucleic acid sequence shown in SEQ ID NO.2, the ALFA-Nb nanobody encoded by the nucleic acid sequence shown in SEQ ID NO.8, and the mcherry-Nb nanobody encoded by the nucleic acid sequence shown in SEQ ID NO.9 are connected to an exogenous protein or polypeptide.

7. An expression vector for promoting the expression and secretion of foreign proteins or polypeptides, characterized in that: Comprising a polynucleotide sequence for encoding the nanobody fusion protein according to any one of claims 1 to 6 and a vector backbone.

8. The expression vector for promoting the expression and secretion of foreign proteins or polypeptides according to claim 7, characterized in that: The vector backbone comprises a recombinant adeno-associated virus (rAAV) vector; preferably, it also comprises an inserted promoter nucleotide sequence; further preferably, it comprises a CAG promoter nucleotide sequence.

9. A host cell for promoting the expression and secretion of foreign proteins or polypeptides, characterized in that: The expression vector according to any one of claims 7 or 8 is expressed in a host cell.

10. A method for preparing the nanobody fusion protein according to any one of claims 1 to 6, comprising culturing the host cell according to claim 9 and isolating the nanobody fusion protein according to any one of claims 1 to 6.

11. A nano-antibody-assisted protein expression system for promoting the expression and secretion of foreign proteins or polypeptides, characterized in that: Comprising the expression vector according to claim 7 or 8.

12. The nano-antibody-assisted protein expression system for promoting the expression and secretion of foreign proteins or polypeptides according to claim 11, characterized in that: In vitro expression systems and / or in vivo expression systems are included.

13. A method for constructing a nano-antibody-assisted protein expression system for promoting the expression and secretion of foreign proteins or polypeptides as described in claim 11 or 12, comprising cloning the nucleotide sequence of the fusion protein described in any one of claims 1 to 6 into a vector backbone and then transfecting a host cell to construct the system.

14. According to the method for constructing a nano-antibody auxiliary protein expression system for promoting the expression and secretion of foreign proteins or polypeptides as described in claim 13, the vector backbone will include a recombinant adeno-associated virus (rAAV) vector, preferably, also including a nucleotide sequence for inserting a promoter; further preferably, including a nucleotide sequence for inserting a CAG promoter.

15. A method for promoting the expression and secretion of foreign proteins or polypeptides by nanoantibodies, characterized in that: The nucleotide sequence comprising the fusion protein according to any one of claims 1 to 6 is cloned into an expression vector.

16. Use of the fusion protein according to any one of claims 1 to 6, the expression vector according to claim 7, and the protein expression system according to claim 11 or 12, characterized in that: Nanobodies promote the expression and / or secretion of foreign proteins or polypeptides fused thereto.

17. Use of the fusion protein, expression vector and protein expression system according to claim 16, characterized in that: The application includes in biomedicine; preferably, the application includes in gene therapy and preparation of macromolecular biological products.

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