A method for preparing a fusion protein for targeted treatment of Alzheimer's disease and its delivery system

Abstract

The present invention discloses a method for preparing a fusion protein for targeted treatment of Alzheimer's disease and a delivery system thereof. The present invention connects the RING type + B box type domain in the TRIM21 protein with an antibody or nanobody targeting Aβ oligomers through a linker, constructs a bispecific fusion protein, selects AAV9 with neurophagocytic properties as a delivery vector for the nucleic acid sequence of the bispecific fusion protein, and mediates the expression of the anti-Aβ oligomer bispecific fusion protein to achieve long-term stable expression in vivo, and its expression product can specifically recognize Aβ oligomers and activate the ubiquitination degradation system, so as to achieve the purpose of long-term specific removal of Aβ oligomers in vivo, thereby achieving the treatment of AD.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology and drug delivery, and in particular to a method for preparing a fusion protein for targeted treatment of Alzheimer's disease and a delivery system thereof. Background Art

[0002] Alzheimer's disease (AD) is a degenerative disease of the central nervous system, and its pathological feature is progressive cognitive impairment. The main neuropathological feature is the deposition of extracellular amyloid beta (Aβ) to form plaques and intracellular neurofibrillary tangles. Aβ deposition plaques are formed by the aggregation of Aβ monomers into oligomers, fibrils, and finally fibers. Aβ exists in various forms, including monomers, oligomers, fibrils, and fibers, among which oligomers are the most toxic.

[0003] At present, the hot spots of AD drug research and development are small molecule inhibitor drugs and immune antibody drugs targeting Aβ aggregation. Among them, small molecule inhibitor drugs have poor targeting and greater neurotoxicity; antibody drugs require long-term repeated administration, which imposes a heavy burden on patients physically and economically. At present, there are many monoclonal antibodies in the form of IgG for AD treatment in clinical practice, but due to their low blood-brain barrier permeability, the treatment effect is not good. Moreover, IgG can also cause side effects such as meningoencephalitis and cerebral microbleeds in the body. At present, most of the nanoantibodies used in clinical treatment of AD are nanoantibodies in the form of scFv, which have disadvantages such as a short retention time in the body. Summary of the invention

[0004] In view of the defects of the above-mentioned AD antibody drug treatment, the present invention provides an innovative method for preparing a fusion protein for targeted treatment of Alzheimer's disease and its delivery system.

[0005] The concept of designing a bispecific fusion protein molecule of the present invention adopts the form of "Ring-Bait". The principle is to use the "Ring" domain in the TRIM21 protein to activate the E3 ubiquitin ligase and the protein degradation system to degrade the oligomers of amyloid beta (Aβ) that specifically bind to the "Bait" domain, so as to achieve the purpose of treating Alzheimer's disease. Among them, the "Ring" domain refers to the "Ring" domain in TRIM21 associated with E3, and the "Bait" domain refers to an antibody or nanobody targeting Aβ oligomers. The two functional molecules of "Ring" and "Bait" are connected by a linker, so that the fusion protein molecule has the function of specifically recognizing Aβ oligomers and activating the ubiquitination system.

[0006] Specifically, the present invention adopts the following technical solutions:

[0007] A bispecific fusion protein comprises a RING type + B box type domain in a TRIM21 protein, a linker, and an antibody or nanobody targeting Aβ oligomers; the RING type + B box type domain in the TRIM21 protein and the antibody or nanobody targeting Aβ oligomers are connected by a linker; the linker is a protein linker or a small molecule compound.

[0008] The present invention uses the RING type + B box type domain in the TRIM21 protein as the "Ring" domain. When two or more "Ring" domains are close to and enriched, the ubiquitination tag system can be activated to degrade the target protein. Preferably, the amino acid sequence of the RING type + B box type domain in the TRIM21 protein is shown in SEQ ID NO.15.

[0009] The present invention uses antibodies or nanobodies targeting Aβ oligomers as the "Bait" domain. Preferably, the antibodies targeting Aβ oligomers include IgG antibodies; IgG antibodies are derived from antibodies obtained by immunizing animals with antigens prepared by coupling the full length of Aβ42 or its polypeptides with other proteins, and the other proteins mainly include bovine serum albumin (BSA), chicken ovalbumin (OVA) and hemocyanin (KLH), and further specifically KLH.

[0010] Preferably, the nanobody targeting Aβ oligomers includes nanobodies in the form of scFv, Fab or VHHs. The nanobody is derived from other forms of nanobodies such as scFv, Fab and VHHs obtained by phage display or yeast display screening. Further preferably, the nanobody is derived from VHHs nanobody obtained by phage display panning of alpaca immune library. Further preferably, the amino acid sequence of the nanobody targeting Aβ oligomers is shown in any one of SEQ ID NO.6-10.

[0011] The "Ring" and "Bait" domains of the present invention are connected by a protein linker or a small molecule compound. Preferably, the protein linker includes a rigid or flexible linker. Rigid linkers include (NANP)3, A(EAAAK)nA, EPKIPQPQPKPQPQPQPQPQPQPKPQPKPEP; further preferably, the value of n in A(EAAAK)nA ranges from 2 to 6, and the more preferred value is 3. Flexible linkers include (GGGGS)n, and further preferably, the value of n in (GGGGS)n ranges from 3 to 5, and the more preferred value is 3.

[0012] Preferably, the small molecule compound comprises an alkyl linker, PEG or a triazole linker.

[0013] The present invention designs different bispecific fusion proteins by combining different VHH nanobodies with different linkers, namely "Ring" + Linker1 + NB1 (SEQ ID NO.16), "Ring" + Linker1 + NB2 (SEQ ID NO.17), "Ring" + Linker1 + NB3 (SEQ ID NO.18), "Ring" + Linker1 + NB4 (SEQ ID NO.19), "Ring" + Linker1 + NB5 (SEQ ID NO.20), "Ring" + Linker2 + NB1 (SEQ ID NO.21), "Ring" + Linker2 + NB2 (SEQ ID NO.22), "Ring" + Linker2 + NB3 (SEQ ID NO.23), "Ring" + Linker2 + NB4 (SEQ ID NO.24), "Ring" + Linker2 + NB5 (SEQ ID NO.25), "Ring" + Linker3 + NB1 (SEQ ID NO.26), "Ring" + Linker3 + NB2 (SEQ ID NO.27). NO.27), "Ring"+Linker3+NB3 (SEQ ID NO.28), "Ring"+Linker3+NB4 (SEQ ID NO.29), "Ring"+Linker3+NB5 (SEQ ID NO.30), "Ring"+Linker4+NB1 (SEQ ID NO.31), "Ring"+Linker4+NB2 (SEQ ID NO.32), “Ring”+Linker4+NB3 (SEQ ID NO.33), “Ring”+Linker4+NB4 (SEQ ID NO.34), “Ring”+Linker4+NB5 (SEQ ID NO.35).

[0014] The present invention also provides a drug delivery system for the targeted treatment of Alzheimer's disease, wherein the coding gene of the above-mentioned bispecific fusion protein is constructed into an AAV vector to prepare an AAV vector delivery system, or the above-mentioned bispecific fusion protein or its coding gene is prepared into a protein corona delivery system through nanomaterial encapsulation technology.

[0015] Preferably, the AAV vector comprises AAV2, AAV5 or AAV9.

[0016] The present invention also provides the use of the above-mentioned bispecific fusion protein or drug delivery system in the preparation of drugs for the targeted treatment of Alzheimer's disease.

[0017] Preferably, the bispecific fusion protein is administered in the form of a protein or its mRNA drug or DNA vaccine by delivery or injection.

[0018] In the first aspect, the present invention uses chemically synthesized Aβ1-16 and hemocyanin (KLH) conjugate KLH-Aβ1-16 as an antigen to immunize alpacas, construct a phage display library, and conduct 4 rounds of panning with Aβ oligomers as the target, and finally obtain 5 high-affinity antibodies, which are named NB1 (SEQ ID NO.6), NB2 (SEQ ID NO.7), NB3 (SEQ ID NO.8), NB4 (SEQID NO.9) and NB5 (SEQ ID NO.10).

[0019] Preferably, the NB2 (SEQ ID NO. 7) sequence is selected from the above 5 Nanobodies as the "Bait" domain in the bispecific molecule, and its sequence is used to design a bispecific fusion protein molecule for heterologous expression.

[0020] Specifically, the codon-optimized gene sequence of “Ring”+Linker1+NB2+6His fusion protein was inserted into pcDNA3.4 and heterologously expressed in mammalian cells HEK293.

[0021] In the second aspect, the linkers involved in the present invention include flexible linkers: usually composed of amino acids such as glycine or serine, these linkers allow greater movement between two functional domains; rigid linkers: these linkers provide a fixed distance between components; in vivo cleavable linkers: designed to be cleaved by specific enzymes or conditions in a biological environment.

[0022] Preferably, in the present invention, a rigid protein linker is selected to connect the "Ring" and "Bait" structures, and A(EAAAK)3A is further selected.

[0023] The third aspect is the design of a bispecific fusion protein, in which "Ring" refers to the RING type + Bbox type domain in the TRIM21 protein, and its amino acid sequence is from segment N to segment C; "Bait" is the nanoantibodies NB1, NB2, NB3, NB4 and NB5 targeting Aβ oligomers, and the amino acid sequence order of the nanoantibodies in the present invention is from segment C to segment N.

[0024] Preferably, NB2 is the VHH screened out with the highest affinity to the target. The codon-optimized gene sequence of the "Ring"+A(EAAAK)3A+NB2+6His (NB2 amino acid sequence from segment C to segment N) fusion protein is inserted into pcDNA3.4 and heterologously expressed in mammalian cells HEK293.

[0025] Fourthly, the ability of the nanoantibodies in the purified fusion protein to bind to Aβ oligomers was detected by a sandwich ELISA method, and in vitro experiments demonstrated that the "Bait" domain of the bispecific fusion protein molecule of the present invention has a high affinity for Aβ oligomers.

[0026] The fifth aspect involves delivery methods, including "protein crown" and AAV vector delivery system. Direct injection mainly includes intravenous injection and intrathecal injection. AAV vector delivery system mainly includes AAV5 and AAV9.

[0027] Preferably, AAV9 is used in the present invention to deliver the nucleic acid sequence of the bispecific fusion protein.

[0028] Sixthly, through brain tissue section staining analysis and determination of target gene transcription levels, the results showed that the rAAV9-Ring-Linker1-NB2 delivery system is effective and can degrade Aβ polymers in vivo, and therefore can be used as a candidate drug for the treatment of Alzheimer's disease.

[0029] In summary, the present invention uses AAV9 vector as a delivery tool, and mediates the expression of anti-Aβ oligomer bispecific fusion protein by delivering the nucleic acid sequence of bispecific fusion protein in vivo, so as to achieve long-term stable expression in vivo, and its expression product can specifically recognize Aβ oligomers and activate the ubiquitination degradation system, so as to achieve the purpose of long-term specific clearance of Aβ oligomers in vivo, thereby realizing the treatment of AD. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is an agarose gel electrophoresis diagram of two rounds of PCR amplification in Example 1 of the present invention; M: DNA Marker; Lane 1: the result of the first round of PCR amplification (IgG), the band gel recovery product in the red frame is used as the template for the second round; Lane 2: VHHs.

[0031] Figure 2 The results of colony PCR identification in Example 3 of the present invention; M: DNA Marker, lanes 1-3: different monoclonal strains No. 1-3.

[0032] Figure 3 This is a map for constructing the plasmid pcDNA-Ring-Linker1-NB2-6His in Example 3 of the present invention.

[0033] Figure 4 The map is constructed for the plasmid pcDNA-Ring-Linker1-NB2-6His in Example 3 of the present invention; M: protein marker; lane 1: sample solution after membrane; lane 2: flow solution; lane 3: equilibrium solution Buffer A; lane 4: eluent; lane 5: sample after desalting.

[0034] Figure 5 This is the Elisa test result of the RL2H fusion egg in Example 4 of the present invention.

[0035] Figure 6 This is a map of the plasmid pAAV-RL2 constructed in Example 5 of the present invention.

[0036] Figure 7 This is a statistical chart of the number of times the experimental mice crossed the original platform in Example 6 of the present invention.

[0037] Figure 8 This is the open field test result in Example 6 of the present invention.

[0038] Fig. 9 The results of immunofluorescence staining of hippocampus. DETAILED DESCRIPTION

[0039] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solution of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] Example 1: Acquisition of VHH immune library

[0041] Chemically synthesized Aβ1-16 and hemocyanin (KLH) conjugate KLH-Aβ1-16 was used as the antigen. The antigen and adjuvant were emulsified at a ratio of 1:1 and then immunized alpacas. The alpacas were immunized by three-point subcutaneous injection in the neck, with immunizations every 3 weeks for a total of 6 immunizations. 20 mL of peripheral blood was collected from the alpacas one week after the 6 immunizations for subsequent experiments.

[0042] The lymphocytes in the peripheral blood of alpacas were extracted using the kit and separated according to the instructions. The number of lymphocytes after extraction was greater than 2.5×10 7 The total RNA from lymphocytes was extracted using an RNA extraction kit. The total RNA was stored at -80°C for subsequent reverse transcription experiments. The extracted RNA was reverse transcribed into cDNA using a reverse transcription kit according to the instructions, and stored at -20°C for subsequent experiments.

[0043] The cDNA obtained by reverse transcription was used as a template for nested PCR reaction. Two rounds of amplification were performed. The primer sequences of the first round of PCR were CALL001 (GTCCTGGCTGCTCTTCT, SEQ ID NO.1) and CALL002 (GGTACGTGCTGTTGAACTGTTCC, SEQ ID NO.2); the primer sequences of the second round of PCR were VHH-F1 (GGCCATTACGGCCGATGTGCAGCTGCAGGAGTCTGGRGGA, SEQ ID NO.3), VHH-R1 (ACCCAGGTCACCGTCTCCAGCGGCCGATTAGGCC, SEQ ID NO.4) and VHH-R2 (ACCCAGGTCACCGTCTCCTCAGGCCGATTAGGCC, SEQ ID NO.5), and the PCR product of about 700 bp in the first round of PCR product was used as a template for further amplification. The PCR products of the two rounds of amplification were analyzed by agarose gel electrophoresis. The results are shown in Figure 1 .

[0044] Example 2: VHH phage construction, panning and sequencing

[0045] The present invention digested the VHHs in Example 1 with the phagemid vector pComb3XSS with SfiI, connected them, and electroporated them into TG1 competent cells; added helper phage, and prepared the KLH-Aβ1-16 nanoantibody phage library after PEG / NaCl precipitation. The library capacity was identified to be 3.6×10 8 CFU / mL, and the positive rate of sequencing analysis was 96%. With Aβ42 oligomers as the target antigen, 122 monoclonal colonies were randomly picked after 4 rounds of panning and identified by sandwich Phase-ELISA with a positive rate of 89.6%. Among them, the neutralization rate of 21 monoclonal phage supernatants was ≥95%. After sequencing, 5 high-affinity antibodies were finally obtained, named NB1 (SEQ ID NO.6), NB2 (SEQ ID NO.7), NB3 (SEQ ID NO.8), NB4 (SEQ ID NO.9) and NB5 (SEQ ID NO.10). Among them, the binding constant of NB2 is 2.1×10 7 L / moL and KD value are 1.2×10 -4 M, has the highest affinity to the target.

[0046] SEQ ID NO.6:

[0047] QVQLQESGGGSVQAGGSLRLSCAASASGFTMFSQGWFRQPPGKEREGVANAIRDGRSIYADSVKGRFTISQDNAKNTLYLQMNSLKPEDTAMYYCALSGYSAKGYSFDGQGSYFWGQGTQVTVSS(NB1);

[0048] SEQ ID NO.7:

[0049] QVQLQESGGGSVQAGGSLRLSCAASASGFTMFSQGWFRQPPGKEREGVANAIRRDGRSIYADSVKGRFTISQDNAKNTLYLQMNSLKPEDTAMYYCALSGYSAKYSFDGQGSYFWGQGTQVTVSS(NB2);

[0050] SEQ ID NO.8:

[0051] QVQLQESGGGSVQAGGSLRLSCAASASGFTMFSQGWFRQPPGKEREGVANAIRDGRSIYA

[0052] DSVKGRFTISQDNAKNTLYLQMNSLKPEDTAMYYCALSGYSAKGYSFDMQGSYFWGQGTQVTVSS(NB3);

[0053] SEQ ID NO.9:

[0054] QVQLQESGGGSVQAGGSLRLSCAASASGFTFFSQGWFRQPPGKEREGVANAIRDGRSIYADSVKGRFTISQDNAKNTLYLQMNSLKPEDTAMYYCALSGYSAKGYSFDGQGSYFWGQGTQVTVSS(NB4);

[0055] SEQ ID NO.10:

[0056] QVQLQESGGGSVQAGGSLRLSCAASASGFTMFSQGWFRQPPGKEREGVANAIRDGRSIYADSVKGRFTISQDNAKNTLYLQMNSLKPEDTAMYYCALSGYSAKGYSSDGQGSYFWGQGTQVTVSS(NB5).

[0057] Example 3: Design, Heterologous Expression, and Purification of Bispecific Fusion Proteins

[0058] 1. Design of bispecific fusion protein molecules

[0059] In the present invention, linker1 (SEQ ID NO.11), linker2 (SEQ ID NO.12), linker3 (SEQID NO.13) and linker4 (SEQ ID NO.14) are used to connect the "Ring" (SEQ ID NO.15) with different screened high-affinity nanoantibodies NB; in order to make the two domains in the bispecific fusion protein have their own complete functions, not only the protein linker is used, but also the C segment of the amino acid sequence of the nanoantibody referred to by "Bait" is connected to the linker.

[0060] By combining different VHH nanobodies with different linkers, different bispecific fusion protein molecules were designed, namely "Ring" + Linker1 + NB1 (SEQ ID NO.16), "Ring" + Linker1 + NB2 (SEQ ID NO.17), "Ring" + Linker1 + NB3 (SEQ ID NO.18), "Ring" + Linker1 + NB4 (SEQ ID NO.19), "Ring" + Linker1 + NB5 (SEQ ID NO.20), "Ring" + Linker2 + NB1 (SEQ ID NO.21), "Ring" + Linker2 + NB2 (SEQ ID NO.22), "Ring" + Linker2 + NB3 (SEQ ID NO.23), "Ring" + Linker2 + NB4 (SEQ ID NO.24), "Ring" + Linker2 + NB5 (SEQ ID NO.25), "Ring" + Linker3 + NB1 (SEQ ID NO.26), and "Ring" + Linker3 + NB2 (SEQ ID NO.27). NO.27), "Ring"+Linker3+NB3 (SEQ ID NO.28), "Ring"+Linker3+NB4 (SEQ ID NO.29), "Ring"+Linker3+NB5 (SEQ ID NO.30), "Ring"+Linker4+NB1 (SEQ ID NO.31), "Ring"+Linker4+NB2 (SEQ ID NO.32), “Ring”+Linker4+NB3 (SEQ ID NO.33), “Ring”+Linker4+NB4 (SEQ ID NO.34), “Ring”+Linker4+NB5 (SEQ ID NO.35).

[0061] SEQ ID NO.11:AEAAAKEAAAKEAAAKA(linker1);

[0062] SEQ ID NO.12:NANPNANPNANP(linker2);

[0063] SEQ ID NO.13:EPKIPQPQPKPQPQPQPQPQPKPQPKPEP(linker3);

[0064] SEQ ID NO.14:GGGGSGGGGSGGGGS(linker4);

[0065] SEQ ID NO.15:

[0066] MASAARLTMMWEEVTCPICLDPPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQLANMVNNLKEISQEAREGTQGERCAVHGERLHLFCEKDGKALCWVCAQSRKHRDHAMV(RING type+B boxtype+MV);

[0067] SEQ ID NO.16:

[0068] MASAARLTMMWEEVTCPICLDPPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQLANMVNNLKEISQEAREGTQGERCAVHGERLHLFCEKDGKALCWVCAQSRKHRDHAMVAEAAAKEAAAKEAAKAS SVTVQTGQGWFYSGQGDFSYGKASYGSLACYYMATDEPKLSNMQLYLTNKANDQSITFRGKVSDAYISRGDRIANAVGEREKGPPQRFWGQSFMTFGSASAACSLRLSGGAQVSGGGSEQLQVQ(“Ring”+Linker1+NB1)

[0069] SEQ ID NO.17:

[0070] MASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQLANMVNNLKEISQEAREGTQGERCAVHGERLHLFCEKDGKALCWVCAQSRKHRDHAMVAEAAAKEAAAKEAAAKASSVTVQTGQGWFYSGQGDFSYKASYGSLACYYMATDEPKLSNMQLYLTNKANDQSITFRGKVSDAYISRGDRRIANAVGEREKGPPQRFWGQSFMTFGSASAACSLRLSGGAQVSGGGSEQLQVQ(“Ring”+Linker1+NB2);

[0071] SEQ ID NO.18:

[0072] MASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQLANMVNNLKEISQEAREGTQGERCAVHGERLHLFCEKDGKALCWVCAQSRKHRDHAMVAEAAAKEAAAKEAAAKASSVTVQTGQGWFYSGQMDFSYGKASYGSLACYYMATDEPKLSNMQLYLTNKANDQSITFRGKVSDAYISRGDRIANAVGEREKGPPQRFWGQSFMTFGSASAACSLRLSGGAQVSGGGSEQLQVQ(“Ring”+Linker1+NB3);

[0073] SEQ ID NO.19:

[0074] MASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQLANMVNNLKEISQEAREGTQGERCAVHGERLHLFCEKDGKALCWVCAQSRKHRDHAMVAEAAAKEAAAKEAAAKASSVTVQTGQGWFYSGQGDFSYGKASYGSLACYYMATDEPKLSNMQLYLTNKANDQSITFRGKVSDAYISRGDRIANAVGEREKGPPQRFWGQSFFTFGSASAACSLRLSGGAQVSGGGSEQLQVQ(“Ring”+Linker1+NB4);

[0075] SEQ ID NO.20:

[0076] MASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQLANMVNNLKEISQEAREGTQGERCAVHGERLHLFCEKDGKALCWVCAQSRKHRDHAMVAEAAAKEAAAKEAAAKASSVTVQTGQGWFYSGQGDSSYGKASYGSLACYYMATDEPKLSNMQLYLTNKANDQSITFRGKVSDAYISRGDRIANAVGEREKGPPQRFWGQSFMTFGSASAACSLRLSGGAQVSGGGSEQLQVQ(“Ring”+Linker1+NB5);

[0077] SEQ ID NO.21:

[0078] MASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQLANMVNNLKEISQEAREGTQGERCAVHGERLHLFCEKDGKALCWVCAQSRKHRDHAMVNANPNANPNANPSSVTVQTGQGWFYSGQGDFSYGKASYGSLACYYMATDEPKLSNMQLYLTNKANDQSITFRGKVSDAYISRGDRIANAVGEREKGPPQRFWGQSFMTFGSASAACSLRLSGGAQVSGGGSEQLQVQ(“Ring”+Linker2+NB1);

[0079] SEQ ID NO.22:

[0080] MASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQLANMVNNLKEISQEAREGTQGERCAVHGERLHLFCEKDGKALCWVCAQSRKHRDHAMVNANPNANPNANPSSVTVQTGQGWFYSGQGDFSYKASYGSLACYYMATDEPKLSNMQLYLTNKANDQSITFRGKVSDAYISRGDRRIANAVGEREKGPPQRFWGQSFMTFGSASAACSLRLSGGAQVSGGGSEQLQVQ(“Ring”+Linker2+NB2);

[0081] SEQ ID NO.23:

[0082] MASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQLANMVNNLKEISQEAREGTQGERCAVHGERLHLFCEKDGKALCWVCAQSRKHRDHAMVNANPNANPNANPSSVTVQTGQGWFYSGQMDFSYGKASYGSLACYYMATDEPKLSNMQLYLTNKANDQSITFRGKVSDAYISRGDRIANAVGEREKGPPQRFWGQSFMTFGSASAACSLRLSGGAQVSGGGSEQLQVQ(“Ring”+Linker2+NB3);

[0083] SEQ ID NO.24:

[0084] MASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQLANMVNNLKEISQEAREGTQGERCAVHGERLHLFCEKDGKALCWVCAQSRKHRDHAMVNANPNANPNANPSSVTVQTGQGWFYSGQGDFSYGKASYGSLACYYMATDEPKLSNMQLYLTNKANDQSITFRGKVSDAYISRGDRIANAVGEREKGPPQRFWGQSFFTFGSASAACSLRLSGGAQVSGGGSEQLQVQ(“Ring”+Linker2+NB4);

[0085] SEQ ID NO.25:

[0086] MASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQLANMVNNLKEISQEAREGTQGERCAVHGERLHLFCEKDGKALCWVCAQSRKHRDHAMVNANPNANPNANPSSVTVQTGQGWFYSGQGDSSYGKASYGSLACYYMATDEPKLSNMQLYLTNKANDQSITFRGKVSDAYISRGDRIANAVGEREKGPPQRFWGQSFMTFGSASAACSLRLSGGAQVSGGGSEQLQVQ(“Ring”+Linker2+NB5);

[0087] SEQ ID NO.26:

[0088] MASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQLANMVNNLKEISQEAREGTQGERCAVHGERLHLFCEKDGKALCWVCAQSRKHRDHAMVEPKIPQPQPKPQPQPQPQPQPKPQPKPEPSSVTVQTGQGWFYSGQGDFSYGKASYGSLACYYMATDEPKLSNMQLYLTNKANDQSITFRGKVSDAYISRGDRIANAVGEREKGPPQRFWGQSFMTFGSASAACSLRLSGGAQVSGGGSEQLQVQ(“Ring”+Linker3+NB1);

[0089] SEQ ID NO.27:

[0090] MASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQLANMVNNLKEISQEAREGTQGERCAVHGERLHLFCEKDGKALCWVCAQSRKHRDHAMVEPKIPQPQPKPQPQPQPQPQPKPQPKPEPSSVTVQTGQGWFYSGQGDFSYKASYGSLACYYMATDEPKLSNMQLYLTNKANDQSITFRGKVSDAYISRGDRRIANAVGEREKGPPQRFWGQSFMTFGSASAACSLRLSGGAQVSGGGSEQLQVQ(“Ring”+Linker3+NB2);

[0091] SEQ ID NO.28:

[0092] MASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQLANMVNNLKEISQEAREGTQGERCAVHGERLHLFCEKDGKALCWVCAQSRKHRDHAMVEPKIPQPQPKPQPQPQPQPQPKPQPKPEPSSVTVQTGQGWFYSGQMDFSYGKASYGSLACYYMATDEPKLSNMQLYLTNKANDQSITFRGKVSDAYISRGDRIANAVGEREKGPPQRFWGQSFMTFGSASAACSLRLSGGAQVSGGGSEQLQVQ(“Ring”+Linker3+NB3);

[0093] SEQ ID NO.29:

[0094] MASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQLANMVNNLKEISQEAREGTQGERCAVHGERLHLFCEKDGKALCWVCAQSRKHRDHAMVEPKIPQPQPKPQPQPQPQPQPKPQPKPEPSSVTVQTGQGWFYSGQGDFSYGKASYGSLACYYMATDEPKLSNMQLYLTNKANDQSITFRGKVSDAYISRGDRIANAVGEREKGPPQRFWGQSFFTFGSASAACSLRLSGGAQVSGGGSEQLQVQ(“Ring”+Linker3+NB4);

[0095] SEQ ID NO.30:

[0096] MASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQLANMVNNLKEISQEAREGTQGERCAVHGERLHLFCEKDGKALCWVCAQSRKHRDHAMVEPKIPQPQPKPQPQPQPQPQPKPQPKPEPSSVTVQTGQGWFYSGQGDSSYGKASYGSLACYYMATDEPKLSNMQLYLTNKANDQSITFRGKVSDAYISRGDRIANAVGEREKGPPQRFWGQSFMTFGSASAACSLRLSGGAQVSGGGSEQLQVQ(“Ring”+Linker3+NB5);

[0097] SEQ ID NO.31:

[0098] MASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQLANMVNNLKEISQEAREGTQGERCAVHGERLHLFCEKDGKALCWVCAQSRKHRDHAMVGGGGSGGGGSGGGGSSSVTVQTGQGWFYSGQGDFSYGKASYGSLACYYMATDEPKLSNMQLYLTNKANDQSITFRGKVSDAYISRGDRIANAVGEREKGPPQRFWGQSFMTFGSASAACSLRLSGGAQVSGGGSEQLQVQ(“Ring”+Linker4+NB1);

[0099] SEQ ID NO.32:

[0100] MASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQLANMVNNLKEISQEAREGTQGERCAVHGERLHLFCEKDGKALCWVCAQSRKHRDHAMVGGGGSGGGGSGGGGSSSVTVQTGQGWFYSGQGDFSYKASYGSLACYYMATDEPKLSNMQLYLTNKANDQSITFRGKVSDAYISRGDRRIANAVGEREKGPPQRFWGQSFMTFGSASAACSLRLSGGAQVSGGGSEQLQVQ(“Ring”+Linker4+NB2);

[0101] SEQ ID NO.33:

[0102] MASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQLANMVNNLKEISQEAREGTQGERCAVHGERLHLFCEKDGKALCWVCAQSRKHRDHAMVGGGGSGGGGSGGGGSSSVTVQTGQGWFYSGQMDFSYGKASYGSLACYYMATDEPKLSNMQLYLTNKANDQSITFRGKVSDAYISRGDRIANAVGEREKGPPQRFWGQSFMTFGSASAACSLRLSGGAQVSGGGSEQLQVQ(“Ring”+Linker4+NB3);

[0103] SEQ ID NO.34:

[0104] MASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQLANMVNNLKEISQEAREGTQGERCAVHGERLHLFCEKDGKALCWVCAQSRKHRDHAMVGGGGSGGGGSGGGGSSSVTVQTGQGWFYSGQGDFSYGKASYGSLACYYMATDEPKLSNMQLYLTNKANDQSITFRGKVSDAYISRGDRIANAVGEREKGPPQRFWGQSFFTFGSASAACSLRLSGGAQVSGGGSEQLQVQ(“Ring”+Linker4+NB4);

[0105] SEQ ID NO.35:

[0106] MASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQLANMVNNLKEISQEAREGTQGERCAVHGERLHLFCEKDGKALCWVCAQSRKHRDHAMVGGGGSGGGGSGGGGSSSVTVQTGQGWFYSGQGDSSYGKASYGSLACYYMATDEPKLSNMQLYLTNKANDQSITFRGKVSDAYISRGDRIANAVGEREKGPPQRFWGQSFMTFGSASAACSLRLSGGAQVSGGGSEQLQVQ(“Ring”+Linker4+NB5);

[0107] SEQ ID NO.36:

[0108] ATGTACAGAATGCAGCTGCTGTCTTGCATCGCCCTGAGCCTGGCCCTGGTGACCAACTCCATGGCCAGCGCTGCCAGACTGACCATGATGTGGGAGGAGGTGACCTGCCCCATCTGTCTGGACCCTTTCGTTGAGCCAGTGAGTATTGAGTGCGGCCATAGCTTCTGCCAGGAGTGCATCTCCCAGGTGGGAAAGGGGGGCGGGTCTGTGTGCCCCGTGTGTAGACAGAGATTCCTGCTGAAAAACCTGAGACCTAACCGGCAGCTGGCCAACATGGTGAACAACCTGAAAGAAATCTCTCAGGAAGCTAGAGAGGGTACCCAGGGCGAGCGGTGCGCCGTGCACGGCGAGCGGCTGCATCTGTTTTGCGAGAAGGATGGGAAGGCACTCTGTTGGGTGTGCGCCCAGAGCAGAAAGCATAGAGATCACGCCATGGTGGCCGAAGCCGCCGCCAAGGAGGCCGCTGCCAAGGAAGCCGCCGCCAAGGCTTCTTCCGTGACCGTGCAGACCGGCCAGGGCTGGTTTTATTCCGGCCAGGGAGACTTCAGCTACAAGGCCTCCTACGGCTCACTGGCCTGCTACTATATGGCCACAGATGAGCCCAAGCTGAGCAACATGCAGCTGTACCTGACCAACAAGGCCAACGACCAGTCAATCACCTTCCGGGGCAAGGTGAGCGATGCCTACATTAGCCGGGGGGACAGACGGATTGCCAACGCCGTGGGGGAGCGGGAGAAGGGCCCCCCTCAGAGGTTCTGGGGCCAGAGCTTTATGACATTTGGCTCTGCCTCCGCCGCATGCTCTCTGCGGCTGAGCGGCGGCGCCCAGGTGAGCGGAGGAGGCTCCGAGCAGCTGCAGGTGCAGCACCATCACCACCACCATTGA(RL2H);

[0109] SEQ ID NO.37:GGATCCATGTACAGAATGCAGCTGCTG;

[0110] SEQ ID NO.38: GCGGCCGCTCAATGGTGGTGGTGATGGTG;

[0111] SEQ ID NO.39: ATGGCCAGCG CTGCCAGACT GACCA;

[0112] SEQ ID NO. 40: ACCTGCAGCTGCTCGGAGCCTCCTC.

[0113] 2. Construction of the expression vector pcDNA-Ring-Linker1-NB2-6His and preparation and identification of endotoxin-free plasmid

[0114] The amino acid sequence of Ring-Linker1-NB2 (SEQ ID NO.17) was optimized for codon preference according to Homo sapiens after adding human IL-2 signal peptide at the N-terminus, and the Ring-Linker1-NB2-6His (abbreviated as RL2H) gene fragment was synthesized by Shanghai Shenggong Gene Co., Ltd. The sequence is shown in SEQ ID NO.36, and the restriction sites BamHI and NotI were contained at both ends of the target fragment by PCR amplification. The Ring-Linker1-NB2-6His gene fragment was constructed into the plasmid puc57 to obtain a plasmid containing the target gene fragment Ring-Linker1-NB2-6His. The plasmid containing the target gene fragment Ring-Linker1-NB2-6His was synthesized as a template, and the RL2H gene was amplified with primers BamHI-RL2H-F (SEQ ID NO.37) and NotI-RL2H-R (SEQ ID NO.38), and the PCR product was recovered by gel. The pcDNA3.4 vector backbone plasmid and RL2H gene were double-digested with EcoRI and XhoI restriction endonucleases, respectively. The gel-recovered products were ligated with T4 DNA ligase at 16°C for 4-6h. After ligation and transformation, the ligation product was added to Escherichia coli DH5a competent cells, ice-bathed for 30min, heat-shocked at 42°C for 90s, and ice-bathed for 2min, then 1mL of LB liquid culture medium was added and incubated at 250rpm and 37°C for 1h. The bacteria were collected by centrifugation at 4000rpm, 800μL of supernatant was removed and resuspended, and the cells were coated with ampicillin (Amp)-resistant LB culture medium plates and cultured at 37°C overnight. Different single colonies were picked for colony PCR identification. There was 800bp between the colony PCR identification primers F (SEQ ID NO.39 and R (SEQ ID NO.40) and the upstream and downstream primers. The PCR product was identified by agarose gel electrophoresis bands (see the results). Figure 2) was consistent with the designed 800 bp. After the positive clones were identified by colony PCR, sequencing analysis was performed. The plasmid with the correct sequencing was named pcDNA-Ring-Linker1-NB2-6His (see the map for details). Figure 3 ).

[0115] The clones with correct sequencing were used for the preparation of endotoxin-free plasmids. The positive clones were picked and inoculated into 3 mL LB medium (containing a final concentration of 100 μg / mL Amp), and cultured at 37°C and 220 RPM for 8 h; the above seed solution was inoculated into 500 mL LB medium at a volume fraction of 1% and cultured overnight; the bacterial liquid cultured overnight was centrifuged and collected and used for endotoxin-free plasmid extraction kit to extract plasmids, and the extracted plasmid concentration was determined by Nanodrop and finally concentrated to 1 mg / mL for transfection experiments.

[0116] 3. Expression vector transfection and target protein expression and purification

[0117] The transfection method of the present invention adopts Expi293 TM Expression System User Guide, the specific steps are as follows:

[0118] 1) Resuscitation and activation of Expi293F TM The cells were cultured until the day before transfection (day -1) when the viable cell density reached approximately 3-5×10 6 After overnight culture, the final viable cell density reached 4.5-5.5×10 6 Cells / mL and viability ≥95% were used for transfection.

[0119] 2) Preheat Expi293 with fresh TM Dilute the cells to 3 × 10 6 viable cells / mL, and then gently shake the culture flask to mix the cells.

[0120] 3) Before use, add ExpiFectamine TM Gently invert the 293 reagent bottle 4 to 5 times to ensure thorough mixing.

[0121] 4) Use Opti-Plex TM Complexation buffer (or Opti-MEM TM I culture medium) and then gently shake or invert to mix.

[0122] 5) Using Opti-Plex TM Complexation buffer (or Opti-MEM TM I medium - if used in step 4) dilute ExpiFectamine TM 293 Reagent and then gently shake or invert to mix.

[0123] 6) Incubate at room temperature for 5 minutes.

[0124] 7) Dilute ExpiFectamine TM Add the 293 reagent to the diluted plasmid DNA and then gently shake or invert to mix.

[0125] 8) Incubate ExpiFectamine at room temperature TM 293 / plasmid DNA complex for 10-20 min.

[0126] 9) Slowly transfer the complex to the cells, gently shake the culture bottle during the addition process, and then place it on an orbital shaker and culture the cells in an incubator with relative humidity ≥80% and 8% CO2 at 37°C.

[0127] 10) 18-22 hours after transfection, add ExpiFectamine to the transfection bottle TM 293 Transfection Enhancer 1 and Expi-Fectamine TM 293 Transfection Enhancer 2, gently shake the culture bottle during the addition process.

[0128] 11) After 5 days of transformation, centrifuge at 4°C, 10,000 RPM for 15 min to collect the supernatant for protein purification.

[0129] 4. The specific steps for purification of the target protein are as follows:

[0130] 1) Sample pretreatment: The cell culture medium was centrifuged at 10000 RPM for 15 min to collect the supernatant, which was filtered through a 0.22 μm filter and placed in a chromatography cabinet at 4°C for protein purification;

[0131] 2) NTA-Ni column equilibration: Pass the column with Buffer A (20 mM PB, pH 7.4, 150 mM NaCl);

[0132] 3) Sample loading: Slowly pass the supernatant after passing through the membrane through the column at a flow rate of 2 mL / min. When the UV value is less than 20, discard the flow-through;

[0133] 4) Washing: Use 10 column volumes of Buffer B (20 mM PB, pH 7.4, 150 mM NaCl, 10 mM imidazole) to elute nonspecific impurities;

[0134] 5) Elution: Use 10 column volumes of Buffer C (20mM PB, pH7.4, 150mM NaCl, 300mM imidazole) to elute the target protein and collect the eluate. The eluate is subjected to ultrafiltration to remove imidazole. The replacement buffer is Buffer A (20mM PB, pH7.4, 150mM NaCl). After removing imidazole, store it in a -80℃ refrigerator for subsequent experiments.

[0135] 6) Samples were taken during protein purification and desalting for SDS-PAGE electrophoresis analysis (results are shown in Figure 4 ), the results showed that the purified protein band was consistent with the theoretical molecular weight of RL2H fusion protein of about 30kDa, and the purity after purification and desalting was greater than 95% ( Figure 4 Lane 5 in the figure).

[0136] Example 4: Elisa detection of RL2H fusion protein

[0137] (1) Antigen coating: Aβ42 oligomers (prepared by diluting Aβ monomer stock solution and incubating at 37°C for 3-5 h) were used as antigens for coating, 100 ng / well / 100 μL, and placed in a 4°C refrigerator for overnight coating;

[0138] (2) Washing: On the second day, aspirate the coating solution and wash the cells three times with PBST, each time for 5 min.

[0139] (3) Blocking: add 200 μL of blocking solution (5% skim milk powder in PBST) to each cavity and block for 2 h;

[0140] (4) Washing: Aspirate the blocking solution and wash three times with PBST, each wash for 5 min;

[0141] (5) Incubation of fusion protein RL2H: Use PBST to dilute the fusion protein RL2H to 12 ng / mL, 10 ng / mL, 8 ng / mL, 6 ng / mL, 4 ng / mL, 2 ng / mL, 1 ng / mL, 0.5 ng / mL and 0.25 ng / mL, respectively, and incubate at 37°C for 1.5 h;

[0142] (7) Washing: Aspirate the primary antibody dilution solution and wash three times with PBST, each wash for 5 min;

[0143] (8) Secondary antibody incubation: dilute the secondary antibody (RING finger protein 81 antibody (HRP conjugated)) to the working concentration range using antibody diluent and incubate at 37°C for 1 h.

[0144] (9) Washing: Aspirate the secondary antibody incubation solution and wash three times with PBST, each wash for 5 min;

[0145] (10) Color development: Add TMB color developing solution, color development for 5 min, and then use 2 M sulfuric acid solution to terminate the reaction;

[0146] (11) Using a microplate reader, read the absorbance at 450 nm and indirectly calculate the titer of IgY;

[0147] (12) Linear regression analysis was performed using the concentration (ng / mL) as the independent variable (x) and the OD450 absorbance value as the dependent variable (y) to obtain the curve (see Figure 5 ) and the model formula is: y = 0.0235x + 0.293, the results show that ( Figure 5 ) The fusion protein RL2H binds to Aβ oligomers at a concentration of 12.46 ng / mL, indicating that RL2H has a high affinity for Aβ oligomers.

[0148] Example 5: Delivery system design and packaging of rAAV9-RL2

[0149] The plasmid containing the target gene fragment Ring-Linker1-NB2-6His was used as a template, and the primers RL2-NheI-F (GCTAGCATGGCCAGCGCTGCCAGACTG, SEQ ID NO.41) and RL2-XhoI-R (CTCGAGCTGCACCTGCAGCTGCTCGGA, SEQ ID NO.42) were used to amplify the target gene RL2 containing the restriction enzyme sites NheI and XhoI, and the PCR product was recovered by gel. The pAAV-MCS vector backbone plasmid and the RL2 gene were double-digested with NheI and XhoI restriction endonucleases, respectively, and the enzyme digestion products were connected overnight by T4 DNA ligase, and the ligation products were transformed into Escherichia coli DH5a competent cells, and the positive clones were selected for sequencing. The plasmid with correct sequencing was named pAAV-RL2 (see the map) Figure 6 ).

[0150] A large amount of correctly identified pAAV-RL2 plasmid was prepared and used to co-transfect HEK-293T cells with mature and stable three plasmids for virus packaging. The other two plasmids were pAAV-RC9 and the auxiliary plasmid pHelper. After 72 hours of transfection, the virus was purified by cesium chloride density gradient centrifugation to remove the empty shell virus and finally obtain a titer of 8×10 13 / mL recombinant rAAV9-Ring-Linker1-NB2.

[0151] Example 6: In vivo efficacy evaluation of rAAV9-Ring-Linker1-NB2

[0152] Based on the pathological characteristics of Alzheimer's disease, the present invention selected 12-month-old Tg-3XFAD transgenic mice (purchased from Wuhan Youdu Biotechnology Co., Ltd.) as an animal pathological model. A total of 42 Tg-3XFAD transgenic mice were selected and randomly divided into a model control group (group B) of 6 mice, an intrathecal injection of rAAV9-Ring-Linker1-NB2 high-dose group (2 μL (1×10 11 vp / μL, group C) 18, low-dose group (2μL (1×10 10 vp / μL), group D) 18 mice, all male. The blank control group (group A) used WT-C57BL / 6 mice, 6 mice; the specific grouping and experimental design are shown in Table 1.

[0153] Table 1 Grouping and experimental design

[0154]

[0155] 1. Behavioral Experiment

[0156] (1) Morris water maze test

[0157] Morris water maze was used to evaluate the spatial learning and memory ability of mice treated for 8 weeks. Positioning navigation test: The water maze is divided into four quadrants, which are quadrants I, II, III, and IV in clockwise order. The four quadrants are used as the water entry for the animals in turn. Training is carried out for 5 consecutive days, and 4 different starting quadrants are trained every day. The underwater platform was removed the next day after the positioning navigation training. The mice were placed in the water at the same entry point. The number of times the mice crossed the original platform within 60 seconds was recorded to evaluate the learning and memory ability of the mice. Compared with the normal group, the number of times the mice in the model group crossed the original platform was significantly reduced (P<0.05), indicating that the transgenic mouse model was successfully established. Compared with the model group, the number of times the low-dose group and the high-dose group crossed the original platform was significantly increased (P<0.05 or P<0.01); compared with the normal group, there was no significant difference in the number of times the low-dose group and the high-dose group crossed the original platform (P>0.05) ( Figure 7 ).

[0158] (2) Open field test

[0159] The experimental system consists of a 0.5m×0.5m×0.4m plastic box and a video acquisition system. The video acquisition software has an automatic zoning function. The test experiment is: the experimental mice of each group that have been treated for 8 weeks are gently placed in the box facing one wall. After 6 minutes of free movement, the camera system automatically records the total distance of the mice in the box within 5 minutes. Compared with the normal group, the total movement distance of the model group, low-dose group and high-dose group in the open field test ( Figure 8) had no significant difference (P>0.05), indicating that there was no significant difference in the spontaneous activity of mice in each group.

[0160] The above experiments show that rAAV9-Ring-Linker1-NB2 can improve the anxiety-like behavior of transgenic Alzheimer's mice.

[0161] 2. Determination of the in vivo transcription level of the target gene RL2

[0162] The experimental mice in group C and group D were euthanized at 2 weeks, 4 weeks and 8 weeks of drug administration, respectively. Appropriate amount of mouse brain tissue samples were taken, 1 mL of Trizol was added and quickly ground, and then RNA was extracted using a kit and reverse transcribed into cDNA. The cDNA of the reverse transcription product was used as a template, and the reaction system was prepared according to the instructions of the RealTime PCR MasterMix (SYBR Green) kit, and amplification was performed using a Real-time PCR instrument. The primer pairs in the qPCR system were β-actin-F (CAAGAGAGGTATCCTGACCCT, SEQ ID NO.43) and β-actin-R (TGATCTGGGTCATCTTTTCAC, SEQ ID NO.44), RL2-F (AGCGATGCCTACATTAGCCG, SEQ ID NO.45) and RL2-F (ATGTCATAAAGCTCTGGCCCC, SEQ ID NO.46); the reaction procedure was: 95℃ pre-denaturation for 2 min, 95℃ denaturation for 5 s, 60℃ annealing for 20 s, and 40 cycles, according to 2 －ΔΔCt The formula was used to calculate the relative transcription level of the target gene mRNA in the sample. By intrathecal injection of rAAV9-Ring-Linker1-NB2 into experimental mice, the transcription level of the target gene RL2 reached the highest level in the fourth week and remained at a high level after 8 weeks of administration (see Table 2), proving that rAAV9-Ring-Linker1-NB2 can achieve long-term expression in vivo.

[0163] Table 2 Relative transcription level of gene Ring-Linker1-NB2 (%)

[0164]

[0165] 3. Elisa test of Aβ content in brain

[0166] After the 8-week experiment, the A, B, C and D experimental groups were euthanized after 8 weeks of administration, and the homogenates of the hippocampus and cortex were collected, weighed and added with 4 times the volume of PBS. After centrifugation, the supernatant was collected and the Aβ content was detected using a mouse Aβ1-42 Elisa detection kit.

[0167] Table 3 Elisa statistical comparison of Aβ content in cerebral cortex and hippocampus of mice treated for 8 weeks

[0168]

[0169] T test between group B and group A ## P<0.01; indicates statistically significant difference, and the model was successfully established;

[0170] T test between group C and group B ** P<0.01; indicates that there is a statistically significant difference in the high-dose group and there is a therapeutic effect;

[0171] T test between group D and group B * P<0.05; indicates that there is a statistically significant difference in the low dose and there is a therapeutic effect;

[0172] The results in Table 3 indicate that the model mice were successfully established and that rAAV9-Ring-Linker1-NB2 has the effect of reducing Aβ content.

[0173] 4. Immunofluorescence staining of brain tissue sections for pathological examination

[0174] The mice in experimental groups A, B, C and D were euthanized 8 weeks after administration, and the sections were prepared and immunofluorescence staining was performed to detect the distribution of Aβ.

[0175] Compared with the tissue sections of group A (wild type group), group B (AD model group) had a significant number of stained red spots, indicating that the model was successfully established; the number of stained spots in group C (high dose group) and group D (low dose group) showed a significant progressive decrease compared with group B ( Fig. 9 ), which preliminarily suggested that the test substance has the effect of clearing Aβ plaques.

[0176] 5. Conclusion

[0177] Based on the above results, the transcriptome results (Table 2) showed that the target gene RL2 was still transcribed 8 weeks after administration. Moreover, the results of tissue sections ( Fig. 9 ) showed that after 8 weeks of administration, the amyloid plaques in the brain tissue of the low-dose and high-dose groups were significantly reduced. Therefore, the rAAV9-Ring-Linker1-NB2 of the present invention has the function of long-term transcriptional expression and degradation of Aβ polymers, thereby achieving the effect of treating Alzheimer's disease.

[0178] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A bispecific fusion protein, characterized in that: It includes a RING type+B box type domain in a TRIM21 protein, a linker, and an antibody or nanobody targeting Aβ oligomers; the RING type+B box type domain in the TRIM21 protein and the antibody or nanobody targeting Aβ oligomers are connected by a linker; the amino acid sequence of the RING type+B box type domain in the TRIM21 protein is shown in SEQ ID NO.15, and the linker is a protein linker or a small molecule compound.

2. The bispecific fusion protein according to claim 1, characterized in that The antibody targeting Aβ oligomers includes IgG antibody; the nanobody targeting Aβ oligomers includes nanobody in the form of scFv, Fab or VHHs structure.

3. The bispecific fusion protein according to claim 2, characterized in that: The amino acid sequence of the nanobody targeting Aβ oligomers is shown in any one of SEQ ID NOs. 6-10.

4. The bispecific fusion protein according to claim 1, characterized in that The protein linker includes (NANP)3, A(EAAAK)nA, EPKIPQPQPKPQPQPQPQPQPQPKPQPKPEP or (GGGGS)n; the small molecule compound includes an alkyl linker, PEG or a triazole linker.

5. The bispecific fusion protein according to claim 4, characterized in that The numerical range of n in the A(EAAAK)nA is 2-6, and the numerical range of n in the (GGGGS)n is 1-5.

6. The bispecific fusion protein according to any one of claims 1 to 5, characterized in that: The amino acid sequence of the bispecific fusion protein is shown in any one of SEQ ID NOs. 16-35.

7. A drug delivery system for targeted treatment of Alzheimer's disease, characterized in that: The coding gene of the bispecific fusion protein according to any one of claims 1 to 5 is constructed into an AAV vector to prepare an AAV vector delivery system, or the bispecific fusion protein or its coding gene according to any one of claims 1 to 5 is prepared into a protein corona delivery system through nanomaterial encapsulation technology.

8. The drug delivery system according to claim 7, characterized in that The AAV vector includes AAV2, AAV5 or AAV9.

9. Use of the bispecific fusion protein according to any one of claims 1 to 5 or the drug delivery system according to any one of claims 7 to 8 in the preparation of a drug for the targeted treatment of Alzheimer's disease.

10. The use according to claim 9, characterized in that: The bispecific fusion protein is administered in the form of protein or its mRNA drug or DNA vaccine through delivery or injection.

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