Nucleic acid sequence construct for co-expression of TRIM11 and ApoE2 and application of nucleic acid sequence construct

Abstract

The invention belongs to the technical field of biology, and particularly relates to a nucleic acid sequence construct for co-expressing TRIM11 and ApoE2 and application of the nucleic acid sequence construct. According to the invention, the TRIM11 gene and the ApoE2 gene are connected by utilizing a self-processing unit, and the TRIM11 can prevent misfolding and aggregation of tau protein by promoting SUMOylation and proteasome degradation of the tau protein, and can also be used as a depolymerases to dissolve existing tau aggregates at the same time; the apolipoprotein ApoE2 is a secretory protein with a signal peptide, can be combined with A beta, and promotes A beta clearance in a lipid state and ApoE subtype dependence mode. The recombinant adeno-associated virus vector provided by the invention realizes simultaneous down-regulation of Tau protein and beta-like protein through efficient co-expression of TRIM11 and ApoE2. The compound can be used for research and development of medicines for treating dementia.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a nucleic acid sequence construct for co-expressing TRIM11 and ApoE2 and an application thereof. Background Art

[0002] Dementia is a syndrome that can be caused by a variety of diseases that destroy nerve cells and damage the brain over time, often leading to a deterioration in cognitive function (i.e. the ability to process thoughts), a persistent decline in memory, thought, behavior and social skills, and changes that affect a person's ability to care for themselves. Currently, more than 55 million people worldwide suffer from dementia, more than 60% of whom live in low- and middle-income countries. There are nearly 10 million new cases each year. Dementia is caused by a variety of diseases and injuries that affect the brain. Alzheimer's disease (AD) is the most common form of dementia, which may account for 60-70% of cases, and the risk of developing the disease increases with age.

[0003] Alzheimer's disease is a neurodegenerative disease, with nearly 70% of risk factors related to genetics; other risk factors include head trauma, depression and hypertension. AD symptoms include memory impairment, aphasia, apraxia, agnosia and visual-spatial impairment. In addition, the patient's abstract thinking and calculation ability will also be impaired, often accompanied by changes in personality and behavior.

[0004] The true cause of Alzheimer's disease remains unknown. There are many hypotheses that attempt to explain the pathogenesis of AD, such as the Aβ cascade hypothesis, the cholinergic hypothesis, the abnormal phosphorylation of Tau protein hypothesis, the neuroinflammation hypothesis, and the metal ion disorder hypothesis. The characteristics of neuropathological changes in the AD brain include amyloid plaques formed by Aβ protein deposition, neurofibrillary tangles (intracellular aggregates composed of hyperphosphorylated tau protein), synaptic loss, atrophy, selective depletion of neurotransmitter systems (such as acetylcholine), and Lewy bodies (in a few cases), all of which lead to information communication disorders between neurons and even neuronal death, ultimately leading to AD. Among them, the β-amyloid protein (Aβ) deposition hypothesis and the abnormal phosphorylated Tau protein hypothesis are the most classic pathologies and hypotheses.

[0005] There is currently no treatment that can stop or reverse the course of the disease. Only a few methods may temporarily relieve or improve symptoms. Current drug development ideas mainly focus on hot areas such as clearing β-amyloid protein (Aβ), regulating Tau protein, and acetylcholinesterase inhibitors (AChE).

[0006] Recent studies on the tripartite motif (TRIM) protein family have found that several TRIM molecules have a strong ability to prevent tau aggregation. They found that the expression level of TRIM11 was significantly downregulated in Alzheimer's disease brains. In addition, there is a strong negative correlation between its expression level and tau aggregation. In brains with high expression of TRIM11, the level of tau aggregation is low, and vice versa. Further mechanistic studies have found that TRIM11 can proteasome degrade defective tau proteins and excessive normal tau proteins, thereby linking tau proteins to the ubiquitin proteasome pathway. TRIM11 can also act as a molecular chaperone to prevent tau misfolding and aggregation, and play the role of tau protein depolymerase to decompose tau protein aggregates that have already formed. TRIM11 prevents spontaneous and induced aggregation of tau proteins in cells, keeping tau proteins in a soluble state. Therefore, TRIM11 is an important neuroprotective factor.

[0007] The protein encoded by the ApoE gene is called apolipoprotein E. It has multiple functions in the body, one of which is involved in lipid metabolism and cholesterol transport. There are three common ApoE alleles (E2, E3, and E4). The difference between them lies in the changes in two sites at the ApoE gene level. The reaction at the protein level is whether these two sites (amino acid 112 and amino acid 158) are cysteine ​​or arginine. If both sites of ApoE encoded on a chromosome are Cys, this chromosome is ApoE2. If the 112th site is Cys and the 158th site is Arg, then this apolipoprotein is ApoE3. If both are Arg, then it is ApoE4. The three alleles can be combined into six genotypes, ApoE2 / 2, 2 / 3, 2 / 4, 3 / 3, 3 / 4, and 4 / 4, thereby encoding three ApoE protein isomers, namely ApoE2, ApoE3, and ApoE4. AD is closely related to the polymorphism of this gene. The AD incidence of ApoE3 homozygous people is set as the benchmark. The AD incidence of people with ApoE4 is higher than that of ApoE3 homozygous people. The incidence of two heterozygous people containing E4 / E2 and E4 / E3 is 2.6 and 3.6 times that of ApoE3 homozygous people, respectively; while the AD incidence of pure E4 / E4 soars to 15.5 times. E2 can reduce the incidence of AD.

[0008] Gene therapy is an emerging treatment method that applies modern molecular biology technology and precision medicine theory and practice. It is one of the most promising biomedical fields with promising future development prospects. AAV-based gene therapy is actively promoted as a very promising alternative, and recent clinical trials have successfully highlighted its practicality. LX1001 is an AAV-based gene therapy developed by LEXEO Therapeutics, which aims to deliver a transgene expressing protective ApoE2 to the central nervous system (CNS) of Alzheimer's patients carrying two ApoE4 alleles to prevent or slow disease progression. In February 2022, positive data from a Phase 1 / 2 clinical trial of LX1001 in the treatment of ApoE4 homozygous AD patients were announced. Clinical data showed that in two patients who were followed up for more than 12 months after a single treatment, the levels of Tau protein and phosphorylated Tau protein in the cerebrospinal fluid were significantly reduced compared with baseline, and no serious safety and tolerability issues were observed. Recently, scientists used AAV to deliver TRIM11 into the brains of various tauopathy mouse models. It was found that intracranial delivery of TRIM11 can effectively prevent the pathological formation of tau protein and neuroinflammation. In addition, TRIM11 can also effectively prevent cognitive and motor function decline. Summary of the invention

[0009] Based on this, the present invention provides a recombinant adeno-associated virus vector that co-expresses TRIM11 and ApoE2, achieving the co-expression of TRIM11 protein and ApoE2 protein, while down-regulating Tau protein and β-like protein. It can be used in the research and development of drugs for treating Alzheimer's disease.

[0010] The technical solution adopted by the present invention is as follows:

[0011] A nucleic acid sequence construct, characterized in that it comprises a composite sequence, wherein the composite sequence is composed of a nucleotide sequence encoding triple helical protein 11 (TRIM11) and human apolipoprotein E2 (ApoE2), and a self-processing unit sequence (APU) connecting the nucleotide sequences encoding the two proteins.

[0012] Preferably, the amino acid sequence of the triple helical structure protein 11 is shown in SEQ ID NO.1; the amino acid sequence of human apolipoprotein E2 is shown in SEQ ID NO.3; the self-processing unit sequence includes but is not limited to an internal ribosome entry site sequence (IRES), a sequence encoding a 2A peptide or a 2A-like peptide, Intein, a type B bacterial Intein-like domain (BIL), a Furin sequence and its derivatives.

[0013] Preferably, the self-processing unit sequence 2A peptide or 2A-like peptide in the composite sequence includes but is not limited to F2A derived from hand, foot and mouth disease virus, E2A derived from equine rhinitis virus, P2A derived from porcine teschovirus and T2A derived from Lymantriasis fasciitis virus.

[0014] Preferably, the self-processing unit sequence in the composite sequence is P2A derived from porcine teschovirus.

[0015] Preferably, the nucleotide sequence of the composite sequence has a nucleic acid having a nucleotide sequence as shown in any one of SEQ ID NOs. 7 to 11.

[0016] The composite sequence described in the present invention may also be a nucleic acid having one or more nucleotides substituted, deleted or added in any fragment of the sequence described in SEQ ID NO.7 to 11; or a nucleic acid having at least 85% homology with the sequence of the nucleic acid and encoding TRIM11 (SEQ ID NO.1) and ApoE2 (SEQ ID NO.3) proteins; or a nucleic acid that is partially complementary or fully complementary to any of the above items.

[0017] The present invention also provides a eukaryotic expression vector comprising a nucleic acid having a nucleotide sequence as shown in any one of SEQ ID NOs. 7 to 11.

[0018] Preferably, the vector is one of a virus, a non-virus, and naked DNA.

[0019] Preferably, the non-viral vector is selected from one of nanocarriers and polymer carriers, such as cationic polymer carriers, liposome carriers and nanoparticle carriers, and the nucleotide sequence construct is encapsulated and delivered to target cells and tissues for expression; the viral vector is selected from at least one of a lentiviral vector, an adenoviral vector and an adeno-associated viral vector.

[0020] Preferably, the serotype of the adeno-associated virus vector includes but is not limited to AAV1, AAV2, AAV4, AAV5, AAV8, AAV9, PHP.eB and AAV-retro.

[0021] The present invention also provides a use of the nucleic acid sequence construct or the eukaryotic expression vector in the preparation of a drug for treating dementia.

[0022] The nucleotide sequence construct of the present invention can be administered via in vivo and in vitro routes to achieve the expression of the target gene in target cells and tissues. The target gene is introduced into the human body's own or allogeneic cells (or heterologous cells) in vitro, and then returned to the body after in vitro cell amplification. Alternatively, the nucleotide sequence is assembled into a specific eukaryotic expression vector and directly introduced into the body. This vector can be a virus or a non-virus, or even naked DNA. The administration routes of the virus, non-viral vector or naked DNA include but are not limited to intravenous systemic administration, intracranial or intrathecal injection.

[0023] The nucleotide sequence construct provided by the present invention connects the sequence encoding TRIM11 protein and the sequence encoding apolipoprotein ApoE through a self-processing unit (taking P2A as an example) to construct a recombinant adeno-associated virus vector pAAV-3×FLAG-TRIM11-P2A-ApoE2-HA (wt). The main construction idea is: connect the TRIM11 gene and the ApoE2 gene through P2A, wherein TRIM11 prevents the misfolding and aggregation of tau protein by promoting the SUMOylation and proteasome degradation of tau protein, and can also act as a depolymerase to dissolve existing tau aggregates. Apolipoprotein ApoE2 is a secretory protein with a signal peptide that can bind to Aβ and promote the clearance of Aβ in a lipid state and ApoE subtype-dependent manner. The sequence schematic diagram of the target gene in the constructed recombinant adeno-associated virus vector pAAV-3×FLAG-TRIM11-P2A-ApoE2-HA (wt) is shown in Figure 1 The amino acid sequence information of TRIM11 is shown in SEQ ID NO.1, and the nucleotide sequence encoding TRIM11 is shown in SEQ ID NO.2; the amino acid sequence information of ApoE2 is shown in SEQ ID NO.3; the nucleotide sequence encoding ApoE2 is shown in SEQ ID NO.4; the amino acid sequence information of P2A is shown in SEQ ID NO.5, and the nucleotide sequence information encoding P2A is shown in SEQ ID NO.6.

[0024] The sequences of SEQ ID NO.7 to 11 in the present invention are optimized by selecting 5 different online codon optimization platforms: GenSmart, IDT, GeneArt, VectorBuilder and Tsingke, and then transfecting 293 cells. It is found that the expression levels of the sequences optimized by IDT, GeneArt and VectorBuilder (VB) are significantly improved, among which the optimization effect of VertorBuilder is the best. The expression levels of TRIM11 after optimization are 1.91 times that of the wild type, and ApoE2 is 2.57 times that of the wild type.

[0025] Studies have found that Aβ promotes the hyperphosphorylation and protein accumulation of Tau protein by activating JNK kinase. On the other hand, Tau protein can also inhibit the degradation of Aβ protein. Therefore, Aβ and Tau toxicity can interact to form a snowball loop and continue to aggravate the progression of AD. This mechanism suggests that the previous drug development for single targets of Aβ or Tau may not effectively inhibit the progression of AD, and using Aβ and Tau as combined targets may be the most effective way to slow down AD. Therefore, the present invention pioneered the construction of AAV expression vectors for TRIM11 and ApoE2, and optimized the codons to obtain gene fragments that efficiently co-express TRIM11 and ApoE2 in cells, thereby constructing recombinant adeno-associated virus vectors that efficiently co-express TRIM11 and ApoE2 and drugs for the treatment of Alzheimer's disease. Experiments show that AAV viruses carrying TRIM11 and ApoE2 can directly infect SH-SY5Y neuroblasts, and overexpressing AAV vectors carrying target genes in target cells can simultaneously downregulate Tau protein and β-amyloid protein.

[0026] The present invention connects the target gene TRIM11 and ApoE2 through an auto-processing unit (APU), constructs a nucleotide sequence construct, verifies the expression of the target gene TRIM11 and ApoE2 by Western blot and ELISA, and co-transfects Tau, finding that the expression of Tau is negatively correlated with the vector, and the expression of tau is downregulated with the expression of TRIM11, and presents a dose-dependent effect. Then, the codons of the target gene are optimized by different codon optimization platforms, and the gene fragments with high expression efficiency are obtained by Western blot screening. The AAV vector is constructed with the optimized sequence, the AAV recombinant virus is packaged and purified, and after infecting the neuroblastoma SH-SY5Y, the expression of the target gene in the host cell is verified. At the same time, after the vector after sequence optimization is co-transfected with Tau, it is found that the optimized vector can efficiently inhibit the formation of Tau.

[0027] Compared with the prior art, the present invention has the following advantages: the nucleotide sequence construct provided by the present invention can achieve efficient co-expression of TRIM11 and ApoE2, providing an important basis for the research of drugs for treating Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 A diagram of the vector structure constructed for the present invention;

[0029] Figure 2This is the Western blot expression result of the target genes TRIM11 and ApoE2 after transient transfection of the expression vector;

[0030] Figure 3 This is the standard curve of ELISA detection of ApoE;

[0031] Figure 4 Real-Time PCR standard curve for AAV virus titer detection;

[0032] Figure 5 The results of Western blot detection of TRIM11 and ApoE2 expression in target cells after recombinant AAV virus infected human neuroblastoma cells SH-SY5Y;

[0033] Figure 6 This is a Western blot result showing the induction of Tau protein degradation by overexpressing TRIM11 and ApoE2 wild-type vectors;

[0034] Figure 7 This is the Western blot test result of protein expression after transient transfection using vectors optimized by different optimization platforms;

[0035] Figure 8 This is a diagram of Western blot analysis using ImageJ;

[0036] Fig. 9 The results of Western blot detection of target gene expression after neuroblastoma cells SH-SY5Y were infected with different recombinant adeno-associated viruses AAV before and after the sequence was optimized by VectorBuilder platform;

[0037] Fig.10 This is the Western blot detection result of the vector optimized by VectorBuilder platform, which overexpressed and induced Tau protein degradation;

[0038] Fig.11 The Western blot results of the sequence-optimized vector co-expressing with BACE1 and APP show the expression of the target gene;

[0039] Fig.12 This is the standard curve of Aβ40 ELISA detection for the sequence-optimized vector co-expressed with BACE1 and APP;

[0040] Fig.13 This is an analysis chart of ELISA results. DETAILED DESCRIPTION

[0041] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than 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.

[0042] The PEI was purchased from Polyscience, catalog number 23966-100; the RIPA cell lysate was purchased from Sangon Biotechnology, catalog number C500007-0100; the TRIM11 and ApoE primary antibodies were purchased from Boster Biotechnology, catalog numbers BM4412 and A08971-2, respectively; the FLAG primary antibody was purchased from Proteintech, catalog number 66008-4-Ig; the HA primary antibody was purchased from Affinity, catalog number T0008; the GFP antibody was purchased from Proteintech, catalog number 66002-1; the secondary antibody HRP-conjugate daffinipure Goat anti Rabbit IgG (H+L) was purchased from Proteintech, catalog number SA0001-2; the Goat Abto Ms IgG (HRP) was purchased from Abcam, catalog number ab205719; the ApoE ELISA kit was purchased from Elabscience, catalog number E-EL-H0470; the 10kDa MWCO ultrafiltration tubes were purchased from Millipore, catalog number UFC901008.

[0043] Example 1 Construction of AAV viral vector pAAV-3×FLAG-TRIM11-P2A-ApoE2-HA (wt)

[0044] The construction process of the AAV viral vector pAAV-3×FLAG-TRIM11-P2A-ApoE2-HA (wt) is as follows:

[0045] The wild-type TRIM11 and ApoE2 ORFs were connected through P2A, and the AgeI restriction site and 3×FLAG tag were fused to the N-terminus of TRIM11 in sequence, and the HA tag and EcoRI restriction site were fused to the C-terminus of ApoE2 in sequence. Nanjing Qingke Biotechnology was commissioned to synthesize the AgeI-3×FLAG-TRIM11-P2A-ApoE2-HA-EcoRI full gene. The synthesized full gene fragment and pAAV vector backbone were double-digested with AgeI and EcoRI, respectively, and detected by agarose gel electrophoresis. The target gene fragment and the linearized vector backbone fragment were then recovered by cutting the gel, and the recovered target gene and the linearized vector backbone fragment were connected overnight by T4 DNA ligase. The ligation product was transformed into Stbl3 Escherichia coli competent cells, LB plates were coated, monoclonal colonies were selected, and then the colonies identified as recombinant plasmids were sent to Nanjing Qingke Biotechnology for sequencing by PCR. The recombinant plasmid with correct sequencing results was the constructed recombinant vector.

[0046] TRIM11 prevents the misfolding and aggregation of tau protein by promoting SUMOylation and proteasomal degradation of tau protein, and can also dissolve existing tau aggregates as a depolymerase. Apolipoprotein ApoE2 is a secretory protein with a signal peptide that can bind to Aβ and promote Aβ clearance in a lipid state and ApoE isoform-dependent manner.

[0047] The sequence diagram of the target gene in the recombinant adeno-associated virus vector pAAV-3×FLAG-TRIM11-P2A-ApoE2-HA (wt) constructed by the present invention is as follows: Figure 1 The amino acid sequence information of TRIM11 is shown in SEQ ID NO.1, and the nucleotide sequence encoding TRIM11 is shown in SEQ ID NO.2; the amino acid sequence information of ApoE2 is shown in SEQ ID NO.3; the nucleotide sequence encoding ApoE2 is shown in SEQ ID NO.4; the amino acid sequence information of P2A is shown in SEQ ID NO.5, and the nucleotide sequence information encoding P2A is shown in SEQ ID NO.6.

[0048] Experimental Example 1 Identification of protein expression

[0049] 1. Experimental process:

[0050] (1) HEK293 cells in logarithmic growth phase with cell viability > 98% were selected, diluted with DMEM + 10% FBS complete medium and plated on 6-well plates, and recombinant adeno-associated virus vector pAAV-3×FLAG-TRIM11-P2A-ApoE2-HA (wt) was transfected into the cells with PEI, and recombinant adeno-associated virus vector pAAV-EGFP was transfected as a control, wherein the transfection ratio of DNA:PEI was 1:3 (μg:μL). After transfection, the cells were placed in a 37°C, 5% CO2 incubator for static culture;

[0051] (2) After 48 h, cells and culture supernatant (Supernatant, SP) were collected, and untransfected HEK293 cells and their supernatant were used as blank controls;

[0052] (3) Lyse the cells with RIPA lysis buffer, centrifuge at 12000 rpm for 15 min, discard the precipitate, and take the supernatant for Western blot to detect the expression of TRIM11, FLAG, ApoE, HA, GFP, and β-actin;

[0053] (4) ApoE ELISA (Elabscience, E-EL-H0470) was used to detect the ApoE2 content in the cell culture supernatant.

[0054] 2. Experimental results: The experimental results are as follows Figure 2 , Figure 3 shown. Figure 2 In the figure, 1 is a pAAV-EGFP vector, 2 is a pAAV-3×FLAG-TRIM11-P2A-ApoE2-HA vector of the present invention; Figure 2 It can be seen that endogenous TRIM11 and ApoE2 are expressed in HEK293 cells. After transfection with the recombinant adeno-associated virus vector pAAV-3×FLAG-TRIM11-P2A-ApoE2-HA (wt) of the present invention for 48 hours, it was found that compared with HEK293 cells not transfected with the plasmid, the amount of TRIM11 and ApoE2 in the transfected group cells was significantly increased. The Western blot results of FLAG and HA further proved that the recombinant adeno-associated virus vector pAAV-3×FLAG-TRIM11-P2A-ApoE2-HA (wt) mediated the expression of the target gene TRIM11; at the same time, the expression of ApoE2 was also detected in the cell lysate (Whole cell lysate, WCL) and the cell culture supernatant, but it was not detected in the control group transfected with the pAAV-EGFP vector, indicating that ApoE2 was successfully expressed and secreted into the extracellular space after transfection; thus, it can be shown that the target genes TRIM11 and ApoE2 are both expressed.

[0055] Depend on Figure 3 It can be seen that the ApoE ELISA kit was used to detect the ApoE content in the cell culture supernatant. The data analysis was performed using the online ELISA data analysis tool GainData developed by Arigo. The standard curve is shown in Figure 3 shown.

[0056] ELISA results showed that 48 hours after transfection, the concentration of ApoE2 in the cell supernatant was 932.29 ng / mL, while in the control group transfected with pAAV-EGFP, the concentration of ApoE2 was the background level of 0.451 ng / mL, indicating that ApoE2 was successfully expressed and secreted outside the cells after transfection.

[0057] It can be seen that Western blot can simultaneously detect TRIM11 and ApoE2 in cell lysates, indicating that the pAAV-3×FLAG-TRIM11-P2A-ApoE2-HA (wt) vector can co-express TRIM11 and ApoE2; and the secretory expression of ApoE2 was further verified by ELISA. This indicates that the vector can co-express TRIM11 and ApoE2, and ApoE2 can be secreted extracellularly.

[0058] Experimental Example 2 Construction of Type II Recombinant Adeno-Associated Virus Vector and Protein Detection

[0059] 1. Experimental process: HEK293T cells in the logarithmic growth phase were passaged into 10 15 cm cell culture dishes, and when the degree of fusion reached 80%, the pAAV-3×FLAG-TRIM11-P2A-ApoE2-HA (wt) expression vector (i.e., the wild-type vector constructed by the present invention), capsid protein plasmid AAV2 and auxiliary packaging plasmid pHelper were co-transfected into the cells at a molar ratio of 1:1:1 under PEI mediation, and the mass ratio of total plasmid DNA (i.e., the total DNA of the vector of the present invention and AAV2) to PEI was 1:3. The cells were collected 72 hours after transfection, resuspended with RIPA cell lysis buffer, and lysed by repeated freezing and thawing in liquid nitrogen and 37°C water bath for 3 times. The supernatant was collected by centrifugation at 12000 rpm for 20 min and filtered with a 0.45 μm filter to obtain the filtered AAV virus liquid. The AAV virus liquid was purified by iodixanol density gradient centrifugation. The specific steps were as follows: 5 mL of 60% iodixanol, 5 mL of 40% iodixanol, 6 mL of 25% iodixanol and 8 mL of 15% iodixanol were added to the Beckman quick-seal tube in sequence, and finally the filtered AAV virus liquid was carefully added on top of the iodixanol. The centrifuge tube was heat-sealed and centrifuged at 35,000 g for 90 min. The virus liquid layer containing AAV was removed from the side of the centrifuge tube with a syringe. The AAV virus layer was located at the junction of 60% and 40% iodixanol. The virus liquid was then filtered with a 0.45 μm filter, and the AAV virus was concentrated and replaced with a Millipore 100 kDa MWCO ultrafiltration tube (Cat. No. UFC9100).

[0060] Taqman qPCR was used to detect the virus titer. The primers used for AAV virus titer determination were: F: 5'-GGAACCCCTAGTGATGGAGTT-3', R: 5'-CGGCCTCAGTGAGCGA-3', and probe: 5'-FAM-CACTCCCTCTCTGCGCGCTCG-BBQ-3'. The gradient dilution plasmid pAAV-3×FLAG-TRIM11-P2A-ApoE2-HA (wt) (2×10 9 , 2×10 8 , 2×10 7 , 2×10 6 , 2×10 5 At the same time, qPCR was performed to make a standard curve. The qPCR enzyme was Taq Pro U+Multiple Probe qPCR Mix, purchased from Novozymes, catalog number QN213-01. The qPCR system was prepared according to the system and conditions recommended in the instruction manual. The instrument used was the QuantStudio1 real-time fluorescence quantitative PCR system from Thermo Fisher Scientific.

[0061] Recombinant adeno-associated virus AAV-3×FLAG-TRIM11-P2A-ApoE2-HA (wt) was used to infect human neuroblastoma cells SH-SY5Y (MOI=10000), and recombinant adeno-associated virus AAV-EGFP was used as a control. After 4 days, the cells and culture supernatant were collected, and the cells were lysed with RIPA lysis buffer. Western blot was used to detect the expression of TRIM11 and ApoE2 in the cells, and ApoE ELISA was used to detect the secretory expression of ApoE in the cell supernatant.

[0062] 2. Experimental results: The experimental results are as follows Figure 4-Figure 5 As shown, Figure 4 The Real-Time PCR standard curve was constructed after gradient dilution of the pAAV-3×FLAG-TRIM11-P2A-ApoE2-HA(wt) plasmid. The adeno-associated virus AAV-3×FLAG-TRIM11-P2A-ApoE2-HA(wt) (250-fold dilution) amplified at the same time had a Ct value of 21.391. The virus titer calculated by the standard curve equation was 1.16E+12vg / mL.

[0063] Figure 5 In the figure, 1 is AAV-EGFP, 2 is AAV-3×FLAG-TRIM11-P2A-ApoE2-HA; Figure 5 Western blot results showed that endogenous TRIM11 and ApoE2 were expressed in human neuroblastoma cells SH-SY5Y. Four days after SH-SY5Y cells were infected with type 2 recombinant adeno-associated virus AAV-3×FLAG-TRIM11-P2A-ApoE2-HA (wt), the expression of TRIM11 and ApoE2 in the cells was significantly increased. The ApoE ELISA results showed that the concentration of ApoE2 in the supernatant was 281.75 ng / mL, while the concentration of ApoE2 in the supernatant of the control group cells infected with AAV-EGFP was 1.508 ng / mL, which was the background expression. This showed that after rAAV infected human neuroblastoma cells SH-SY5Y, ApoE2 was successfully expressed and secreted into the extracellular space.

[0064] Experimental Example 3: Detection of Tau protein levels

[0065] 1. Experimental process: tau P301L is a common variant tau protein in familial tauopathy. Animal models that simulate tau protein pathology based on overexpression of tau mutant P301L are widely used in Alzheimer's disease research.

[0066] Different amounts of plasmid pAAV-TRIM11-P2A-ApoE2 and pRK5-EGFP-Tau-P301L vector were co-transfected into HEK293 cells. After 48 hours, the cells were collected and lysed with RIPA cell lysis buffer, and the expression of the target protein in the cells was detected by Western blot.

[0067] 2. Experimental results: The specific experimental results are as follows Figure 6 As shown, Figure 6 In the table, band 1 corresponds to the first column in the table, i.e., pRK5-EGFP-Tau-P301L 1μg, pAAV-EGFP 4μg, and the following bands 2-5 correspond to the 2-5 columns of the table, respectively. The pAAV vector is used as an empty vector to fill the total DNA transfected in each well to keep it consistent. Figure 6 It can be seen that the results show that after transfection of different amounts of pAAV-3×FLAG-TRIM11-P2A-ApoE2-HA (wt) vector, TRIM11 and ApoE2 proteins were expressed, and were positively correlated with the amount of transfected plasmid, while tau P301L aggregates were significantly degraded, and showed a dose-dependent effect. This shows that our target protein can downregulate the protein level of intracellular Tau.

[0068] Experimental Example 4: Sequence Optimization

[0069] 1. Experimental process: 5 different online codon optimization platforms were selected: GenSmart, IDT, GeneArt, VectorBuilder and Tsingke, and the TRIM11-P2A-ApoE2 gene of the present invention was optimized. During the optimization, a 3×FLAG tag was added to the front end of TRIM11, and an HA tag was added to the back of ApoE2. The nucleotide sequence optimized by VectorBuilder is shown in SEQ ID NO.7, the nucleotide sequence optimized by GenSmart is shown in SEQ ID NO.8, the nucleotide sequence optimized by IDT is shown in SEQ ID NO.9, the nucleotide sequence optimized by GeneArt is shown in SEQ ID NO.10, and the nucleotide sequence optimized by Tsingke is shown in SEQ ID NO.11. Then the optimized sequence was transfected into HEK293 cells together with other gene sequences, and the cells were collected and lysed after 48 hours, and Western blot detection was performed according to the method of Experimental Example 1. Due to the expression of endogenous genes, in order to remove the background, FLAG was used to reflect the expression of TRIM11 and HA was used to reflect the expression of ApoE2 during the experiment.

[0070] 2. Experimental results: The test results are as follows Figure 7 As shown, Figure 7In the figure, 1 is the original sequence of 3×FLAG-TRIM11-P2A-ApoE2-HA (wt) of the present invention; 2 is the vector optimized by IDT software; 3 is the vector optimized by GeneArt software; 4 is the vector optimized by Tsingke software; 5 is the vector optimized by VectorBuilder software; 6 is the vector optimized by GenSmart software. The Western blot results were analyzed by ImageJ. Figure 8 As shown, the expression levels of the sequences optimized by IDT, GeneArt and VectorBuilder (VB) were significantly improved, among which the optimization effect of VectorBuilder was the best. The expression levels of TRIM11 after optimization were 1.91 times that of the wild type, and ApoE2 was 2.57 times that of the wild type.

[0071] Experimental Example 5 Protein Expression Comparison

[0072] 1. Experimental samples: sequence-optimized AAV-3×FLAG-TRIM11-P2A-ApoE2-HA (VB), wild-type AAV-3×FLAG-TRIM11-P2A-ApoE2-HA (wt);

[0073] 2. Experimental method: AAV-3×FLAG-TRIM11-P2A-ApoE2-HA (VB) was packaged and purified, and the neuroblastoma cells SH-SY5Y were infected with wild-type AAV-3×FLAG-TRIM11-P2A-ApoE2-HA (wt) (refer to the method described in Experimental Example 2) at MOI = 10000. The AAV-EGFP group was infected with the same MOI as a control. After 5 days of infection, the cells were collected and lysed, and the detection was performed according to the method of Experimental Example 2.

[0074] 3. Experimental results: Western blot results are as follows Fig. 9 As shown, Fig. 9 Among them, 1 is AAV-EGFP, 2 is AAV-3×FLAG-TRIM11-P2A-ApoE2-HA (wt), and 3 is AAV-3×FLAG-TRIM11-P2A-ApoE2-HA (VB). It can be seen that in the AAV group after target sequence optimization, the expression levels of TRIM11 and ApoE2 are significantly higher than those in the wild group, indicating that after the sequence-optimized AAV infects the target cells, the expression of the target gene is significantly increased.

[0075] Experimental Example 6 Detection of tau protein content

[0076] 1. Experimental process: tau P301L is a common tau family mutation. Overexpression of tau P301L is usually used as an animal model to simulate Alzheimer's disease. Different amounts of TRIM11-P2A-ApoE2 (VB) and tau P301L were co-transfected into 293T cells. After 48 hours, the cells were collected and lysed, and the test was performed according to the method of Experimental Example 3.

[0077] 2. Experimental results: Western blot results are as follows Fig.10 As shown, it can be seen that overexpression of the target genes TRIM11 and ApoE2 promoted the obvious degradation of tau P301L aggregates, and the degradation showed a dose-dependent effect.

[0078] Test Example 7 Aβ protein detection

[0079] 1. Experimental process: β-amyloid protein (Aβ) is a polypeptide containing 39-43 amino acids, which is derived from amyloid precursor protein (APP). APP is a transmembrane glycoprotein with two degradation pathways in vivo: non-amyloid and amyloid pathways. In the amyloid pathway, APP is cleaved by BACE1 (beta-site APP cleavingenzyme-1, an aspartic acid protease, also known as β-secretase) and γ-secretase in sequence to produce a series of Aβ of varying lengths, mainly Aβ40 and Aβ42, which are easily aggregated to form insoluble substances and are the main components of senile plaques. APP, BACE1 and pAAV-3×FLAG-TRIM11-P2A-ApoE2-HA (VB) plasmids were co-transfected into HEK293T cells, and APP, BACE1 and pAAV-EGFP were co-transfected in the control group. After 48 hours, the cells were collected and lysed for Western blot and ELISA detection. The standard curve of ELISA was drawn according to the method of Experimental Example 1.

[0080] 2. Experimental results: The experimental results are as follows Figure 11-13 As shown, Fig.11 and Fig.13 In the figure, 1 is pAAV-EGFP, and 2 is pAAV-3×FLAG-TRIM11-P2A-ApoE2-HA. Fig.11 Western blot was used to verify the expression of the target genes. The results showed that after transfection with the pAAV-3×FLAG-TRIM11-P2A-ApoE2-HA(VB) plasmid, the target genes APP, BACE1, TRIM11 and ApoE2 were all expressed. The ELISA standard curve was as follows Fig.12 As shown, pAAV-EGFP was used as the control group, and the ELISA results were analyzed as shown in Fig.13 As shown, overexpression of the target genes TRIM11 and ApoE2 can significantly inhibit the production of Aβ40.

Claims

1. A nucleic acid sequence construct, characterized in that: The invention comprises a composite sequence, which is composed of a nucleotide sequence encoding a triple helical structure protein 11 and a human apolipoprotein E2 and a self-processing unit sequence connected between the nucleotide sequences encoding the two proteins.

2. The nucleic acid sequence construct according to claim 1, characterized in that: The amino acid sequence of the triple helical structure protein 11 is shown in SEQ ID NO.1; the amino acid sequence of the human apolipoprotein E2 is shown in SEQ ID NO.3; the self-processing unit sequence is one of an internal ribosome entry site sequence, a sequence encoding a 2A peptide or a 2A-like peptide, an Intein, a B-type bacterial Intein-like domain, a Furin sequence or a derivative thereof.

3. The nucleic acid sequence construct according to claim 2, characterized in that: The self-processing unit sequence 2A peptide or 2A-like peptide in the composite sequence is one of F2A derived from hand, foot and mouth disease virus, E2A derived from equine rhinitis virus, P2A derived from porcine teschovirus and T2A derived from tricholoma clarkii virus.

4. The nucleic acid sequence construct according to claim 3, characterized in that The self-processing unit sequence in the composite sequence is P2A derived from porcine Teschovirus.

5. The nucleic acid sequence construct according to claim 4, characterized in that The nucleotide sequence of the composite sequence has a nucleic acid with a nucleotide sequence as shown in any one of SEQ ID NOs. 7 to 11.

6. A eukaryotic expression vector, characterized in that: A nucleic acid comprising the nucleotide sequence shown in any one of SEQ ID NOs. 7 to 11 as described in claim 5.

7. The eukaryotic expression vector according to claim 6, wherein The vector is one of virus, non-virus and naked DNA.

8. The eukaryotic expression vector according to claim 7, wherein The non-viral vector is selected from one of a nano-vector and a polymer vector, and the viral vector is selected from at least one of a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector.

9. The eukaryotic expression vector according to claim 8, wherein The serotypes of the adeno-associated virus vector include but are not limited to AAV1, AAV2, AAV4, AAV5, AAV8, AAV9, PHP.eB and AAV-retro.

10. Use of the nucleic acid sequence construct according to any one of claims 1 to 5, or the eukaryotic expression vector according to any one of claims 6 to 9 in the preparation of a drug for treating dementia.

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