CN113373176A

CN113373176A - Construction method of gene therapy vector and application of gene therapy vector in Alzheimer disease drugs

Info

Publication number: CN113373176A
Application number: CN202110466482.XA
Authority: CN
Inventors: 利时雨; 孙晗笑; 孙含蓄; 冯丽霞; 邓建善; 刘淑婷; 钟志颖; 耿程旭
Original assignee: Guangzhou Hongrun Biotechnology Co ltd
Current assignee: Guangzhou Hongrun Biotechnology Co ltd
Priority date: 2021-04-28
Filing date: 2021-04-28
Publication date: 2021-09-10

Abstract

The invention discloses a pIRES-Rt-sirt2-Tet-nap recombinant autophagy vector, which expresses Rt-sirt2 in mammalian cells through the connection of internal ribosome entry sites IRES, introduces DNA nucleic acid autophagy peptide nap into the vector, controls the expression of the DNA nucleic acid autophagy peptide nap in a tetracycline regulation system, and activates a molecular mechanism of molecular chaperone mediated autophagy in cells. An Alzheimer's disease mouse model experiment proves that after the treatment of pIRES-Rt-sirt2-Tet-nap, the ATP content in cortex and hippocampus tissues is increased, the ROS level is reduced, the cytochrome c oxidase activity is increased, and the mitochondrial function is improved; mitochondrial fusion proteins Mfn1 and Mfn2, autophagy proteins PINK1, Parkin and LC3I/LC3II are increased in expression level, the expression level of split protein Drp1 is slightly reduced, a mitochondrial network is recovered at a certain level, and meanwhile, the autophagy carrier can generate molecular chaperone mediated autophagy in vivo and then autophagy apoptosis, so that a new strategy is provided for treating diseases related to mitochondrial dysfunction such as Alzheimer's disease and the like by using sirt2 protein.

Description

Construction method of gene therapy vector and application of gene therapy vector in Alzheimer disease drugs

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a recombinant autophagy vector capable of recovering mitochondrial function and improving symptoms of mice with Alzheimer's disease, and a preparation method and application thereof.

Background

Alzheimer's Disease (AD) is an age-related neurodegenerative disease in which affected mitochondrial dynamics-related proteins include transcription factors involved in mitochondrial fusion and fission and mitochondrial biogenesis. One study showed that increased levels of mitochondrial fission protein Drp1 and decreased levels of mitochondrial fusion proteins Mfn1, Mfn2, and OPA1 in brain tissue of AD patients, suggesting that increased mitochondrial fission may be associated with neuronal dysfunction in AD. Analysis of brain tissue from AD and healthy subjects showed a decrease in expression of genes associated with mitochondrial biogenesis such as PGC-1 α, TFAM and NRF 2.

Research shows that PINK1 can directly activate mitochondrial autophagy by recruiting autophagy receptors, accelerate impaired mitochondrial clearance and play a role in protecting cell homeostasis, and PINK1 down-regulation can cause mitochondrial dysfunction, oxidative stress and neurological dysfunction. Fang et al found that AD model mice can reverse memory impairment through PINK-1, Parkin or DCT-1 dependent pathways under stimulation by activators of mitochondrial autophagy, reduce the content of Α β protein, prevent cognitive impairment in AD model mice, and further, they found that enhancement of mitochondrial autophagy in tau transgenic mice eliminates hyperphosphorylation of tau protein associated with AD and improves memory impairment in mice, indicating that impairment of mitochondrial autophagy is a key event in the pathogenesis of AD and represents a potential therapeutic intervention in AD through improvement of mitochondrial autophagy function.

Molecular chaperone-mediated autophagy (CMA) occurs by first requiring heat shock protein 70(HSC70) to recognize recognition sequences in substrate proteins and target them to lysosome membranes, and this recognition process requires co-assistance of other co-chaperones including Hsp90, Hsp40, Hip, Hop and Bag-1, which are involved in the unfolding of the substrate proteins and the transport of the latter substrate proteins into the lysosomal lumens; when the substrate binds to the cytoplasmic tail of the LAMP-2A protein (lysosome-associated membrane protein type 2A) on the lysosomal membrane, it triggers multimerization of LAMP2A to form a complex that mediates translocation of the substrate; finally, the substrate enters the lyso-chamber and is degraded.

Tetracycline-induced regulatory systems, namely the tetracycline-suppressible system (Tet-Off) and the tetracycline-inducible system (Tet-On). In the Tet-Off system, transactivator (TetR) binds to TetO in the absence of tetracycline to induce gene expression, and in the presence of tetracycline, the two separate and gene expression is inhibited. In contrast to the Tet-Off system, TetR binds to its TetO sequence and induces downstream gene expression in the presence of tetracycline.

Internal Ribosome Entry Sites (IRES) have been widely used in the construction of gene vectors, translation of an IRES upstream target gene follows the translation rule of eukaryotic proteins, and a downstream target gene can recruit ribosomes through the IRES to realize translation of the downstream gene, so that the two genes connected by the IRES can be expressed simultaneously and independently, and the problems of instability and difference of gene expression levels when two exogenous genes are co-transfected by two separate vectors in the transgenic field are solved.

Disclosure of Invention

Objects of the invention

A novel autophagy vector pIRES-Rt-sirt2-Tet-nap is constructed and applied to in vivo experiments of an AD mouse model, the effect of Rt-sirt2 on treating AD is clarified, and a novel strategy is provided for preventing and treating diseases of mitochondrial dysfunction such as AD and the like while the high-efficiency expression of exogenous genes in cells is effectively controlled.

(II) technical scheme

In order to solve the problems, the invention provides a method for controlling molecular chaperone mediated autophagy by using a tetracycline regulation system, connecting an autophagy peptide segment nap with a tetracycline regulation element, introducing the autophagy peptide segment nap into a plasmid pIRES-Rt-Sirt2 with an internal ribosome entry site, constructing an autophagy vector capable of efficiently expressing rhodotorula glutinis recombinant Sirt2 protein, injecting the autophagy vector into an AD mouse model through tail veins, and carrying out mitochondrial index detection and animal behavior experiments in mouse brain tissues to prove the treatment effect of the Sirt2 protein on aging diseases, which comprises the following steps:

s1, in the invention, an internal ribosome binding site IRES is introduced into an expression vector to successfully construct a plasmid vector pIRES-Rt-sirt2, and then a DNA nucleic acid autophagy peptide nap is successfully designed and synthesized through analysis of ProtParamol and NCBI databases, wherein the DNA nucleic acid autophagy peptide nap consists of a structural domain for connecting histone H1 and DNA and a recognition motif for mediating a CMA autophagy pathway, the nucleotide sequence is (atg) tcg cag atc aag ttg tcc atc aag cgc ctg gtc acc aag ttc gag cgccag (tga), the amino acid sequence is 'sqiklsikrlvtkfrq', the isoelectric point is pI: 11.10, in addition, cloning the nap peptide fragment into a response element Tet-on of a tetracycline induction regulation and control system, introducing the whole Tet-on induction regulation and control system carrying the nap peptide fragment into a plasmid pIRES-Rt-sirt2, and successfully constructing an autophagy vector pIRES-Rt-sirt 2-Tet-on;

s2, the prepared cationic liposome wrapped with the Rt-sirt2 autophagy vector is injected into an AD model mouse body through tail veins, mitochondrial function indexes in brain tissues of the AD model mouse are detected, and animal behavior experiments are carried out. The result shows that the transferrin modified cationic liposome carrying the Sirt2 gene autophagy vector can successfully pass through the BBB of a mouse to reach cortex and hippocampal tissues, and after treatment by Rt-Sirt2, the ATP content and cytochrome c oxidation (Cyt c) enzyme activity in the cortex and hippocampal tissues of an AD model mouse are increased, the ROS level is reduced, and the mitochondrial function is improved; in addition, the mtDNA content is increased, the expression level of related transcription factors is up-regulated, the mitochondrial quality is improved, and further detection shows that the expression levels of mitochondrial dynamics protein and autophagy protein are improved to different degrees, so that the damaged mitochondrial network in the brain tissue of the AD model mouse is recovered, and the results improve the learning and cognitive abilities of the AD model mouse. In summary, the experiment proves that Rt-sirt2 can improve the quality, function and mitochondrial network of mitochondria in cells by regulating the biogenesis of mitochondria, so as to reverse the damaged mitochondrial function in brain tissues of AD model mice, and further improve the learning and cognitive functions of AD model mice.

Drawings

FIG. 1 is a pIRES-Rt-sirt2-Tet-nap plasmid map.

FIG. 2 shows the electrophoresis of PCR amplification of Rt-sirt2 gene fragment, wherein M is Marker, lane 1 is Rt-sirt 2.

FIG. 3 is a PCR (left) and plasmid double restriction enzyme digestion (right) verification chart of target gene colony in Escherichia coli transformed strain, wherein the left chart is M: Marker, Lane 1 is strain transfected with pIRES-Rt-sirt2 plasmid, Lane 2 is strain transfected with pIRES2-EGFP plasmid; on the right, M: Marker, lane 1 and lane 2 pIRES-Rt-sirt2 and pIRES2-EGFP plasmids were subjected to Nhe I/BamHI double digestion, respectively.

FIG. 4 is a diagram showing the sequencing result of sirt2 of the target gene.

FIG. 5 shows the PCR (left panel) and double restriction enzyme (right panel) identification of plasmid clones, the left panel being M: Marker, lane 1: strain transfected with pMD18-T-nap plasmid, lane 2: strain transfected with pMD18-T vector; on the right, M: Marker, lane 1 and lane 2 pMD18-T-nap and pMD18-T plasmids were subjected to EcoRI/EcoRV double digestion.

FIG. 6 shows the identification of the bacterial colony PCR (left) and double digestion (right) of the Ptight-MSC fragment and rtTA-SV40polyA fragment, wherein M is Marker, Lane 1 and Lane 2 are derived from the bacterial liquid lysate of transfection plasmids pMD18-T-Ptight-MSC and pMD18-T, respectively, and Lane 3 and Lane 4 are derived from the bacterial liquid lysate of transfection pMD18-T-rtTA-SV40polyA and pMD18-T, respectively; on the right, M: Marker,

lanes

1 and 2, pMD18-T-Ptight-MCS and pMD18-T plasmid were subjected to BamHI/XbaI double digestion,

lanes

3 and 4, pMD18-T-rtTA-SV40polyA and pMD18-T plasmid were subjected to KpnI/BamHI double digestion.

FIG. 7 shows the PCR (left panel) and double digestion (right panel) identification of plasmid colonies, wherein the left panel shows M: Marker, Lane 1 and Lane 2 are derived from the lysate of transfection plasmids pMD18-T-Ptight-nap and pMD18-T-Ptight-MCS, respectively, and the right panel shows M: Marker, Lane 1 and Lane 2, pMD18-T-Ptight-nap and pMD18-T-Ptight-MCS, respectively, are subjected to EcoRI/EcoRV double digestion

FIG. 8 shows the PCR (left) and double digestion (right) identification of plasmid colonies, wherein M: Marker, lane 1 and lane 2 are derived from the lysate of transfected plasmids pcDNA3.1-Ptight-nap and pcDNA3.1, respectively, and M: Marker, lane 1 and lane 2, and plasmid pcDNA3.1-Ptight-nap and pcDNA3.1 are subjected to BamH I/EcoRV double digestion.

FIG. 9 shows PCR (left) and double-restriction enzyme digestion (right) identification of plasmid clones, wherein M: Marker, lane 1 and lane 2 are derived from lysate of plasmids pcDNA3.1-rtTA-SV40polyA-Ptight-nap and pcDNA3.1-Ptight-nap, respectively, and Kpn I/BamH I double-restriction enzyme digestion is performed on plasmids pcDNA3.1-rtTA-SV40polyA-Ptight-nap and pcDNA3.1-Ptight-nap.

FIG. 10 is a transmission electron micrograph of liposomes.

FIG. 11 shows the expression of the target gene in the mouse cortex (left panel) and hippocampal tissue (right panel).

Figure 12 is ATP content in mouse cortex and hippocampal tissue, p × < 0.01 vs.control; p # < 0.05vs.

Figure 13 is ROS content in mouse cortex and hippocampal tissues, p < 0.05vs. control; p # < 0.05vs.

Figure 14 is Cyt c oxidase activity in mouse cortex and hippocampal tissues, p × < 0.01, p × < 0.001 vs.control; p # < 0.05, p # # < 0.01vs.

Figure 15 is the relative content of mtDNA in mouse cortex and hippocampal tissues, p × < 0.01 vs.control; p # < 0.05vs.

FIG. 16 is a graph of the protein levels of mitochondrial biogenesis-associated transcription factors in mouse cortex and hippocampal tissues, 1: Control; 2, AD; 3, pIRES-Rt-sirt 2-Tet-nap; 4, Tf-pIRES-Rt-sirt2-Tet-nap, p is less than 0.01vs. control; p # < 0.05vs.

FIG. 17 shows the expression levels of Mfn1, Mfn2, and Drp1 proteins in mouse cortex and hippocampal tissues, 1: Control; 2, AD; 3, pIRES-Rt-sirt 2-Tet-nap; 4, Tf-pIRES-Rt-sirt2-Tet-nap, p is less than 0.05, p is less than 0.001vs. control; p # < 0.05vs. AD; ns (no significant) vs.

FIG. 18 shows the expression levels of PINK1, Parkin and LC3 II/LC 3I proteins in mouse cortex and hippocampal tissues, 1: Control; 2, AD; 3, pIRES-Rt-sirt 2-Tet-nap; 4, Tf-pIRES-Rt-sirt2-Tet-nap, p is less than 0.01, p is less than 0.001vs. control; p # < 0.05, p # # < 0.01vs

Figure 19 is the change in learning and cognitive function in mice, p < 0.05vs. control; p # < 0.05vs.

FIG. 20 shows the results of expression of a target protein in brain tissue after autophagy.

Detailed Description

The present invention will be described in further detail with reference to examples.

Example 1: construction, identification and expression of vector pIRES-Rsirt2-Tet-nap

1 materials of the experiment

Escherichia coli DH5 alpha and Rhodotorula glutinis strain Rhodosporidium toruloides, laboratory preservation. pIRES2-EGFP and pTet-On were purchased from International Biogene technology, Inc. of the union of Beijing village; pTRE-Tight is available from Huaao Biotechnology Ltd; pcDNA3.1(+) was purchased from Guangzhouding Biotechnology, Inc.; PMD18-T vector was purchased from Ed technologies (Beijing) Ltd; pIRES-Rt-sirt2-EGFP, pMD18-T-nap, pMD18-T-Ptight-MCS, pMD18-T-Ptight-nap, pcDNA3.1-Ptight-nap, pMD18-T-rtTA-SV40polyA, pcDNA3.1-rtTA-SV40polyA-Ptight-nap, pIRES-Rt-sirt2-Tet-nap were prepared by the laboratory.

2 method of experiment

2.1 isolation and amplification of the target Gene of Rhodotorula glutinis

(1) The extraction of Rhodotorula glutinis DNA was performed according to the kit protocol.

(2) According to the sequence of the Rt-sirt2 gene in NCBI, the upstream primers of the introduced Nhe I/BamH I at the two ends of the Rt-sirt2 gene are 5'-ATGTCCGCACCTTCGTCCAAGCCGA-3', the lower primer is 5'-CTACAAAGCCGGTTTCTTCTCCTCG-3', and a PCR instrument program is set to amplify the sequence of the Rt-sirt 2.

2.2 gel electrophoresis and recovery of PCR products

(1) Agarose gel electrophoresis

Dissolving 0.25g of agarose in 25mL of 1 XTAE electrophoresis buffer, and adding 1. mu.L of 10mg/mL EB solution; cooling the gel to 70 ℃ to prepare the gel. The solidified gel was placed in an electrophoresis tank, and 1 XTAE electrophoresis buffer was added thereto. After adding 5. mu.L of 10 XLoading Buffer to the sample, electrophoresis was performed at 160V, and the sample was stored by photography.

(2) Recovery of PCR fragments

The PCR fragments were recovered according to the gel recovery kit instructions and stored at-20 ℃ for future use.

2.3 construction of pIRES-Rt-sirt2 by homologous recombination

(1) Carrying out Nhe I/BamH I double enzyme digestion on plasmid pIRES2-EGFP, carrying out agarose gel electrophoresis, and carrying out recovery and purification according to the operation instruction of the gel recovery kit, wherein the components and the dosage of the enzyme digestion reaction are as follows: pIRES2-EGFP, 15 μ L; 10 XQuickCut Buffer4 μ L; 1 mu L of QuickCutNhe I; 1 mu L of QuickCut BamH I; the volume of the sterilized water is up to 40 mu L. After mixing, centrifugation was carried out and the whole reaction was maintained at 30 ℃ for 5 min.

(2) Recovered sirt2 fragment (4. mu.L) and pIRES2-EGFP fragment (1. mu.L) were subjected to Treidef^TMThe operation was described by SoSoSoSoo Cloning kit, using 2X SoSoSoo Mix at 5. mu.L, and the whole reaction was reacted at 50 ℃ for 30min to construct pIRES-Rt-sirt2-EGFP plasmid.

2.4 transfection and characterization of plasmid pIRES-Rt-sirt2

(1) Preparation of E.coli DH 5. alpha. competence: recovering Escherichia coli DH5 alpha by LB culture plate at 37 ℃; inoculating a single colony in an LB liquid culture medium; when OD is reached₆₀₀When the concentration is approximately equal to 0.4, centrifuging; adding pre-cooled 0.1mol/L CaCl into the precipitate₂5mL of solution is subjected to ice bath and secondary centrifugation; 1mL of CaCl containing 0.1mol/L was added₂To resuspend the strain; subpackaging the strain on ice (100 μ L/portion), freezing at-80 deg.CStoring for later use.

(2) Transformation of pIRES-Rt-sirt2 plasmid: 2 mu L of plasmid is taken to be put into 200 mu L of susceptible strain, evenly shaken and iced, and then is thermally shocked in water bath at 42 ℃ and iced immediately; adding 500. mu.L of nonreactive LB liquid culture medium, shaking-culturing at 37 ℃, centrifuging and discarding the supernatant. Adding 50 mu g/mL Kan (1:100) into LB solid culture medium; the cells were spread on a solid medium containing resistance, cultured at 37 ℃ for 1 hour, and then cultured in an inverted state. Single colonies with good morphology were picked, inoculated into 5mL liquid medium containing kan, and shake-cultured overnight at 37 ℃.

(3) PCR detection of pIRES-Rt-sirt2 plasmid: the above-mentioned strain OD₆₀₀When the concentration is approximately equal to 2, taking 1mL for centrifugation (12000rpm), adding 100 mu L of TAE buffer solution into the collected strain precipitate, carrying out heavy suspension in a water bath, and immediately transferring to-20 ℃ for storage for a moment; after thawing and centrifugation, collecting supernatant for colony PCR detection.

(4) The extraction of pIRES-Rt-sirt2 plasmid was performed according to the protocol of the plasmid extraction kit.

(5) The positive clone is respectively enzyme-digested by Nhe I/BamH I, and the enzyme-digested reaction components and the dosage are as follows: pIRES-Rt-sirt27.5 μ L; 2 mu L of 10X QuickCut Buffer; 0.5 mu L of QuickCutNhe I; 0.5 mu L of QuickCutBomH I; the volume of the sterilized water is up to 20 mu L.

(6) The pIRES-Rt-sirt2 plasmid with correct enzyme digestion verification is sent to Dalianbao bio-corporation for sequencing.

2.5 design, Synthesis and characterization of DNA autophagy peptide nap

(1) Analyzing a connecting histone H1 structure by using ProtParamol software, locking a structural domain of the connecting histone H1 structure, and inquiring a nucleic acid sequence corresponding to the structural domain through NCBI; a recognition motif ` KFERQ ` mediating CMA autophagy was ligated to the above-mentioned tail of the nucleotide sequence to construct a DNA Nucleic acid autophagy peptide nap (nap).

(2) The designed nap peptide fragment is sent to Dalianbao company for synthesis.

(3) EcoRI/EcoRV was introduced at both ends of the synthesized nap fragment and cloned into pMD18-T by homologous recombination to construct the pMD18-T-nap plasmid as described in 2.3 and 2.4.

2.6 construction of pcDNA3.1-rtTA-SV40polyA-Ptight-nap plasmid

(1) Construction and characterization of plasmids

The pTRE-Tight plasmid is used as a template, primers are designed, a Ptight-MSC fragment is amplified, BamHI/XbaI, an upstream primer 5'-CTCGAGTTTACTCCCTATCAGTGAT-3' and a downstream primer 5'-CTAGAGATATCGTCGACAAGCTTAT-3' are introduced at two ends of the Ptight-MSC fragment. The amplified Ptight-MSC fragment was cloned into pMD18-T to construct pMD18-T-Ptight-MSC plasmid.

(2) Plasmid pMD18-T-nap and pMD18-T-Ptight-MSC were ligated after EcoRI/EcoRV double digestion to construct pMD18-T-Ptight-nap plasmid.

(3) The pcDNA3.1 plasmid and the pMD18-T-Ptight-nap plasmid were digested simultaneously with EcoRV/BamHI, and ligated to construct pcDNA3.1-Ptight-nap plasmid.

(4) The amplification of rtTA-SV40polyA fragment and the construction of plasmid pMD18-T-rtTA-SV40polyA refer to (1) in 2.6, an upstream primer 5'-ATGTCTAGATTAGATAAAAGTAAAG-3' and a downstream primer 5'-ATGTCTAGATTAGATAAAAGTAAAG-3', and Kpn I/BamHI are introduced at both ends of the rtTA-SV40polyA fragment.

(5) The pMD18-T-rtTA-SV40polyA and pcDNA3.1-Ptight-nap plasmids are respectively subjected to double enzyme digestion by Kpn I/BamH I and then are connected to construct pcDNA3.1-rtTA-SV40polyA-Ptight-nap plasmid.

Construction of 2.7pIRES-Rt-sirt2-Tet-nap plasmid

Firstly, a CMV promoter-rtTA-SV40polyA-Ptight-nap-BGH polyA fragment in a pcDNA3.1-rtTA-SV40polyA-Ptight-nap plasmid is amplified, Afl II is introduced into two ends of the fragment, then the fragment is cloned into a plasmid pIRES-Rt-sirt2 to construct a pIRES-Rt-sirt2-Tet-nap plasmid, and the constructed plasmid is directly sent to a Dalibao organism for sequencing.

3 results of the experiment

3.1 identification of plasmid pIRES-Rt-sirt2

(1) PCR detection of amplified target gene fragment

The amplified Rt-sirt2 fragment is subjected to agarose gel electrophoresis by using Rhodotorula glutinis DNA as a template, and a band (figure 1) exists at 1600bp, the size of the band accords with that of the Rt-sirt2, and the Rt-sirt2 gene amplification is successful.

(2) Verification of plasmid pIRES-Rt-sirt2

As shown in FIGS. 1-2, lane 1 shows a band at 1600bp, while lane 2 of the control group shows no band, indicating that the plasmid pIRES-Rt-sirt2 was successfully transferred into E.coli. After the Nhe I/BamHI double digestion, two bands of 1600bp and 6000bp appear in lane 1, while only one band of 5300bp is found in lane 2 (FIG. 2).

(3) Plasmid pIRES-Rt-sirt2 sequencing

The pIRES-Rt-sirt2 plasmid which is verified to be correct is sent to a Dalibao organism for sequencing, and after the sequencing result is compared with the nucleotide sequence of the target gene in the NCBI database, the two are completely consistent (figure 3), which shows that the Rt-sirt2 fragment is successfully cloned.

3.2 Synthesis of DNA autophagy peptide nap and identification of plasmid pMD18-T-nap

The nucleotide sequence of nap peptide fragment synthesized by Dalibao was 5 'tcgcagatcaagttgtccatcaag cgcctggtcaccaagttcgagcgccag 3', and the amino acid sequence was "sqiklsikrlvttferq". The identification of the plasmid pMD18-T-nap revealed that lane 1 had a fragment of about 50bp, while lane 2 of the empty plasmid group had no band of interest (FIG. 4), and the double digestion indicated that the nap peptide was successfully cloned into the pMD18-T vector.

3.3 identification of plasmid pcDNA3.1-rtTA-SV40polyA-Ptight-nap

(1) Identification of pMD18-T-Ptight-MSC plasmid and plasmid pMD18-T-rtTA-SV40polyA

The plasmid identification analysis of pMD18-T-Ptight-MCS and pMD18-T-rtTA-SV40polyA found that the Ptight-MSC fragment and rtTA-SV40polyA fragment were successfully cloned (FIG. 5).

(2) Identification of pMD18-T-Ptight-nap plasmid

After plasmids pMD18-T-nap and pMD18-T-Ptight-MCS are subjected to double enzyme digestion by EcoRI/EcoRV, plasmids pMD18-T-Ptight-nap are constructed by ligation, the identification result is shown in figure 6, a band of about 50bp exists in a lane 1 in the left picture, bands of about 50bp and 3000bp exist in a lane 1 in the right picture, the sizes of the bands are consistent with the sizes of a nap fragment and pMD18-T + Ptight respectively, and the nap fragment is successfully cloned.

(3) Identification of pcDNA3.1-Ptight-nap plasmid

The identification and analysis of the plasmid pcDNA3.1-Ptight-nap revealed a band of about 380bp in lane 1 (FIG. 7), a band of about 380bp and about 5400bp in lane 1, and a band of about 5400bp in lane 2 (FIG. 7).

(4) Identification of pcDNA3.1-rtTA-SV40polyA-Ptight-nap plasmid

The results of the identification of pcDNA3.1-rtTA-SV40polyA-Ptight-nap plasmid are shown in FIG. 8, which shows that rtTA-SV40polyA was successfully cloned into pcDNA3.1-Ptight-nap plasmid.

(5) Identification of double-gene autophagy vector pIRES-Rt-sirt2-Tet-nap

Sequencing pIRES-Rt-sirt2Tet-nap plasmid shows that the sequence of the fragment cloned into the pIRES-Rt-sirt2 plasmid is completely consistent with the sequence in the database (figure 9), and the successful construction of the double-gene autophagy vector pIRES-Rt-sirt2-Tet-nap is shown.

Example 2: effect of Alzheimer's disease mouse phenotype

1 laboratory animal

The AD mouse model and the normal mouse are purchased from the Guangdong province medical experimental animal center, the weight of the AD mouse model and the normal mouse model is 18-20 g/mouse, and the AD mouse model and the normal mouse are 6 months old. Before the experiment, the mice are raised in an SPF animal laboratory, the raising temperature is 20-25 ℃, the humidity is 60%, the mice receive illumination for 12 hours every day, and fresh food and water are replaced once every day.

2 cationic liposomes

2.1 preparation of cationic liposomes

Dissolving 10mg of DC-chol, 20mg of DOPE and 4mg of DSPE-PEG 2000 in dichloromethane solution; respectively rotating and evaporating to prepare uniform films; adding 25mL of dichloromethane into the film for dissolving, adding 1mL of phosphoric acid buffer solution containing 0.2mg/mL of plasmid, mixing in a vortex mode, and carrying out ultrasonic water bath treatment; rotary evaporation again, wherein the uncoated double-gene autophagy vector is degraded by DNase I (10U) and Exonuclease III (25U); the liposomes and the plasmid degraded by nuclease were separated by gel column chromatography with phosphate buffer (pH 7.4) as an eluent, and the collected eluent was used for the following determination of encapsulation efficiency.

2.2 determination of encapsulation efficiency

Weighing Reagent B5 mu L, placing the Reagent B in a centrifuge tube, adding Reagent tA25 mu L, and fully and uniformly mixing to prepare a dyeing working solution;

the preparation of the standard solution is shown in table 1, and each prepared solution is placed in an ice tank for later use;

TABLE 1 Standard solution preparation

Serial number	ReagentB/μL	ReagentC/μL	DNA/μg
				1	500	500	1
2	500	The diluted ReagentC solution 500 was removed from tube No. 1	0.5
				3	500	The diluted ReagentC solution 500 was removed from tube 2	0.25
4	500	Diluted ReagentC solution 500 was removed from tube 3	0.125
				5	500	Empty contrast	0

Measuring 500 mu L of dyeing working solution in a cuvette, adding 500 mu L of standard solution, incubating for 5min in a dark place, measuring the light absorption value at 260nm, drawing a standard curve, and calculating the DNA content in the sample according to the standard curve;

the envelope rate calculation formula is as follows: the encapsulation ratio (%) (addition amount-free amount) ÷ addition amount × 100%.

2.3 preparation of Targeted cationic Liposome (Tf-pIRES-Rt-sirt2-Tet-nap)

Measuring borate buffer solution (pH is 8.0) to dissolve and convert iron; mixing the transferrin solution and a sulfhydrylation reagent 2-iminothiolane hydrochloride according to a molar ratio of 1:80, and incubating for 1 h; removing redundant 2-iminothiolane hydrochloride by using a phosphoric acid buffer solution to obtain thiolated transferrin; coupling the cationic liposome coated with the plasmid prepared in the step 2.2 with thiolated transferrin, and reacting overnight; unreacted thiolated transferrin is separated from the liposome coupled with transferrin by agarose CL-4B, and the eluent is collected for subsequent detection.

2.4 determination of the coupling ratio

Transferrin coupling rate was determined according to the BCA kit protocol.

2.5 Observation of Liposome morphology

Taking a proper amount of liposome, carrying out negative staining for 30s by using a 1% tungsten phosphate solution, then sucking a sample, dropwise adding the sample on a copper net, naturally drying at room temperature, and then observing under a transmission electron microscope.

2.6 measurement of particle diameter and zeta potential

The particle size and zeta potential of the liposomes were measured by a nanometer particle size analyzer and a zeta potential meter.

2.7 statistical analysis

Data were analyzed using Graph PadPrism 5 software, two comparisons were tested for t, and comparisons between multiple mean groups were analyzed using One-wayANOVA, with p < 0.05 considered to be of significance.

3 animal experiments

3.1Morris Water maze experiment

(1) Animal grouping: animals were randomized into four groups (N ═ 9): a normal group, an Alzheimer's disease group (AD group), AD + Rt-sirt2(Rt-sirt2 group) and AD + Tf-Rt-sirt2(Tf-Rt-sirt2 group);

(2) animal treatment: injecting 1ml of cationic liposome tail vein of the prepared double-gene autophagy vector into AD mice, wherein the concentration of the plasmid is 50 mug/ml, and the control group is given with physiological saline with the same volume;

(3) experimental apparatus: a circular water pool with the diameter of 120cm and the height of 40cm, a transparent platform with the diameter of 10cm and the height of 25cm, wherein the transparent platform is fixed at the position 2cm under water, the water pool is artificially divided into four quadrants, a proper amount of milk powder is added into the water to cover the platform, and the water temperature is kept at 24-28 ℃ in the whole experiment process. The first day of the experiment mice were placed in water for free swimming for 60s to become familiar with the environment; and (3) navigation experiment: the following day after tail vein injection, mice were placed in water from any quadrant; the mouse found the platform underwater within 60s and stayed on the platform for 10s, which was considered to be the platform found, otherwise, the platform was not found, at which time the mouse was artificially brought to the platform and the latency was recorded. Each mouse was tested 4 times a day for 5 days, and the average time of each group of mice to reach the platform each day was calculated to evaluate its spatial learning ability. Space exploration experiment: after the experiment is finished, the platform is taken away, the mouse is put into water from the direction opposite to the original direction, the times of passing through the position of the original platform within 60s are recorded, and the spatial memory capacity of the mouse is evaluated.

3.1.2NORT experiments

Experimental apparatus: the length, width and height of the glass box are respectively 50cm, 40cm and 25 cm.

(1) An adaptation period: 2 days before starting the experiment, the mice were placed in an empty box to move freely to become familiar with the environment;

(2) in the familiarity stage: placing two articles with the same shape and appearance in a glass box, placing a mouse in the box at the same distance from the articles, freely exploring for 6min, and familiarizing with the surrounding environment;

(3) and (3) testing period: after the end of the familiarity period, the mice were returned to their cages for 24h and placed in the cage and allowed to explore freely for 6 min. Then, one article in the box was replaced, search times of the mouse for the new and old articles within 6min were recorded and expressed as T1 and T2, respectively, and the Recognition Index (RI) was calculated from the recorded times as represented by RI ═ T1/(T1+ T2) × 100%.

3.2 identification of the protein of interest

(1) After 24h of tail vein injection, mice were anesthetized by intraperitoneal injection of 10% chloral hydrate;

(2) opening the brain of the mouse to take out the hippocampus and cortex, and washing the taken out tissue with precooled physiological saline to remove blood and impurities; weighing after blotting with filter paper;

(3) respectively taking 0.1g of cortex and hippocampus tissues, adding 1mL of lysate containing phosphatase inhibitor, protease inhibitor and PMSF into each tissue, grinding, and paying attention to the fact that the whole operation is carried out on ice;

(4) when ground to no apparent tissue mass, the homogenate was transferred to a clean EP tube and centrifuged (4 ℃, 12000rpm, 5min) to collect the supernatant, which was then used for Rt-sirt 2.

3.3 measurement of ATP content in brain tissue

Adding lysis solution into cortex and Hippocampus tissue, and grinding on ice; 10000g of centrifugal grinding homogenate, collecting supernatant after the homogenate is finished, and measuring the ATP content in cortex and hippocampus tissues according to the operation instruction of an ATP kit.

3.4 determination of ROS levels in brain tissue

Taking out the cortex and the hippocampus of the mouse, and adding precooled homogenate buffer solution according to the proportion (1: 10); after mixing evenly, 10000g of the mixture is centrifuged, and the supernatant is collected; determination of ROS levels was performed according to ROS kit protocol.

3.5 measurement of cytochrome c oxidase Activity in brain tissue

(1) Extraction of mitochondria in cortex and hippocampal tissues

Cooling cortex or hippocampus tissue, and grinding to suspension state; centrifuging to collect supernatant, grinding and centrifuging the precipitate for the second time, combining the two supernatants, centrifuging the combined supernatants to obtain precipitate, and obtaining the precipitate as crude mitochondria; adding separation buffer solution to suspend the crude mitochondria for gradient centrifugation, and collecting liquid on 25% Percoll and 40% Percoll solution to obtain the mitochondria; the collected mitochondria are subjected to resuspension and centrifugal washing, and the obtained precipitate is the purified mitochondria obtained by separation.

(2) Measurement of cytochrome c Activity in cortex and Hippocampus tissues

Cytochrome c oxidase activity in cortex and hippocampus was determined according to the cytochrome c oxidase kit procedure.

3.6 measurement of mitochondrial DNA content

(1) The extraction of total DNA from brain tissue was performed according to the DNA extraction kit instructions.

(2) The measurement of mitochondrial DNA content in cortex and hippocampus tissues was performed with the primers shown in Table 2:

TABLE 2PCR primers

Target	Forwardprimer(5’-3’)	Reverseprimer(5’-3’)
			Human mtDNA	CAAACCTACGCCAAAATCCA	GAAAATGAATGAGCCTACAGA
Mouse mtDNA	ACACCAAGGTTAATGTAGC	TTGAATCCATCTAAGCATT
			Human nDNA	ACGGACCAGAGCGAAAGCA	GACATCTAAGGGCATCACAGAC
Mouse nDNA	CAGTACTTTAAGTTGGAAACG	ATCAACATAATTGCAGAGC

3.7 measurement of transcription factor expression

The method comprises the steps of taking cerebral cortex and hippocampus tissues of each group of mice, cracking, collecting supernatant, carrying out protein quantification by a BCA method, and detecting the expression level of mitochondrial biogenesis related transcription factor proteins in the cortex and hippocampus tissues of AD model mice by Westernbolt, wherein the used primary antibodies are PGC1 alpha, NRF1 and NRF2 respectively.

3.8 determination of mitochondrial kinetic and autophagic proteins

Detection of the expression levels of the mitochondrial dynamics proteins (Mfn1, Mfn2 and Drp1) and autophagy protein levels (PINK1, Parkin and LC3) was performed as described in 3.7 above, using Mfn1, Mfn2, Drp1, PINK1, Parkin, LC3I and LC3II as primary antibodies.

3.9 detection of expression of target Gene in Hippocampus tissues after Induction of autophagy

Mice were tested for Rt-sirt2 gene expression in mouse cortex and hippocampal tissues 72h after drinking water (pH 7.0) containing 7.5mg/mL 4-epidoxycycline.

3.10 statistical analysis

4. Results of the experiment

4.1 characterization of Targeted Liposome morphology, particle size and Zeta potential

The morphology rule of the prepared targeted cationic liposome is observed under a transmission electron microscope and is spherical (figure 10), and the particle size, the polydispersity index (PDI) and the Zeta potential of the prepared targeted cationic liposome are measured according to the important in-vitro evaluation indexes of the liposome. As shown in Table 3, the cationic liposomes of the plasmids pIRES-Rt-sirt2-Tet-nap and Tf-pIRES-Rt-sirt2-Tet-nap had average particle sizes of 162nm and 182nm, PDI of 0.202 and 0.212, and Zeta potentials of 4.6mV and 7.9mV, respectively.

TABLE 3 characterization of liposome particle size, PDI and Zeta potential

Plasmids	Particle size (nm)	Polydispersity index (PDI)	Zeta potential (mV)
				pIRES-Rt-sirt2-Tet-nap	162.5±5.72	0.202±0.015	4.6±2.9
Tf-pIRES-Rt-sirt2-Tet-nap	182.3±6.09	0.212±0.037	7.9±1.8

4.2 measurement of encapsulation efficiency and coupling efficiency of targeting cationic liposome

Based on the plotted standard curves, the encapsulation efficiency and the coupling efficiency of the prepared cationic liposome are calculated, and the results show that the encapsulation efficiency of the two plasmids is more than 85 percent (table 4), and the coupling efficiency of the targeted cationic liposome reaches more than 50 percent (table 4).

TABLE 4 measurement of Liposome encapsulation and coupling rates

Plasmids	Encapsulation efficiency (%)	Coupling ratio (%)
			pIRES-Rt-sirt2-Tet-nap	85.5±2.4	----
Tf-pIRES-Rt-sirt2-Tet-nap	89.2±3.5	51.3±8.2

4.1 identification of Rt-sir2 expression in mouse cortex and hippocampal tissues

We took cortex and hippocampus tissues of AD model mice injected with plasmid DNA targeted cationic liposome, and found that Rt-sirt2 protein exists in hippocampus and cortex tissues by Western felt detection analysis (FIG. 11), which indicates that the transferrin-modified cationic liposome coated with the two-gene autophagy vector can pass through blood brain barrier through transferrin receptor-mediated transcytosis and enter into brain parenchyma of AD model mice.

4.2 Effect of Rt-sir2 on ATP levels in brain tissue of AD mice

The ATP content in the cortex and the hippocampus of the AD model mouse is obviously reduced compared with that of a normal mouse, while in the Tf-Rt-sirt2 group, the ATP content in the cortex and the hippocampus of the mouse is obviously increased (figure 12), which shows that the treatment of Rt-sirt2 can obviously enhance the brain tissue metabolism level of the AD model mouse and the mitochondrial function.

4.3 Effect of Rt-sir2 on ROS levels in brain tissue of AD mice

The ROS levels in the cortex and hippocampus of AD model mice were significantly higher than those of normal mice, whereas in the Tf-Rt-sirt2 group, the ROS levels in the cortex and hippocampus of mice were significantly decreased (fig. 12), indicating that treatment with Rt-sirt2 was able to significantly reduce the oxidative stress level in brain tissue of AD model mice.

4.4 Effect of Rt-sir2 on mitochondrial enzymatic Activity in brain tissue of AD mice

Cytochrome c oxidase (Cyt c) is an enzyme involved in mitochondrial respiratory electron transfer chain, and the activity of Cyt c in mitochondria isolated from cortex and hippocampus of AD model mice is reduced, on the contrary, tail vein injection of Tf-Rt-sirt2 can significantly improve the activity of Cyt c in hippocampal tissue cells, and the two are significantly different compared with AD group (fig. 14).

4.5 Effect of Rt-sir2 on mitochondrial DNA content in hippocampal tissue of AD mice

In order to verify whether the improvement of the mitochondrial function of the AD model mouse is caused by mitochondrial biogenesis, the mtDNA content in the cortex and the hippocampus of the AD model mouse is further detected, and the experimental result is shown in fig. 15, compared with the normal group of mice under the same background condition, the mtDNA/nDNA ratio in the AD model mouse is significantly reduced, which indicates that the mitochondrial biogenesis in the AD model mouse is reduced, while after Tf-Rt-sirt2 is injected into the tail vein, the mtDNA/nDNA ratio in the cortex and the hippocampus of the AD model mouse is significantly increased, the mitochondrial biogenesis is increased, and compared with the AD model group of mice, the mtDNA/nDNA ratio and the nrna ratio have significant difference, which indicates that Rt-sirt2 can improve the mitochondrial biogenesis in the brain tissue of the AD model mouse, so as to improve the quality and the mitochondrial function.

4.6 Effect of Rt-sir2 on mitochondrial biogenesis-associated transcription factors in AD mice

Consistent with the results of in vitro cytology experiments, the expression levels of mitochondrial biogenesis-associated transcription factors PGC1 α, NRF1 and NRF2 in the cortex and hippocampus of AD model mice were upregulated to different degrees on average and were differentially significant compared to AD group mice (fig. 16).

4.7 Effect of Rt-sir2 on mitochondrial dynamics proteins in brain tissue of AD mice

Western bolt results show that proteins involved in mitochondrial dynamics are changed to different degrees after being treated by Tf-Rt-sirt2, wherein the expression levels of mitochondrial fusion proteins Mfn1 and Mfn2 are obviously increased, but the expression level of mitochondrial fission protein Drp1 is reduced, and compared with an AD model group, the two proteins have no obvious difference (figure 17), and the results show that Rt-sirt2 changes the state of mitochondria in mouse neuronal cells, changes from the original over-fission state to fusion, greatly promotes the balance of mitochondrial fusion and fission, and restores the mitochondrial network.

4.8 Effect of Rt-sir2 on mitophagy-associated proteins in brain tissue of AD mice

We then examined the expression level of the mitophagy-related protein in AD model mice, and we found that the expression level of the mitophagy-related protein PINK1 and its downstream activator protein Parkin protein are both up-regulated by Western bolt analysis, and the LC3 ii/LC 3i ratio is also increased (fig. 18), which is consistent with the results of in vitro cytology experiments, indicating that Rt-sirt2 can activate mitophagy in cells to promote the clearance of damaged mitochondria and maintain the homeostasis of mitochondria in cells.

4.9 Effect of Rt-sir2 on learning and memory function of AD mice

The Morris water maze experiment and the NORT experiment are respectively carried out to evaluate the influence of Rt-sir2 on the learning and cognitive functions of the AD model mouse, the experiment results are shown in figures 1-9, in the Morris water maze experiment, the platform escape latency of each group of mice is gradually shortened along with the increase of training days, but the average escape latency of each group of mice is obviously different, after Tf-Rt-sirt2 is treated, compared with the AD model group of mice, the Tf-Rt-sirt2 group of mice has obviously shortened escape latency (figure 19A), the Tf-Rt-sirt2 can enhance the space learning capability of the AD model mouse, and the number of times that the Tf-Rt-sirt2 group of mice passes through the quadrant where the platform is located is increased (figure 19B), the Tf-Rt-sirt2 can enhance the formation and consolidation of the space memory of the AD model mouse; in addition, in NORT experiment, compared with AD model mouse group, the Tf-Rt-sirt2 group mouse recognition index is increased, which shows that the Tf-Rt-sirt2 can enhance the recognition and memory ability of AD model mouse to new object (FIG. 19C).

4.10 autophagy ablation of the Gene of interest

After finishing the behavioural experiments, we administered exogenously 4-epidoxycycline to induce nap expression. After administration of 4-epidoxycycline for 72h, we found that expression of Rt-sirt2 gene was not detected in mouse cortex and hippocampal tissues (fig. 20), which is consistent with the results of in vitro cytological experiments, indicating that DNA autophagy peptide nap can also cause autophagic apoptosis of vectors carrying exogenous genes by activating CMA in vivo, thus realizing effective controllable expression of exogenous genes in vivo.

Claims

1. The invention constructs a pIRES-Rt-sirt2-Tet-nap recombinant autophagy vector capable of recovering mitochondrial function and improving Alzheimer's disease.

2. The pIRES-Rt-sirt2-Tet-nap recombinant autophagy vector of claim 1, which is constructed by the following steps: an internal ribosome entry site IRES is introduced to construct a plasmid IRES-Rt-sirt2 plasmid, and a DNA nucleic acid autophagy peptide nap consisting of a structural domain connecting histone H1 and DNA and a recognition motif mediating a CMA autophagy pathway and a tetracycline inducible (Tet-on) system are introduced into the plasmid pIRES-Rt-sirt2, so that the plasmid pIRES-Rsirt2-Tet-nap is constructed.

3. The pIRES-Rt-sirt2-Tet-nap recombinant autophagy vector of claim 1, wherein: the carried DNA nucleic acid autophagy peptide nap can enable the carrier carrying the exogenous gene to generate autophagy apoptosis by activating an intracellular molecular chaperone mediated autophagy way in vivo and in vitro, thereby realizing effective and controllable expression of the exogenous gene in vivo/in vitro.

4. The pIRES-Rt-sirt2-Tet-nap recombinant autophagy vector of claim 1, wherein: the ATP content, ROS level, cytochrome c oxidase activity and mtDNA content in cortex and hippocampus tissues of an AD model mouse are obviously improved, so that the quality and function of mitochondria are improved; in addition, the level of mitochondrial fusion proteins and autophagy proteins in cortical and hippocampal tissues is up-regulated, whereas mitochondrial fission proteins are not significantly changed, and the mitochondrial network is restored at a certain level.

5. A pIRES-Rt-sirt2-Tet-nap recombinant autophagy vector capable of restoring mitochondrial function and improving symptoms of mice with Alzheimer's disease, which is characterized in that: the learning and cognitive functions of an AD mouse model are improved, and after 4-epidoxycycline is exogenously given, the expression of the Rt-sirt2 gene cannot be detected in the cortex and the hippocampus of the AD mouse model, so that the autophagy vector carrying the Rt-sirt2 is subjected to molecular chaperone mediated autophagy and then autophagy ablation and death.

6. A pIRES-Rt-sirt2-Tet-nap recombinant autophagy vector capable of recovering mitochondrial function and improving Alzheimer disease is characterized by being applied to other mitochondrial dysfunction diseases such as Alzheimer disease.