CN113424797A - Method for regulating blood brain barrier permeability and application thereof - Google Patents

Abstract

The invention discloses a method for regulating blood brain barrier permeability and application thereof. According to the invention, the fifth exon, the sixth exon and the seventh exon of the PTEN gene in the mouse cerebral vascular endothelial cells are specifically knocked out, so that the permeability of the blood brain barrier of the mouse is increased, and the blood brain barrier leaks; through constructing recombinant adeno-associated virus expressing NEDD4-2 protein and injecting the virus into mice, the number of vesicles in cerebral vascular endothelial cells of the mice is increased, and the blood brain barrier permeability and the blood brain barrier leakage of the mice are also increased. PTEN deletion was shown to result in blood brain barrier leakage due to overexpression of NEDD4-2-Mfsd2 a. Therefore, the permeability of the blood brain barrier can be regulated and controlled by regulating the content or the activity of the PTEN protein or the NEDD4-2 protein, and the protein can be applied to clinical preparation or development of related drugs for regulating and controlling the permeability of the blood brain barrier or related drugs for cerebrovascular diseases.

Description

Method for regulating blood brain barrier permeability and application thereof

Technical Field

The invention relates to the technical field of medical biology, in particular to a method for regulating blood brain barrier permeability and application thereof

Background

Brain Endothelial Cells (ECs) located within the Central Nervous System (CNS) vessels form the Blood Brain Barrier (BBB), tightly controlling the entry and exit of substances into and out of the CNS, and thus maintaining homeostasis of the CNS microenvironment (Zhao et al, 2015). The properties of the BBB that limit substance entry and exit depend on unique features of brain EC, including specific tight and adhesive junctions, low levels of transcytosis, non-selective fenestrations, and pinocytosis (Andreone et al 2015). The blood brain barrier maintains central nervous system homeostasis, and is one of the biggest obstacles to be overcome in the development of drugs acting on the central nervous system. How to make therapeutic drugs penetrate through the blood-brain barrier and enter the central nervous system is always a great problem in the development of related drugs. Modulation of blood brain barrier permeability to facilitate penetration of drugs in the peripheral blood into the central nervous system is one of the solutions to this disorder.

Under physiological and pathological conditions, protein ubiquitination modification plays an important role by affecting the stability and activity of target proteins. A recent study showed that zika virus increased Mfsd2a ubiquitination and caused proteasome-dependent degradation in Human Brain Microvascular Endothelial Cells (HBMECs) (Zhou et al, 2019), suggesting that Mfsd2a may be regulated by polyubiquitination, while the mechanism by which ubiquitin ligase causes Mfsd2a ubiquitination is unclear. Neural precursor cell expression development down-regulation gene 4-like (NEDD4-2) is a HECT type E3 ubiquitin ligase, which transfers ubiquitin to multiple membrane proteins.

Disclosure of Invention

The technical problem to be solved by the present invention is how to modulate the permeability of the blood-brain barrier or how to increase the blood-brain barrier permeability to facilitate drug delivery to the central nervous system.

In order to solve the above technical problem, the present invention firstly provides an application. The application may be P1 or P2.

The P1 is an application of a non-human mammal which specifically knocks out PTEN gene in a cerebral vascular endothelial cell in the preparation of a product for regulating blood brain barrier permeability or a product for drug delivery.

The P2 is an application of a substance for improving or promoting the expression quantity of NEDD4-2 gene or/and the activity or content of NEDD4-2 protein in the brain vascular endothelial cells of non-human mammals in preparing products for regulating and controlling blood brain barrier permeability or developing drug delivery products.

The drug delivery may be delivery of a drug across the blood brain barrier into the central nervous system.

The genome sequence of the PTEN gene is shown as 32734977-32803560 nucleotide of GenBank Accession No. NC-000085.7 (Update Date 2020-09-22). The 32734977 th nucleotide is denoted as the 1 st nucleotide of the PTEN gene, wherein the 32734977-32735824 th nucleotide is the first exon sequence, the 32753416-32753500 th nucleotide is the second exon sequence, the 32769950-32769994 th nucleotide is the third exon sequence, the 32775775472-32515 th exon sequence is the fourth exon sequence, the 32777262-32777500 th nucleotide sequence, the 32789097-32789238 sixth exon sequence, the 32792818-32792984 seventh exon sequence, the 32795238-32795461 eighth exon sequence, and the 32797244-323560 ninth exon sequence.

In the above application, the non-human mammal in P1 may be a non-human mammal obtained by specifically knocking out PTEN gene in brain vascular endothelial cells using Cre/loxP system.

The substance described in P2 can be a recombinant vector expressing NEDD4-2 protein or a recombinant microorganism expressing NEDD4-2 protein.

In the above application, the specific knockout of the PTEN gene in the cerebral vascular endothelial cell described in P1 may be specific knockouts of the fifth exon, the sixth exon, and the seventh exon in the PTEN gene in the cerebral vascular endothelial cell of the non-human mammal.

The recombinant microorganism described in P2 may be a recombinant adeno-associated virus expressing NEDD4-2 protein.

The amino acid sequence of the NEDD4-2 protein is shown as SEQ ID No.7 in the sequence table.

The recombinant adeno-associated virus contains a coding gene of the NEDD4-2 protein. The nucleotide sequence of the coding chain of the coding gene of the NEDD4-2 protein is a DNA molecule shown in SEQ ID No.3 in a sequence table or 31 th-2961 th site of SEQ ID No.3 in the sequence table.

In order to solve the technical problems, the invention also provides a method for regulating and controlling the blood brain barrier permeability of the non-human mammal. The method comprises modulating blood brain barrier permeability using the method of A1) or A2) described below.

A1) Reducing or inhibiting the expression level of PTEN gene or/and the activity or content of PTEN protein in the brain vascular endothelial cells of the non-human mammal.

A2) Increase or promote the expression level of NEDD4-2 gene or/and the activity or content of NEDD4-2 protein in the brain vascular endothelial cells of the non-human mammal.

In the method described above, the modulation of blood-brain barrier permeability may be an increase in blood-brain barrier permeability.

In the method, the reduction or inhibition of the expression level of the PTEN gene or/and the activity or content of the PTEN protein in the brain vascular endothelial cells of the non-human mammal may be specific knock-out of the PTEN gene in the brain vascular endothelial cells of the non-human mammal.

The improvement or promotion of the expression quantity of the NEDD4-2 gene or/and the activity or content of the NEDD4-2 protein in the cerebral vascular endothelial cells of the non-human mammal can be realized by using a recombinant adeno-associated virus expressing the NEDD4-2 protein. The NEDD4-2 protein is a protein with an amino acid sequence of SEQ ID No.7 in a sequence table.

In the method described above, the specifically knocking out the PTEN gene in the brain vascular endothelial cells of the non-human mammal may comprise specifically knocking out the PTEN gene in the brain vascular endothelial cells of the non-human mammal using a Cre/loxP system.

The recombinant adeno-associated virus contains a coding gene of the NEDD4-2 protein. The nucleotide sequence of the coding chain of the coding gene of the NEDD4-2 protein is a DNA molecule shown in SEQ ID No.3 in a sequence table or 31 th-2961 th site of SEQ ID No.3 in the sequence table.

In the method described above, the non-human mammal may be a mouse. The method for specifically knocking out the PTEN gene in the cerebral vascular endothelial cells of the mouse can be as follows:

to PTEN^flox/floxThe mice were mated with SP-A-Cre mice to give F1 generation mice.

The PTEN^flox/floxThe mice were homozygous mice with replacement of the mouse PTEN gene with the recombinant floxed PTEN allele.

The recombinant floxed PTEN allele is a gene on one chromosome of a mouseThe nucleotide sequence of 32773472-32779472 corresponding to GenBank Accession No. NC-000085.7 (Update Date2020-09-22) in the genome sequence is replaced by the DNA fragment shown in SEQ ID No.2 in the sequence table, and the other nucleotide sequence of the PTEN gene is kept unchanged to obtain the recombinant DNA molecule (namely the recombinant allele PTEN of PTEN)^flox). The SP-A-Cre mouse is ｃA transgenic mouse of cerebrovascular endothelial cell specificity expression Cre recombinase;

selecting ｃA double-positive heterozygote mouse from the F1 generation mice, and naming the mouse as SP-A-Cre; PTEN^flox/+ｃA mouse, the SP-A-Cre; PTEN^flox/+One chromosome of the mouse carries the PTEN wild type allele, the other chromosome carries the PTEN recombinant floxed PTEN allele, and the SP- ｃA-Cre; PTEN^flox/+The mice specifically express Cre recombinase in cerebrovascular endothelial cells;

the SP-A-Cre; PTEN^flox/+Mice and the PTEN^flox/floxMating the mice to obtain F2 generation mice;

selecting ｃA double positive homozygous mouse SP-A-Cre from said F2 mouse; PTEN^flox/floxThe mice are mice which specifically knock out PTEN genes in the brain vascular endothelial cells.

The SP-A-Cre; PTEN^flox/floxBoth chromosomes of the mouse carried the floxed PTEN allele and the SP-A-Cre; PTEN^flox/floxIntegrating ｃA SP-A-Cre-hGH target fragment (SEQ ID No.1 in ｃA sequence table) into ｃA mouse genome, specifically expressing Cre recombinase in cerebrovascular endothelial cells, and carrying out SP-A-Cre recombination on the target fragment; PTEN^flox/floxThe fifth exon (i.e., 32777262-32777500 nucleotides of the PTEN allele in GenBank Accession No. NC-000085.7 (Update Date 2020-09-22)), the sixth exon (i.e., 32789097-32789238 nucleotides of GenBank Accession No. NC-000085.7 (Update Date 2020-09-22)) and the seventh exon (i.e., 32792818-3279298792792792987924 nucleotides of GenBank Accession No. NC-000085.7 (Update Date 2020-09-22)) of the PTEN allele on chromosomes in mouse cerebral vascular endothelial cells were knocked out by the Cre/loxP system.

In the use as described above and/or in the method as described above, the modulating permeability of the blood-brain barrier may be increasing permeability of the blood-brain barrier.

In the above-described use and/or the above-described method, the non-human mammal may be any one selected from a mouse, rat, guinea pig, hamster, pig, dog, sheep, monkey, rabbit, cat, cow, and horse.

Products for modulating blood brain barrier permeability and/or drug delivery as described above and/or recombinant adeno-associated viruses as described above are also within the scope of the invention.

The product that modulates blood brain barrier permeability and/or the product of drug delivery described above may be a cerebrovascular related drug; the cerebrovascular related medicine can be a medicine for treating and/or preventing and/or relieving and/or improving cardiovascular and cerebrovascular related diseases.

Modulating blood brain barrier permeability as described above may be increasing blood brain barrier permeability.

The invention finds that the homologous phosphatase-tensin (PTEN)/Akt signal pathway can regulate the stability of Mfsd2a through E3 ubiquitin ligase NEDD4-2, and ensures the normal BBB function. Through the specific knockout of the fifth exon, the sixth exon and the seventh exon of the PTEN gene in the mouse cerebral vascular endothelial cells, the increase of the blood brain barrier permeability and the blood brain barrier leakage of the mouse can be observed; through constructing recombinant adeno-associated virus expressing NEDD4-2 protein and injecting the virus into mice, the number of vesicles in cerebral vascular endothelial cells of the mice is increased, and the blood brain barrier permeability and the blood brain barrier leakage of the mice are also increased. PTEN deletion was shown to result in blood brain barrier leakage due to overexpression of NEDD4-2-Mfsd2 a. Therefore, the permeability of the blood brain barrier can be regulated and controlled by regulating the content or the activity of the PTEN protein or the NEDD4-2 protein, and the protein can be applied to clinical preparation or development of related drugs for regulating and controlling the permeability of the blood brain barrier or related drugs for cerebrovascular diseases.

Drawings

Figure 1 is the result of PTEN cerebrovascular endothelial specific knock-out resulting in decreased expression of PTEN. (A) Brain tissue immunofluorescence staining displaying PTEN^fl/flPTEN expression specific deletion of brain vascular endothelial cells in mice. (B) Real-time PCR results show PTEN^fl/flMouse divisionRNA levels from PTEN in the resulting primary brain endothelial cells were significantly reduced. (C) Western blot experiment shows that PTEN^fl/flThe protein level of primary brain endothelial cells PTEN obtained by mouse separation is obviously reduced.

Figure 2 is a graph showing the results of cerebrovascular-specific knockdown of PTEN resulting in increased blood brain barrier permeability. (A-F) Evans blue dye (A) cadaverine-Alexa555(CADA-A555) (C) and 10-kDa dextran tracer (10-kDa tracer) (E) were injected to find that permeability of the blood brain barrier of PTEN knockout mice was increased. The bottom is the corresponding quantitative data (B, D, f. data mean ± SEM (lower panel, n ═ 4 mice/genotype,. p < 0.01). scale bar 5 mm.

(G) Brain tissue immunostaining analysis after injection of cadeverine-Alexa 555 and 10-kDa tracer. In PTEN^{fl /+}In the group, the tracer (red) was restricted to brain ECs (lectin, white), whereas in PTEN^fl/flIn the parenchyma of the mice, a large amount of diffuse tracer was detected. The scale bar is 50 μm.

(H and I) transcytosis of brain ECs by electron microscopy (H) and quantification of vesicle density (I). PTEN^fl/flEndothelial cells showed a significant increase in vesicular activity, including vacuoles, blastocysts and vacuoles. The scale bar is 150 nm. Data are mean ± SEM (n ═ 4 mice/genotype,. p<0.01)。

(J and K) at P60 for PTEN^fl/+And PTEN^fl/flMouse brain sections were immunostained with Mfsd2a (J). The corresponding statistics are shown on the right (K). And PTEN^fl/+Mouse phase comparison, PTEN^fl/flMouse brain ECs (green) decreased Mfsd2a (red). The scale bar is 50 μm. Data are mean ± SEM (n ═ 4 mice/genotype,. p<0.001)。

Figure 3 is a demonstration of the results of inducing PTEN loss in endothelial cells resulting in BBB leakage. (A and B) Evans blue injection into iPTEN^fl/+(Cdh5-CreERT2；PTEN^fl/+) And iPTEN^fl/fl(Cdh5-CreERT2；PTEN^fl/fl) Mouse (P10). PTEN deletion increases blood brain barrier permeability. The corresponding quantitative data are shown on the right (C). Data are mean ± sem (n ═ 4 mice/genotype,. p-<0.01). The scale bar is 5 mm. (D) Injection of Sulfo-NHS-biotin into iPTEN of P10^fl/+And iPTEN^fl/flImmunostaining analysis of brain tissue sections of mice. The tracer (grey) was found only in iPTEN^fl/+Detected in brain endothelial cells (CD31) of littermates, iPTEN^fl/flA large amount of diffuse tracer was detectable in the brain parenchyma of the mice. Scale bar 1mm (upper panel); 100 μm (lower panel).

FIG. 4 shows that overexpression of NEDD4-2 in brain ECs results in impaired blood brain barrier, mimicking PTEN^fl/flPhenotypic results of mice are shown. (A) Western blot analysis confirmed PTEN^fl/flThe level of p-NEDD4-2 in mouse brain ECs is higher than that of PTEN^fl/+A mouse. (B) Western blot analysis of p-NEDD4-2, NEDD4-2 and Myc after HEK293T cells were transfected with EGFP vector or Myc-NEDD4-2 plasmid. (C-D) immunostaining of brain tissue treated with EGFP-AAV or Myc-NEDD4-2AAV (C) and statistical results (D). Myc-NEDD4-2AAV injection induced Myc (light grey) and EGFP (light grey) overexpression and Mfsd2a (white) reduction in ECs (CD31, dark grey). Scale bar, 50 μm. (E-F) EGFP-AAV and Myc-NEDD4-2AAV treated brain sections immunostained (E) and statistically analyzed (F) after injection of Cad-A555 tracer. Tracer (red) leaked in the brains of mice overexpressing NEDD 4-2. Mfsd2a was reduced (light gray) in ECs with over-expression of NEDD4-2 (white). Scale bar, 50 μm. (G-H) Observation of transcytosis of brain ECs following therapy with Myc-NEDD4-2AAV (G) and statistics of vesicle density (H). Overexpression of NEDD4-2 increased the vesicular activity of endothelial cells. Data are mean ± SEM (n ═ 4 mice/genotype,. p<0.01). The scale bar is 150 nm.

Fig. 5 shows that overexpression of Mfsd2a rescued blood brain barrier leakage due to PTEN knockout. (A) Strategy for AAV overexpression of Mfsd2 a. (B) Western blot analysis of HA in HEK293T cells transfected with vector or HA-Mfsd2a plasmid. (C) Immunostaining analysis of tdTomato (dark grey), lectin (light grey) and HA (white) in brain tissue. HA positive staining showed successful overexpression of Mfsd2a in brain ECs. The scale bar is 20 μm. (D) AAV overexpresses Mfsd2a in mouse cerebral blood vessels. (E and F) recovery of the blood-brain barrier of the Mfsd2a overexpressing group compared to the control group was found after injection of the Sulfo-NHS-biotin tracer in mouse P60 (30 days after AAV treatment) (E) ((E))E) And quantitative data (F) on the right. Data are mean ± SEM (n ═ 4 mice/genotype,. p<0.01). The scale bar is 100 μm. (G and H) transcytosis of brain ECs by Electron microscopy (G) and quantification of vesicle density (H). Overexpression of Mfsd2a to PTEN^fl/flVesicle activity of endothelial cells was reduced to PTEN^fl/+Normal levels of endothelial cells. The right hand side is the quantitative data (H). Data are mean ± SEM (n ═ 4 mice/genotype,. p<0.01). The scale bar is 150 nm.

Fig. 6 shows that blocking the crypt-mediated transcytosis can rescue the blood brain barrier leakage caused by PTEN deletion. (A) AAV knockout of cerebrovascular Cav-1. (B) Western blot analysis of Cav-1 proteins transfected with vectors or sh-Cav-1 plasmid in bEnd.3 cells. (C) Brain tissue immunostaining analysis showed reduced expression of Caveolin1 (dark grey) in sh-Cav-1 AAV (light grey) treated brain ECs (CD31, white) Scale 10 μm (D) Experimental protocol for CAV cerebral vascular knockout Cav-1 in mice (E and F) injection of Sulfo-NHS-biotin tracer in mouse P60 (30 days after AAV treatment) showed that Cav-1 gene knockout PTEN-N-biotin tracer showed compared to control (E)^fl/flThe blood brain barrier leakage of the mice was saved. The corresponding quantitative data are shown on the right (F). Mean ± SEM (n ═ 4/mouse genotype,. sp.,. p)<0.01). The scale bar is 100 μm. (G and H) transcytosis of brain ECs by electron microscopy (G) and corresponding quantification of vesicle density (H). Cav-1 knockdown to PTEN^fl/flThe level of vesicular activity of the endothelial cells was reduced to normal levels. Data are mean ± SEM (n ═ 4 mice/genotype,. p<0.01). The scale bar is 150 nm.

FIG. 7 shows the results of PTEN/AKT regulating the expression of Mfsd2a protein by NEDD 4-2. (A) Western blot analysis Mfsd2a p-AKT1, AKT1 and GAPDH levels in HEK293T cells expressed in IGF-1 treated or untreated MFSD 2A. MG132 was added 8 hours before collection. IGF-1 treatment increased p-AKT1 and decreased expression of Mfsd 2. (B) In HEK293 cells transfected with MFSD2A-EGFP and NEDD4-2-Myc plasmids, immunostaining analysis of EGFP ((light grey) and Myc (dark grey) Scale 20 μm. (C) Western blot analysis of MFSD 2A-expressed HEK293T cells after IGF-1 or LY294002 treatment MFSD2A levels, MFSD2A, p-AKT1, AKT1, p-NEDD4-2(S342/S448), NEDD4-2 and GAPDH levels, IGF-1 treatment increased p-AKT1 and p-NEDD4-2 (S63448) siRNA, MFSD2A decreased and time-dependent SD 294002 treatment decreased p-AKT1 and p-NEDD4-2(S342/S448) and increased MFSD A (MFSD A) in a dose-dependent manner and Western blot 26-24-MFDD A-9-expressing IGF-293, NEDD 2-342/S-A-expressing HEK 5926-expressing HEK2 cells after IGF-1, p-AKT-598-1, and LY2 treatment, NEDD4-2 and GAPDH levels. siNEDD4-2 treatment increased MFSD2A levels, blocking IGF-1-induced reduction of MFSD2A protein.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The mice used in the present invention were raised and maintained in Specific Pathogen Free (SPF) animal facilities approved by the release force laboratory animal care committee (LAMC) of china, and were lit at 8 a.m. and subjected to a 12 hour reverse light/dark cycle. Mice were given food and water at random. All rearing steps were carried out according to protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the Beijing institute of Life sciences. Mice carrying the floxed PTEN allele (Suzuki et al, 2001), mice carrying the floxed Akt1 allele (Di et al, 2012), SP- ｃA-Cre transgenic mice (Li et al, 2011; Li et al, 2012), and Cdh5-CreERT2 mice (Wang et al, 2010) were used for the experiments. Genotyping was performed as described previously. All experimental animals were backcrossed to a C57BL/6J (Viton Li Hua, cat # 219) genetic background. Healthy mice were used in the experiments, the age of which is specified herein. No mice were used in previously unrelated experiments. The effect of gender was not assessed in this study.

The animals used in the experimental design of the examples described below were randomly assigned to experimental groups and the experimenter had no knowledge of the genotype of the animals until the experiment was completed. No statistical method was used to predetermine the sample size. According to 3Rs, the minimum sample size that can produce significant differences is used. No data or animals were excluded from the analysis.

Tamoxifen injection in the following examples: to induce gene recombination, P4-P5 mice were intraperitoneally injected with tamoxifen (Sigma-Aldrich, T5648) for one day (10mg/kg i.e.,10mg/mL in 10% EtOH/Sesame oil, Sigma-Aldrich) according to published procedures.

The cell lines used in the present invention: human embryonic kidney (HEK293T) cells (CRL-11268, ATCC) were maintained at 37 ℃ with 5% CO₂In (1), the cell culture medium (06-1055-57-1ACS, Biological Industries) contains 10% fetal bovine serum (10099-141, Gibco). A mouse brain endothelial cell line, bEnd.3cells (CRL-2299, ATCC), was maintained in a humidified 5% CO2 culture at 37 ℃ in cell culture medium (06-1055-57-1ACS, Biological Industries) containing 10% FBS (10099-. Cell lines were obtained directly from ATCC without additional cell identification.

The following examples are methods for measuring blood brain barrier permeability and quantifying vascular leakage:

evans blue dye (2% saline, E2129, Sigma Aldrich) or Sulfo-NHS-Biotin (0.5mg/g body weight, 21335, Thermo Fisher Scientific) was intraperitoneally and intravenously, respectively. After postnatal mice P30 and P60 pups were anesthetized, 10 μ L of cadoverine alexa555 or 10-KDA dextran-tetramethylrhodamine (4mg/ml D3312 Invitrogen) was injected into the left ventricle using a Hamilton syringe. After 5 minutes of circulation, dissected brain tissue was fixed by soaking with 4% PFA overnight, cryopreserved with 30% sucrose, and frozen in tissue tek OCT (Sakura). Sections (20 μm) were collected, fixed with 4% PFA for 15min at room temperature, washed with PBS, and vessels were stained with lectin primary and 488-Alexa Fluor conjugated secondary antibodies (1:1000, Invitrogen) or Isonectin B4(1: 500; I21411 molecular probes).

Immunohistochemical experimental methods in the following examples:

histological analysis of mouse brain was performed (Luo et al, 2010). Briefly, mice were euthanized and then perfused with physiological saline through the heart. Tissues were removed, fixed in 4% PFA overnight, and then equilibrated in 30% sucrose. Serial sections (40 μ M thick) of the coronary brain suspended throughout DG were kept in alignment, stored in 96-well plates filled with cryoprotectant solution (glycerol, ethylene glycol and 0.1M phosphate buffer, pH7.4, in volume 1:1: 2), and placed in a-20 ℃ freezer. Tissue sections were blocked with TBS + + (TBS containing 3% donkey serum and 0.3% Triton X-100) for 1 hour at room temperature, stained with primary antibody overnight at 4 ℃ and then fluorescently conjugated with secondary antibody using the corresponding Alexa. Slides were mounted in Prolong (Invitrogen) and viewed with a laser scanning confocal microscope and LSM 880 microscope (Carl Zeiss AG). Immunohistochemistry experiments were repeated at least 3 times.

Electron microscope observation methods in the following examples:

brains were dissected and soaked in 0.1M sodium carbonate buffer mix (2% glutaraldehyde and 4% PFA) for 1 hour (hr) at Room Temperature (RT) followed by 12 hours of soaking in PFA at 4 ℃. After fixation, the tissue was washed 2 times in 0.1M sodium carbonate buffer and then cut into 50 μ M thick free floating sections with a vibrating microtome. Sections were post-fixed with 1% osmium tetroxide and 1.5% potassium ferrocyanide, dehydrated and embedded in epoxy resin.

AAV Vectors (AAV Vectors) for use in the invention:

AAV-BR1 (professor Jakob, et al generously available, 2016), a brain microvascular endothelial cell specific viral vector modified with AAV2, expresses a target gene in brain endothelial cells. Production of recombinant AAV vectors by three transfections of HEK293T cells: (

Etc., 2016). Briefly, HEK293T cells were cultured in cell culture medium supplemented with 10% fetal bovine serum and plasmid DNA, a plasmid encoding a modified AAV2 capsid, and an adenovirus helper plasmid pheler (cell biolabs) were transfected with calcium phosphate. After 3 days of transfection, harvestThe supernatant and cell debris were collected and the vector was purified by iodosaxol density gradient ultracentrifugation.

Recombinant AAV vectors were administered at 5X 10 per mouse¹⁰Doses of gp were injected into the tail vein. In each experiment, at least 4 genotyped mice were used per group.

The experimental method of the immunoprecipitation used in the invention comprises:

cells were lysed with HEPES lysis buffer (20mM HEPES, pH 8.0,50mM NaCl, 0.5% Triton X-100,1mM EDTA,1mM EGTA) and a phosphatase inhibitor and protease inhibitor cocktail (roche) was added. The cell lysate was centrifuged and the protein lysate was incubated with the indicated primary antibody overnight at 4 ℃ to prepare an immune complex. The immunocomplexes were then incubated with protein A/G magnetic beads (Thermo Fisher Scientific) for 1 hour at room temperature. The immunocomplexes bound to the beads were then washed three times with HEPES lysis buffer. Add 50. mu.L of elution buffer. The beads were collected with a magnetic holder and then removed and the supernatant stored. Immunoblotting was performed with the primary antibody to identify the protein of interest. anti-MYC, anti-HA, and anti-His monoclonal antibodies were from Cell Signaling (denfoss, massachusetts, usa).

The Western Blotting experimental method used in the invention comprises the following steps:

brain endothelial cells and cell pellets were lysed with RIPA buffer (50mmol/L Tris pH7.4,150mmol/L NaCl, 1% NP-40,270.25% sodium deoxycholate, 1mmol/L EDTA) containing phosphatase inhibitor and protease inhibitor (mini tablet Roche). The protein was quantified using Pierce BCA protein assay kit (Thermo Fisher Science, 23225). 30ug of cleaved protein was isolated by sodium dodecyl sulfate-gel electrophoresis and transferred to PVDF membrane, followed by overnight incubation with anti-Mfsd 2a (abcam, ab177881, 1:1,000), p-AKT1(Cell Signaling Technology,4060S,1:1,000), AKT1(Cell Signaling Technology,2938S,1:1,000), p-NEDD4-2(Ser342) (Cell Signaling Technology, 12146S, 1:1,000), p-NEDD4-2(Ser448) (Cell Signaling Technology, 8063, 1:1,000), GAPDH (Cell Signaling Technology,5174,1:3,000) antibodies.

The secondary antibody was incubated at room temperature for 1h (Beijing Zhongshan Jinqiao Biotech Co., Ltd.). Western blots were imaged by Image Quant LAS4000Mini (GE Healthcare). Quantification of bands was performed using Quantity One software.

The invention adopts GraphPad Prism 8 statistical software to process data, the experimental result is expressed by mean value plus or minus standard error, and double-tail t test is adopted.

Sources of SP-A-Cre mice: stored in the laboratory. The SP-A-Cre mouse is constructed as follows:

introducing an SP-A-Cre-hGH target segment (shown as SEQ ID No.1 in ｃA sequence table) comprising ｃA lung surfactant protein A (SP-A) gene promoter, ｃA Cre recombinase gene and ｃA human auxin gene polyA into ｃA thoracic pronucleus of ｃA fertilized egg of ｃA Kunming white mouse through microinjection, and transplanting the fertilized egg into an oviduct of ｃA pseudopregnant female mouse so as to obtain ｃA progeny mouse. Mice carrying Cre recombinase genes are screened out by utilizing PCR, and SP-A-Cre mice are obtained by backcrossing with C57 BL/6J. SP-A-Cre mouse only specifically expresses Cre recombinase in cerebrovascular endothelial cells and gastric epithelial cells. In SEQ ID No.1, the SP-A gene promoter sequence is shown as nucleotides 1-1414, the Cre recombinase coding sequence is shown as nucleotides 1415-2615, and the human auxin gene polyA sequence is shown as nucleotides 2616-4824.

Example one cerebrovascular knock-out of the PTEN Gene promotes transcytosis and thus increases blood brain barrier permeability

1.1 preparation of brain vascular endothelial cell-specific PTEN Gene knockout mice

1.1.1 PTEN conditional Gene-Targeted mice (PTEN)^fl/flMouse) construction

PTEN is a dual lipid/protein phosphatase that dephosphorylates the lipid phosphatidylinositol (3, 4, 5) -triphosphate and inhibits Akt activity (Song et al, 2012). Mouse homologous phosphatase-tensin (PTEN) is a protein with an amino acid sequence shown as GenBank Accession No. NP-038796 (Update Date 2021-01-03), and mouse PTEN gene (wild allele, PTEN)^+/+) Including 9 exons, the genome sequence is shown as 32734977-32803560 nucleotide of GenBank Accession No. NC-000085.7 (Update Date2020-09-22), 32734977 th nucleotide is the 1 st nucleotide of PTEN gene, wherein 32734977-32735824 is the first exon sequence, 32753416-32753500 is the second exon sequence, 32769950-32769994 is the third exon sequence, 32775775472-32775515 is the fourth exon sequence, 32777262-32777500 is the fifth exon sequence, 32789097-32789238 is the sixth exon sequence, 32818-32792984 is the seventh exon sequence, 32795238-32795461 is the eighth exon sequence, 32797244-323560 is the ninth exon sequence.

PTEN^flox/+Mice were a Tak w.mak laboratory gift (Suzuki et al, 2001). PTEN^flox/+The DNA fragment shown in SEQ ID No.2 in the sequence table is replaced by the corresponding nucleotide sequence at 32773472-32779472 of GenBank Accession No. NC-000085.7 (Update Date2020-09-22) in the genome sequence of one chromosome of the mouse, and the other nucleotide sequences of the PTEN gene are kept unchanged to obtain the recombinant DNA molecule, namely the PTEN recombinant allele PTEN^flox。

To PTEN^flox/+Mice are selfed to obtain homozygote mice PTEN^flox/floxA mouse. PTEN^flox/floxReplacement of the original PTEN gene sequences on both chromosomes of the mouse into the PTEN recombinant allele PTEN^flox。

1.1.2 preparation of brain vascular endothelial cell-specific PTEN Gene knockout mice

PTEN to be reared in SPF animal housing^flox/floxMating the mouse with SP-A-Cre mouse to obtain F1 generation mouse, and identifying by PCR to obtain cerebrovascular specific PTEN gene knockout heterozygous mouse (SP-A-Cre; PTEN)^flox/+Hereinafter abbreviated as PTEN^{fl /+})。

SP-A-Cre；PTEN ^flox/+Mouse and PTEN^flox/floxMating the mice to obtain F2 generation mice, and screening to obtain ｃA mouse SP-A-Cre of ｃA brain vascular endothelial cell specificity PTEN gene knockout homozygote; PTEN^flox/flox(hereinafter abbreviated as PTEN)^fl/flMouse), namely a mouse which specifically knocks out PTEN gene in cerebrovascular endothelial cells.

SP-A-Cre；PTEN^flox/floxTwo-line staining of miceThe bulk PTEN genes all carry the floxed PTEN allele, and the SP- ｃA-Cre; PTEN^flox/floxIntegrating ｃA SP-A-Cre-hGH target fragment (SEQ ID No.1 in ｃA sequence table) into ｃA mouse genome, specifically expressing Cre recombinase in cerebrovascular endothelial cells, and carrying out SP-A-Cre recombination on the target fragment; PTEN^flox/floxThe fifth exon (i.e., 32777262-32777500 nucleotides of GenBank Accession No. NC-000085.7 (Update Date 2020-09-22)), the sixth exon (i.e., 32789097-32789238 nucleotides of GenBank Accession No. NC-000085.7 (Update Date 2020-09-22)) and the seventh exon (i.e., 32792818-3279298792984 nucleotides of GenBank Accession No. NC-000085.7 (Update Date 2020-09-22)) of chromosomal PTEN allele in mouse cerebral vascular endothelial cells were knocked out by the Cre/loxP system. The specific method comprises the following steps:

mouse genome DNA extraction:

1) a small amount of tissue (ear rim, tail tip or toe) from the mouse to be identified was excised and placed in a centrifuge tube, and about 400. mu.L of tissue lysate was added and placed in a55 ℃ water bath for lysis overnight.

2) 200. mu.L of saturated NaCl solution (6M) was added to the centrifuge tube, shaken vigorously 50-100 times, and then allowed to stand on ice for 10 min.

3) Centrifuge at 13000rpm for 10min at room temperature, pour the supernatant (about 500. mu.L) into a new centrifuge tube, add 800. mu.L of absolute ethanol to each tube, reverse the top and mix well.

4) Centrifugation was carried out at 13000rpm for 10min at room temperature, the supernatant was discarded, 500. mu.L of 75% ethanol was added to each tube, and the DNA was washed by inverting the top and bottom.

5) Centrifuging at 13000rpm for 5min at room temperature, discarding the supernatant, and inverting the centrifuge tube to completely dry at room temperature.

6) 100-200. mu.L of water was added to each tube, placed at 37 ℃ and vortexed to dissolve the DNA.

7) And (3) identifying the genotype by PCR.

Mouse genotype identification: mouse genotype was identified by PCR.

1) Mouse genotype identification primer

PTEN F：5’-CTCCTCTACTCCATTCTTCCC-3’

PTEN R：5’-ACTCCCACCAATGAACAAAC-3’

Cre1：5’-GCCTGCATTACCGGTCGATGC-3’

Cre2：5’-CAGGGTGTTATAAGCAATCCC-3’

2) Reaction System (20. mu.l)

3) PCR amplification procedure

4. Agarose gel electrophoresis

i. Weighing appropriate amount of agarose 2g, adding into 100ml of 1 XTAE microwave oven, heating and boiling, cooling, adding nucleic acid dye, pouring into a gel-making plate, inserting into a comb, and making into agarose gel.

And ii, after the agarose gel is solidified, placing the gel into an electrophoresis tank, dropping a PCR product into an agarose gel hole, placing 140V in the electrophoresis tank for 20min or more, and judging the genotype. The Cre positive band is 487 bp. The PTEN mutation band is

1.1.3 detection of efficiency of PTEN gene knockout of mice by specific PTEN gene knockout of cerebrovascular endothelial cells

For 4 weeks old PTEN^fl/flThe mouse cerebral vessels were examined. Immunofluorescence staining of PTEN and endothelial marker molecule Isolectin B4 (Isonectin B4, IB4) showed, with control PTEN^fl/+Comparison, PTEN^fl/flPTEN positive signals were significantly reduced in cerebrovascular endothelial cells of mice (a in fig. 1). Also in marked contrast to the positive signals widely distributed in non-endothelium surrounding endothelial cells. In addition, 4-week PTEN was obtained by flow sorting^fl/flExtracting RNA and protein from mouse cerebral vascular endothelial cells, and showing PTEN by Real-time PCR detection result^fl/flmRNA level of PTEN in cerebrovascular endothelial cells of mice and PTEN of control group^fl/+The comparison was also significantly reduced (B in fig. 1). The results of protein level detection using the Western Blot experiment indicate that PTEN^fl/flMouse cerebral blood vesselPTEN knockouts in endothelial cells occurred (C in fig. 1). The specific experimental method is as follows:

frozen section immunofluorescence:

1) frozen sections were rehydrated in PBS and washed 3 times for 5min each.

2) Sections were treated with transmembrane fluid (PBS containing 0.5% Triton/3% donkey serum) for 1h at room temperature.

3) The blocking solution was discarded, and 1:200 diluted rabbit anti-PTEN antibody (Cell Signaling, #9559) was added dropwise thereto at 4 ℃ overnight.

4) PBS was washed 4 times for 4min each.

5) Depending on the species of primary antibody, an appropriate donkey-anti-rabbit fluorescent secondary antibody (Invitrogen, # A10042) was selected and incubated for 1h at room temperature.

6) PBS was washed 4 times for 4min each.

7) After all antibody labeling was completed, nuclei were counterstained with DAPI and mounted.

Western Blot：

1) Extraction of Total cellular protein

i. To the cells cultured in the 6-well plate, 60. mu.L of RIPA cell lysate was added. The whole procedure was performed on ice, and the cells were scraped off with a cell scraper.

Collecting cell lysate, fully lysing cells by using an ultrasonic disrupter, centrifuging at 13000rpm at 4 ℃ for 20min, and taking supernatant into a new centrifuge tube.

2) The BCA method determines the protein concentration.

Protein concentration was determined by BCA method (calibration of extracted protein concentration after standard curve drawing). Calculating according to the calibrated concentration, taking a proper volume of sample, proportionally adding 5XLoading Buffer, mixing uniformly, and placing in boiling water for 5min to completely denature the protein.

3) And (4) electrophoresis.

SDS-PAGE gels.

10% separation gel formulation (5 mL): ddH₂O, 2 mL; 30% polyacrylamide solution, 1.67 mL; 1.5M Tris-Cl (pH8.8), 1.25 mL; 10% SDS, 50 μ L; 10% AP, 50 μ L; TEMED, 2 μ L.

5% Cash formulation (1.5 mL): ddH₂O, 1.05 mL; 30% polyacrylamide solution，0.25mL；1M Tris-Cl(PH6.8)，0.19mL；10％SDS，15μL；10％AP，15μL；TEMED，2μL。

ii. loading and electrophoresis

And adding the protein sample into the SDS-PAGE gel sample adding hole. During electrophoresis, 80V electrophoresis is firstly used for 30min, so that a sample is pressed to a straight line and enters separation gel, and then 160V electrophoresis is carried out for about 1h, so that bromophenol blue is electrophoresed to the bottom.

4) And (5) transferring the film.

The proteins were transferred to 0.2 μm PVDF membrane using an electrotransfer apparatus, run at 300mA for 1.5 h.

5) And (5) sealing.

PVDF membrane was blocked with blocking solution (5% skim milk powder in PBS-T) for 0.5h on a shaker at room temperature.

6) Primary antibody incubation.

The desired primary antibody was diluted as required and incubated at 37 ℃ for 2h, or 4 ℃ overnight. The membrane was washed three times with PBS-T solution for 4min each time.

7) And (5) incubating a secondary antibody.

Selecting corresponding secondary antibody according to the species of the primary antibody, adding a secondary antibody diluent (diluted by 5000-10000 times by using a sealing solution), and incubating for 1h in a shaking table at room temperature. The membrane was washed three times with PBS-T, 5min each time.

8) And (4) developing color.

Mixing ECL color developing solution in advance according to the proportion of 1:1, dripping the color developing solution after the film is drained, and imaging by a protein imaging system.

RNA extraction from cells with Trizol and Real-time PCR:

1) total RNA was extracted from the cells.

i.6 well plates cells were lysed by adding 1ml Trizol after media was discarded.

And ii, adding chloroform into 200 mu L of chloroform/mL of Trizol, uniformly mixing by vortex oscillation, and standing for 5min at room temperature.

iii.4 ℃ and centrifugation at 12000rpm for 15 min.

The upper aqueous phase was aspirated off and transferred to another centrifuge tube, isopropanol was added at 0.5mL isopropanol/mL Trizol and mixed by inversion, and left to stand at room temperature for 10 min.

v.4 ℃ and centrifugation at 12000rpm for 10min, the supernatant was discarded and RNA precipitation at the bottom of the tube was visualized.

And vi, adding 1mL of ice-precooled 75% ethanol, blowing and beating the RNA precipitate, and cleaning the RNA.

vii.4 ℃ 7500rpm for 5min, and the supernatant was discarded.

Room temperature drying for 5min (RNA should not be too dry, otherwise difficult to dissolve).

The RNA precipitate was dissolved in 20. mu.L nuclease-free water and blown with a gun, and the mixture was left at 55 ℃ for 10-15min to promote RNA dissolution.

x-70 ℃ preservation of the RNA solution.

Measuring the concentration of RNA by using an ultraviolet spectrophotometer.

2) Reverse transcription of RNA

RNA was inverted to cDNA using the TOYOBO reverse transcription kit.

i. Reverse transcription

The following system (20. mu.l) was prepared on ice

RNA 2μg

5×RT Mix： 4μl

Reverse transcription PCR reaction conditions

37℃：15min

50℃：5min

98℃：5min

4℃：hold

After the reaction, 40. mu.l of RNase-free water was added, and the mixture was stored at 4 ℃ or-20 ℃ until the cDNA was ready for subsequent use.

3)Quantitative Real-Time PCR

i. Reaction system (10. mu.l):

ii, placing the prepared reaction system into a 7500Fast real-time fluorescence quantitative PCR instrument, and setting reaction conditions according to the following procedures:

1.1.4 Permeability phenotype Observation of the blood brain Barrier

Respectively injecting Evans blue dye into 60 days postnatal (P60) brain blood vessel specific PTEN gene knockout mice (SP-A-Cre; PTEN)^flox/floxHereinafter abbreviated as PTEN^fl/fl) And SP-A-Cre; PTEN^fl/+(hereinafter abbreviated as PTEN)^fl/+) Control mice were in the abdominal cavity to test their blood brain barrier integrity. PTEN^fl/flLeakiness of Evans blue was observed in brain parenchyma of mutant mice, but in PTEN^fl/+No leakage was detected in the mice (a in fig. 2). Detecting the absorbance of the brain tissue fluid of the mouse at 620nm after the Evans blue dye is injected by using a full-wavelength microplate reader, and displaying the PTEN^fl/flOD of mouse brain₆₂₀Ratio of nm values to PTEN of same nest^fl/+OD of mouse₆₂₀The nm value is increased by more than 2.8 times (B in FIG. 2), indicating that PTEN^fl/flMice develop blood brain barrier leakage.

To further validate PTEN^fl/flThe blood brain barrier permeability of the mutant mice was increased and the fluorescent dye Cadaverine-Alexa555(A30678, Invitrogen) that failed to pass the BBB and the 10kDa dextran tracer (D3312, Invitrogen) were injected intravenously through the mice (C-F in FIG. 2). Similarly, the results are shown in PTEN^fl/flA large number of fluorescent tracers were observed in the brain parenchyma of the mutant mice, with an increased permeability index (D and F in fig. 2).

In addition, immunostaining analysis of brain tissue sections showed that both tracers were leaked to PTEN^fl/flMutation in brain parenchyma of mice, while in PTEN^fl/+There was no leakage in littermates (G in figure 2). Postnatal PTEN knockdown using Cdh5-CreERT2 transgenic mice (given to Ralf h. adams laboratories, Wang YD et al, Nature,2010) also resulted in disruption of the blood brain barrier of mice, manifested as evans blue dye and leakage of N-hydroxysulfosuccinyl Biotin (Sulfo-NHS-Biotin) (21335, Thermo Fisher Scientific) in brain parenchyma (fig. 3). These data indicate that knockout of the cerebrovascular PTEN gene results in increased blood brain barrier permeability.

EXAMPLE II overexpression of NEDD4-2 in brain ECs leads to impaired blood brain barrier integrity

2.1 construction of NEDD4-2 overexpression vector pAAV-Myc-NEDD4-2 in brain ECs

In order to ectopically overexpress the gene of E3 ubiquitin ligase NEDD4-2 of brain Ecs, the invention adopts a modified brain endothelial specificity AAV-BR1(pAAV-EGFP) system to obtain a recombinant expression vector pAAV-Myc-NEDD4-2 to overexpress NEDD4-2 gene in brain endothelial cells, and simultaneously constructs an overexpression vector of tdTomato gene and tdTomato-2A-Mfsd2A gene: Myc-NEDD4-2 (SEQ ID No.3 in a sequence table), tdTomato and tdTomato-2A-Mfsd2A genes are cloned into pAAV-EGFP (containing a CAG promoter, an EGFP gene and an SV40 Poly-A sequence) (SEQ ID No.4 in the sequence table), and pAAV-Myc-NEDD4-2 plasmid and pAAV-tdTomato, pAAV-tdTomato-2A-Mfsd2A plasmid (SEQ ID No.5 in the sequence table) are respectively generated.

2.1.1 construction of pAAV-Myc-NEDD4-2 recombinant expression vector plasmid

Using primers 5'-ATGGCGACCGGGCTTGGG-3' and 5'-TTAATCCACACCTTCGAAGC-3', and using mouse cerebral vascular endothelial cell cDNA as a template for amplification to obtain a CDS sequence (shown as 31-2961 th nucleotide of SEQ ID No.3 in a sequence table) of E3 ubiquitin ligase NEDD 4-2; the CDS sequence encodes the amino acid sequence of NEDD4-2 protein NP-001107858.1 (SEQ ID No. 7).

The CDS sequence of NEDD4-2 shown in SEQ ID No.3 in the sequence table is cloned to the downstream of a CAG promoter sequence (DNA molecule shown in SEQ ID No.4 in the sequence table) in the pAAV-EGFP plasmid, and the recombinant pAAV-Myc-NEDD4-2 plasmid containing the CDS sequence of pAAV-EGFP is obtained through enzyme digestion glue running verification and sequencing confirmation. The pAAV-Myc-NEDD4-2 vector plasmid is a recombinant adeno-associated virus (rAAV) vector transfer plasmid, comprises two ITR sequences of AAV, and simultaneously has Amp resistance, a CAG promoter (SEQ ID No.4 in a sequence table), a CDS sequence of NEDD4-2 (SEQ ID No.3 in the sequence table) and an SV40 polyA sequence.

In order to knock out Caveolin1 in brain ECs, the invention constructs pAAV-CMV-EGFP-hU6-shRNA vector which comprises an hU6 promoter (SEQ ID No.6 in the sequence table) driving shRNA against Caveolin 1. The sequence of Caveolin1shRNA #1 was 5'-TGAAGCTATTGGCAAGATATT-3'.

2.2 obtaining of brain endothelial cells overexpressing NEDD4-2

2.2.1 packaging and characterization of pAAV-Myc-NEDD4-2 recombinant Virus

2.2.1.1 amplification extraction of plasmid:

the prepared recombinant pAAV-Myc-NEDD4-2 plasmid, helper plasmid pHelper plasmid and pAAV9-rep/cap plasmid (Cell Biolabs) are respectively transformed into Escherichia coli for amplification, and three plasmids are extracted to obtain plasmids with the concentration of more than 1 mug/mul.

Then, the HEK293T cells were revived and passaged.

2.2.1.2 packaging and concentration of recombinant adeno-associated virus pAAV-Myc-NEDD 4-2:

transfection of cells:

the pAAV-Myc-NEDD4-2 plasmid was co-transfected with the helper plasmid pHelper plasmid and pAAV9-rep/cap plasmid into HEK293T cells (10 cm cell culture dish as an example):

the first day: HEK293T cells with a degree of polymerization above 90% were treated as follows: 3 ratio carousel (about 2.5x 10 per disk)⁶) Culture medium Hyclone high sugar DMEM medium (containing 10% FBS);

the next day: changing to serum-free culture medium about 1-2h before plasmid transfer, and transferring the target gene plasmid and the auxiliary plasmid into HEK293T by using a transfection reagent;

and on the third day: after the plasmid is transformed for 24 hours, replacing a new serum-free culture medium;

the fifth day: and (5) performing transfection for 72h for virus recovery.

Collecting viruses:

blowing down cells with the culture medium, and centrifuging; the culture supernatant and the cell pellet were then harvested separately. And (3) precipitating the virus in the culture supernatant by using PEG8000, and obtaining the recombinant adeno-associated virus pAAV-Myc-NEDD4-2 after overnight precipitation.

2.2.1.3 purification and concentration of recombinant adeno-associated virus pAAV-Myc-NEDD 4-2:

1. solid CsCl was added to the virus concentrate until the final density was 1.41g/ml (refractive index 1.372).

2. The sample was added to an ultracentrifuge tube and the remaining volume of the tube was filled with a pre-prepared 1.41g/ml CsCl solution.

3. Centrifugation was carried out at 340000g for 12 hours, thereby forming a density gradient. Samples of different densities were collected sequentially in order. The fractions of the solution containing AAV particles were collected.

4. The purified virus was filled into a10 kDa dialysis bag and desalted overnight at 4 ℃ by dialysis. Thus obtaining purified pAAV-Myc-NEDD4-2 virus.

2.2.1.4 Titer assay

The titer of the virus is detected by a qPCR method, and the packaged recombinant adeno-associated virus pAAV-Myc-NEDD4-2 is obtained, wherein the number of virus particles is 10¹¹vg/. mu.L. The detection method comprises the following steps:

1) AAV viral coat is broken by adeno-associated virus treatment, incubated at 37 deg.C for 30min, and then heated at 95 deg.C for 5min to inactivate enzyme, as follows: 5ul of virus liquid; proteinase K (5ug/ul)1 ul; 4ul of ultrapure water.

2) Centrifuging at 12000rpm for 2min, and collecting supernatant.

3) Copy number of standard, 7 gradient dilution concentrations: 7.2X 10⁸vg/μL,7.2×10⁷vg/μL,7.2×10⁶vg/μL,7.2×10⁵vg/μL,7.2×10⁴vg/μL,7.2×10³vg/μL,7.2×10²vg/. mu.L, and ultrapure water as a negative control.

4) Preparing a PCR reaction system, wherein each sample is 20ul, and the system is as follows:

5) PCR reaction conditions

6) Viral particle number formula: number of virus particles (vg/mL) × 1000 relative to standard

2.2.2 recombinant AAV vector pAAV-Myc-NEDD4-2 injection mice

Recombinant AAV vectors were administered per mouse (including PTEN)^f1/flMouse and control PTEN^f1/+Mouse) 5X 10¹⁰Doses of gp were injected into the tail vein. In each experiment, at least 4 genes were used per groupA type mouse.

2.3 Permeability phenotype Observation of the blood brain Barrier

In agreement with the results of the in vitro observation in the above example I, the mouse NEDD4-2 showed PTEN by Western blot^f1/flp-NEDD4-2 was significantly increased in isolated brain endothelial cells in mice (A in FIG. 4).

Western blot analysis indicated that Myc-NEDD4-2 was successfully expressed (shown by p-Nedd4-2 in B in FIG. 4), and overexpression thereof resulted in a significant increase in p-NEDD4-2 at both Ser342 and Ser448 sites (shown by p-Nedd4-2(Ser342) and p-Nedd4-2(Ser448) in B in FIG. 4). Day P30 wild type mice (Witonglihua, cat # 219) were injected with AAV-Myc-NEDD4-2, and then material was drawn at P60 to observe blood brain barrier permeability.

Consistent with the phenotype of the in vitro results in example one, immunofluorescence staining showed that injection of pAAV-Myc-NEDD4-2 resulted in expression of IB 4-positive endothelial cell Myc-NEDD4-2, and in turn decreased expression of Mfsd2a, as Myc-expressing endothelial cell Mfsd2a levels were significantly decreased (indicated by the arrow in C in fig. 4). Furthermore, by intravenous injection of fluorescently labeled cadiverine-Alexa 555 tracer in mice, it was found that cadiverine-Alexa 555 tracer leaked into brain parenchyma after AAV-Myc-NEDD4-2 treatment (shown in E in FIG. 4), and overexpression of NEDD4-2 in brain ECs could mimic PTEN by increasing permeability of the blood brain barrier^fl/flMouse phenotype, a key role for NEDD4-2 in modulating BBB permeability in vivo was demonstrated (E in figure 4).

In addition, electron microscopy analysis showed that the number of vesicles in endothelial cells overexpressing NEDD4-2 was increased compared to control EGFP empty vector virus infected endothelial cells (G-H in fig. 4), further supporting that NEDD4-2-Mfsd2a is responsible for blood brain barrier leakage due to PTEN deletion.

Example III study on action mechanism of PTEN/Akt/NEDD4-2/Mfsd2a axis in PTEN/Akt Signal pathway

3.1 cerebrovascular knockout of PTEN gene to promote transcytosis

In order to solve the problem that how to destroy the integrity of a blood brain barrier after knocking out a PTEN gene from cerebrovascular endothelium, the invention researches the influence of the PTEN gene on the tight connection and transcytosis of endothelial cells. Electronic microscopeInspection and display, PTEN^fl/flAnd PTEN^fl/+Endothelial cells of littermates have the typical characteristics of capillary endothelial cells and are surrounded by astrocytes and pericytes. Consistent with the findings of the above examples, the brain endothelial cells of PTEN knockout mice exhibited normal levels of claudin expression and localization. No P60 detectable by electron microscopy in 8 PTEN^fl/flMouse and PTEN^fl/+The endothelial tight junction ultrastructure of the littermate mice showed any significant abnormalities (H and J in fig. 2). The tight junctions of the brain endothelial cells of littermate mice were normal, and the electron-dense linear structure showed "kissing points" (H in fig. 2) where adjacent membranes were tightly attached. However, electron microscopy showed a significant increase in the number of plasma membrane-associated and free cytoplasmic vesicles, lateral to and outside the lumen (H in figure 2), indicating PTEN^fl/flThe exocytosis rate of the mutant mice was increased. Of note, control group (PTEN)^fl/+) Mouse comparison, in PTEN^{fl /fl}In mice, more than a 2-fold increase in the number of vesicles was observed at different positions along the transcytosis pathway (I in fig. 2). Taken together, these findings suggest that, in PTEN^fl/flThe blood brain barrier leakage observed in mice may be due to increased endothelial cell transcytosis.

PTEN in a pre-laboratory study related to the present invention^fl/flAnd PTEN^fl/+RNA sequencing of mouse brain endothelial cells showed significant transmembrane protein dysregulation in PTEN knockout endothelial cells (Wang et al, 2019). Among these proteins, Mfsd2a was observed in PTEN^fl/flExpression was down-regulated in brain endothelial cells of mutant mice, and Mfsd2a has been shown to modulate blood brain barrier permeability by inhibiting transcytosis. Consistent with this finding, immunofluorescent staining showed that Mfsd2a had characteristic expression in brain endothelial cells in P60 mice, but that Mfsd2a expression was reduced by 80% in PTEN-deficient endothelial cells relative to control endothelial cells (L-M in fig. 2). This result suggests that a decrease in Mfsd2a may be associated with increased transcytosis due to PTEN knockdown.

3.2 reduction of Mfsd2a results in increased pit-mediated transcytosis, leading to blood brain barrier abnormalities in PTEN knockout mice

To confirm PTEN^fl/flThe invention relates to the relation between reduction of Mfsd2A and blood brain barrier abnormality in a mutant mouse, and the invention uses AAV-tdTomato-2A-HA-Mfsd2A (AAV-Mfsd 2A for short) of adeno-associated virus constructed in the two steps 2.1 of the example to over-express Mfsd2A (A in figure 5) in brain endothelial cells by a brain EC-specific AAV-BR1 system. Western blot experiments confirmed that AAV-Mfsd2a plasmid transfected human embryonic kidney 293T (HEK293T) cells successfully overexpressed Mfsd2a (FIG. 5, B). Immunofluorescent staining analysis showed that AAV-BR1 vector-mediated tdTomato and HA were specifically expressed in isonectin B4(IB4) positive endothelial cells after AAV-Mfsd2a virus treatment, indicating that Mfsd2a was successfully overexpressed in brain endothelial cells (C in fig. 5). Treatment of PTEN from P30 with AAV-Mfsd2a or mock Virus^fl/flAnd PTEN^fl/+Mouse, brain tissue was harvested at P60 (D in FIG. 5). Mice were injected with thiosulfamate biotin (Sulfo-NHS-biotin) to assess blood brain barrier permeability. Immunofluorescence analysis showed that injection of AAV-Mfsd2a not only successfully restored PTEN^fl/flThe reduction of Mfsd2a expression in the mutant mouse endothelial cells also rescued the BBB permeability-increasing phenotype (D and E in fig. 5). Virus-processed PTEN at HA-Mfsd2a^fl/flIn mutant mice, Sulfo-NHS-biotin was only observable in cerebral vessels, while PTEN treated with control AAV-BR1 virus^fl/flThe mutant mice still exhibited a BBB leaky phenotype (E in fig. 5), indicating that Mfsd2a is regulating PTEN^fl/flThe BBB permeability of mutant mice plays a key role.

In addition, EM analysis and corresponding quantitative data showed that over-expression of Mfsd2a enabled PTEN^fl/flVesicle number in endothelial cells of mutant mice was reduced to PTEN^fl/+Comparable levels in mutant mice (F and G in fig. 5), supporting that the reduction of Mfsd2a is responsible for blood brain barrier leakage due to PTEN knockout.

Since previous studies indicate that Mfsd2a in blood-brain barrier can inhibit cell-mediated transcytosis (Andreone et al, 2017), it is possible to interfere with cell-mediated transcytosis by inhibiting caveolin-1 (Cav-1), and it is also possible to rescue blood-brain barrier leakage caused by PTEN knockoutConsistent with the phenotype caused by overexpression of Mfsd2 a. To test this hypothesis, the present invention used a modified brain EC-specific AAV-BR1 system to knock-out Cav-1(AAV-EGFP-U6-shCav-1, hereinafter referred to as AAV-shCav-1) (A in FIG. 6). AAV-shCav-1 plasmid was transfected into bEnd.3cells, which effectively reduced the expression of Cav-1, confirming the successful construction of AAV-shCav-1 plasmid (B in FIG. 6). As expected, the expression of Cav-1 in brain endothelial cells of PTENf1/f1 mice was significantly reduced after treatment with AAV-shCav-1 virus (C in FIG. 6). PTEN^fl/flAnd PTEN^{fl /+}Mice were treated with AAV-shCav-1 or Mock virus at P30 and were drawn at P60 (D in FIG. 6), noting that AAV-shCav-1 treatment effectively blocked PTEN compared to MOCK group mice^fl/flEvans blue leakage in the brain parenchyma of the mutant mice (a in fig. 7 and B in 7). By further examining the localization of Sulfo-NHS-Biotin in the brain, we observed that the blood brain barrier of AAV-shCav-1 virus treated group restored integrity as the tracer was confined to the cerebral vasculature (E in fig. 6), accompanied by a significant decrease in its permeability index compared to control treated group (F in fig. 6). In addition, EM analysis demonstrated that AAV-shCav-1 treatment significantly reduced PTEN^fl/flVesicle density in mouse endothelial cells (G in fig. 6 and H in 6). These data indicate that PTEN deletion leads to leakage of the blood brain barrier by reducing Mfsd2a expression, which in turn leads to increased pit-mediated transcytosis.

3.3 Polyubiquitination of the PTEN/AKT pathway through NEDD4-2 regulates Mfsd2a protein degradation

After confirming that the decrease in Mfsd2a is responsible for blood brain barrier leakage due to PTEN loss, the present inventors next attempted to investigate the underlying mechanism by which PTEN regulates the expression of Mfsd2 a. Since the serine/threonine kinase Akt/PKB is the main downstream target of PTEN, we activated Akt1 signaling pathway in vitro with IGF-1, mimicking the case of PTEN deletion. We constructed a stable HEK293T cell line overexpressing MFSD2A (HEK293T-MFSD2A) to test the possibility of PTEN/AKT1 modulating MFSD2A protein levels (a in fig. 7). Notably, IGF-1 treatment resulted in an increase in phosphorylated AKT1(p-AKT1) and a decrease in Mfsd2a protein expression (a in fig. 7), demonstrating that PTEN/AKT1 can affect expression of Mfsd2A protein. Furthermore, proteasome inhibitor MG132 effectively prevented IGF-1-induced MFSD2A protein decline (a in fig. 7), suggesting that the ubiquitin/proteasome degradation pathway plays a key role in PTEN/AKT 1-induced MFSD2A protein decline. Furthermore, treatment with proteasome inhibitor MG132 effectively prevented IGF-1-induced reduction of MFSD2A (a in fig. 7), suggesting that the ubiquitin/proteasome degradation pathway plays a key role in PTEN/AKT 1-induced reduction of MFSD2A protein expression.

The mechanism that regulates degradation of Mfsd2a ubiquitin/proteasome is not clear. To find the ubiquitination ligase that causes Mfsd2a ubiquitination, the present invention uses UbiBrowser to predict the proteome-wide human E3 substrate network (http:// ubibrowser.ncpsb.org). The predicted E3 ligase was obtained by UbiBrowser using MFSD2A as a substrate. Among the predicted E3 ligases, NEDD4-2 achieved a relatively high likelihood ratio (LR: 6.52), indicating that NEDD4-2 is likely to be a potential regulator of Mfsd2 a.

In addition, the present invention observed that MFSD2A and NEDD4-2 had the same localization in HEK293T cells transfected with both MFSD2A-EGFP and NEDD4-2-Myc plasmids (B in FIG. 7). Whereas NEDD4-2 has been shown to act downstream of the insulin/AKT 1 signaling pathway, directly interacting with its substrate epithelial sodium channel (ENaC) and promoting its ubiquitination (Manning et al, 2018), the present inventors investigated whether NEDD4-2 is involved in degradation of Mfsd2a by the PTEN/AKT1 signaling pathway. Experimental results found that the expression level of phosphorylated NEDD4-2(p-NEDD4-2) at Ser342 and Ser448 residues was significantly increased and the level of MFSD2A protein was decreased in a time-dependent manner after IGF-1 treatment in HEK293T-MFSD2A cell line (C in fig. 7). In contrast, p-AKT1 and p-NEDD4-2 levels were significantly reduced and Mfsd2a protein levels were increased and dose-dependent after inhibition of the AKT1 signaling pathway by PI3K signaling pathway inhibitor LY294002 (C in fig. 7).

To further demonstrate the role of NEDD4-2 in MFSD2A regulation, the present invention uses siRNAs targeting NEDD4-2 to knock down their expression. Notably, NEDD4-2 gene knockdown significantly increased protein levels of MFSD2A in HEK293T-MFSD2A cells (D in fig. 7). Furthermore, NEDD4-2 siRNA treatment prevented IGF-1-induced degradation of MFSD2A protein (D in FIG. 7), indicating that NEDD4-2 is involved in IGF-1 regulated degradation of Mfsd2 a.

Taken together, these results reveal a post-transcriptional regulatory mechanism by which PTEN/AKT1 signals regulate the stability of Mfsd2a through NEDD 4-2-induced ubiquitination.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

<110> military medical research institute of military science institute of people's liberation force of China

<120> method for regulating blood brain barrier permeability and application thereof

<130> GNCSQ210911

<160> 7

<170> PatentIn version 3.5

<210> 1

<211> 4824

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

agtccatttg ttacccacct cctggtctac cctcccacag ttcctcactc ctcttgccct 60

cctgatgtcc ctcccctcgc tgccatcccc cttcccctgc caggcctccc cattccctgg 120

ggcctcacgt ctctcaaggg atagacatgt cttctctcac tgaggccaga tcaggcagtc 180

ctttgcttta tatgtgtcgg gtgggggtgg gggtgggatc cccatgcttt ttaatttaat 240

cttgcttctt tatctttttc ttttccctga tctcccttta tttaatcttc ttgttattat 300

tcccagtatt tttaatccat tttcaacgtt attatctcct ttctgcttct ttcctatggc 360

cgttggtaat agagttatga cagagctata gttttggttg ttctgatgtt ttgctttctt 420

ttctgaatgg cagtgcatct tcaccaggac ctcggaaagg ctttcatttt gtgctgctgg 480

ccacaagtgt taatttagaa tagagggtga ccttgacaag agagagattc ccttcccaca 540

cacttgggag tttgccttct gtggttctgt gccaccctca agggttctaa gtgctcttct 600

tgttaagtgc tctgaaggaa cctgagctgt gtacacacga tggctgcttc ctgtccggcc 660

ctcctttgtg ataggggctc tgagggtggg gaaagctgtc agcttacata tctataaagg 720

atgccgtcca cttgtccact ttgactcgga ggcagacatc cacacagctt gcaggctctg 780

tgtgcgggtg agcttagcca ccagtccctg ggtatccacc agtgtatggg tttctggcag 840

taagagccac acactaattg agggctgact gggtagaaag gtactgcgta cgactaacag 900

tttagcagga ctacgtaccc ttctcgtttc tgtttatctg agaacccaaa acagaataac 960

ttaaaatatt gagggacagg tagagtaacc tggtgactgt acatctccta acagacaacc 1020

ctgttaagat tgtctacaga gcacagcagg attagctaag gtcagagagg tacagtgagt 1080

tccctggggt gacacagcta agttataata gataagaatc tcctgaggac tcaggcctct 1140

gactcacctg agctctcctc ctctattcag ggatctgaag ttggaggcct gcagtgtgat 1200

tgggtaagac accagagatg ctggggattt ctgactgggg atagtttagg tcttcttagt 1260

attctcggct gtacctgcct gtcatgttct tctgtgttcc tgggtcctgc aaagtgtagc 1320

aggaatgggg gtgtcaaagg agagctatgt taatgctaca gattggctgg tggcccttct 1380

gtcctgcagg agaaaccact ggtacagtag ccatatgggc ccaaagaaga agagaaaggt 1440

ttcgaattta ctgaccgtac accaaaattt gcctgcatta ccggtcgatg caacgagtga 1500

tgaggttcgc aagaacctga tggacatgtt cagggatcgc caggcgtttt ctgagcatac 1560

ctggaaaatg cttctgtccg tttgccggtc gtgggcggca tggtgcaagt tgaataaccg 1620

gaaatggttt cccgcagaac ctgaagatgt tcgcgattat cttctatatc ttcaggcgcg 1680

cggtctggca gtaaaaacta tccagcaaca tttgggccag ctaaacatgc ttcatcgtcg 1740

gtccgggctg ccacgaccaa gtgacagcaa tgctgtttca ctggttatgc ggcggatccg 1800

aaaagaaaac gttgatgccg gtgaacgtgc aaaacaggct ctagcgttcg aacgcactga 1860

tttcgaccag gttcgttcac tcatggaaaa tagcgatcgc tgccaggata tacgtaatct 1920

ggcatttctg gggattgctt ataacaccct gttacgtata gccgaaattg ccaggatcag 1980

ggttaaagat atctcacgta ctgacggtgg gagaatgtta atccatattg gcagaacgaa 2040

aacgctggtt agcaccgcag gtgtagagaa ggcacttagc ctgggggtaa ctaaactggt 2100

cgagcgatgg atttccgtct ctggtgtagc tgatgatccg aataactacc tgttttgccg 2160

ggtcagaaaa aatggtgttg ccgcgccatc tgccaccagc cagctatcaa ctcgcgccct 2220

ggaagggatt tttgaagcaa ctcatcgatt gatttacggc gctaaggatg actctggtca 2280

gagatacctg gcctggtctg gacacagtgc ccgtgtcgga gccgcgcgag atatggcccg 2340

cgctggagtt tcaataccgg agatcatgca agctggtggc tggaccaatg taaatattgt 2400

catgaactat atccgtaacc tggatagtga aacaggggca atggtgcgcc tgctggaaga 2460

tggcgattag ccattaacgc ggcgtggtac ctctagagtc gacccgggcg gcctcgagag 2520

atctacgggt ggcatccctg tgacccctcc ccagtgcctc tcctggccct ggaagttgcc 2580

actccagtgc ccaccagcct tgtcctaacc attaacgcgc gaccagcttg atatcgaatt 2640

cctgcagccc gggggatcca ctagtccgat cccaaggccc aactccccga accactcagg 2700

gtcctgtgga cagctcacct agctgcaatg gctacaggta agcgccccta aaatcccttt 2760

gggcacaatg tgtcctgagg ggagaggcag cgacctgtag atgggacggg ggcactaacc 2820

ctcaggtttg gggcttctga atgtgagtat cgccatgtaa gcccagtatt tggccaatct 2880

cagaaagctc ctggtccctg gagggatgga gagagaaaaa caaacagctc ctggagcagg 2940

gagagtgctg gcctcttgct ctccggctcc ctctgttgcc ctctggtttc tccccaggct 3000

cccggacgtc cctgctcctg gcttttggcc tgctctgcct gccctggctt caagagggca 3060

gtgccttccc aaccattccc ttatccaggc tttttgacaa cgctatgctc cgcgcccatc 3120

gtctgcacca gctggccttt gacacctacc aggagtttgt aagctcttgg ggaatgggtg 3180

cgcatcaggg gtggcaggaa ggggtgactt tcccccgctg ggaaataaga ggaggagact 3240

aaggagctca gggtttttcc cgaagcgaaa atgcaggcag atgagcacac gctgagtgag 3300

gttcccagaa aagtaacaat gggagctggt ctccagcgta gaccttggtg ggcggtcctt 3360

ctcctaggaa gaagcctata tcccaaagga acagaagtat tcattcctgc agaaccccca 3420

gacctccctc tgtttctcag agtctattcc gacaccctcc aacagggagg aaacacaaca 3480

gaaatccgtg agtggatgcc ttctccccag gcggggatgg gggagacctg tagtcagagc 3540

ccccgggcag cacagccaat gcccgtcctt cccctgcaga acctagagct gctccgcatc 3600

tccctgctgc tcatccagtc gtggctggag cccgtgcagt tcctcaggag tgtcttcgcc 3660

aacagcctgg tgtacggcgc ctctgacagc aacgtctatg acctcctaaa ggacctagag 3720

gaaggcatcc aaacgctgat gggggtgagg gtggcgccag gggtccccaa tcctggagcc 3780

ccactgactt tgagagctgt gttagagaaa cactgctgcc ctctttttag cagtcaggcc 3840

ctgacccaag agaactcacc ttattcttca tttcccctcg tgaatcctcc aggcctttct 3900

ctacaccctg aaggggaggg aggaaaatga atgaatgaga aagggaggga acagtaccca 3960

agcgcttggc ctctccttct cttccttcac tttgcagagg ctggaagatg gcagcccccg 4020

gactgggcag atcttcaagc agacctacag caagttcgac acaaactcac acaacgatga 4080

cgcactactc aagaactacg ggctgctcta ctgcttcagg aaggacatgg acaaggtcga 4140

gacattcctg cgcatcgtgc agtgccgctc tgtggagggc agctgtggct tctagctgcc 4200

cgggtggcat ccctgtgacc cctccccagt gcctctcctg gccctggaag ttgccactcc 4260

agtgcccacc agccttgtcc taataaaatt aagttgcatc attttgtctg actaggtgtc 4320

cttctataat attatggggt ggaggggggt ggtatggagc aaggggcaag ttgggaagac 4380

aacctgtagg gcctgcgggg tctattggga accaagctgg agtgcagtgg cacaatcttg 4440

gctcactgca atctccgcct cctgggttca agcgattctc ctgcctcagc ctcccgagtt 4500

gttgggattc caggcatgca tgaccaggct cagctaattt ttgttttttt ggtagagacg 4560

gggtttcacc atattggcca ggctggtctc caactcctaa tctcaggtga tctacccacc 4620

ttggcctccc aaattgctgg gattacaggc gtgaaccact gctcccttcc ctgtccttct 4680

gattttaaaa taactatacc agcaggagga cgtccagaca cagcataggc tacctggcca 4740

tgcccaaccg gtgggacatt tgagttgctt gcttggcact gtcctctcat gcgttgggtc 4800

cactcagtag atgcctgttg aatt 4824

<210> 2

<211> 2336

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

tattaaacca aacttttcag agtgtttgtt ccatctttct ctggggtggg accctccctt 60

cccctcctct ccccttccct gcatcacctc cgcaggcaat tgggatccct gaccctagac 120

cagaaagtgt ggcaaactga aaaatctgac ttgtaggaca ctaacaaccg gcttcttagg 180

gtatgtgcct agcttcctct tgtttcctga ttgtatcctt aattcttgac tgtcttccac 240

tgtgggctct tcaccacaca gcacctctca gaagagcaga acctggcttc cctgtgtgga 300

gttctaacac ttggaggtgg agggagaagg gaattcagag ccagtcttgg gtatatgaga 360

tcctgactca aggaaaacca aagaggaagg gaggaaagag aatatagaat atgtgatctt 420

ttgtatatgt gtcagttttc ttcttcctat ctcattttta ggtaagcaga catttagcag 480

agtatttagc aaggatgcat acgtcatcta ataaattttc tcttttcaaa aacagtacat 540

caggtaatac actaaaagaa aaacacatgt gtgtgtccgt gtctgtgtgt gtgtgtgtgt 600

gtgtgtgtgt gtgtgaatac agaagttaat tcccctcagg tctgctccat tgggctgtag 660

tttatggata atttgttcaa tctttgtgtg aactgggttt tgaaatacag ttgagttgta 720

caaattccag atgcccagtg caggcccaca gctatttatt tggaagtctt ggatcagttt 780

tattttggta catagaaaat ttcagttttc aaaaaactaa aaaactaaat aaaacaagaa 840

aatccatatc ttttgtgtta ctctagtatc cactgtggta gactagtcgg tactcagcag 900

gtatgttggt tgaacaacct cagattgggt cctgttcgag ttgagattac ctatttataa 960

ctttggagtt tgagatttgg gctaaggaat aatggaactt tgttttaaaa cactaacttt 1020

tatttttcag taatttcttt ttgtttgttt gtttgttttt tttgagacag ggtcttgtat 1080

cccaggctgg cctaggactc actagatagc aaaggctaat cttaaagata taatctttcc 1140

cagtaactct tctgaagtgc taggattaca gcctgtggta acactcctag cttatttgaa 1200

taatgcttaa gtgtctgatt tccttagtag ttggagtcac caggatgctt ctgaccccac 1260

taatatgtag gatacccttc atagtatcac tgattagtgt tattattgaa aagctaagtg 1320

tttgtcttaa tgtgtcagta ttttactatc agtgggtttt agttatttta ttgtgatctg 1380

gtattaaatt ttgtactctg agagattatt ggaaatgaga tttgtatata aaagagtaaa 1440

ggtctggctt acaattttta gtaagcattg tgttaataat taaattagta tcattcagtt 1500

gtcttttaca tttcctttgt tctttttctt tatttttaac atgtatgttt taagtaatgg 1560

tttaagattg tatgtgatca tctgtcaggt aaagataata gtaagagtag ctatttattc 1620

ataggtattt gtgaaataaa aaatacattc taaagccatg tatagtcttt atccaagaaa 1680

ttacagggtc agtgcagttg aatttacagt gttgcatgtt gatgtcacaa attctgtgaa 1740

caaatatatg cacacaaatt gcatgcatgc gtttaacttt tattaaagct ttggtctcct 1800

taattataag aatgataata gtacctactt cagaattctt gaagttaacg gaaatagtga 1860

ctgtaaaaac acttagcgca gtgtttttac atgatagaaa aggtggtatg atagaaaggg 1920

tggataaata ttgctaatat tgatactctt ccttccagtg tgaaaggtaa ctttatgcca 1980

catttaaact ttcttgtaga tataacttcg tataatgtat gctatacgaa gttatgtaag 2040

ctgtttactt tttccttcct ccctctttgt ggaccaagaa tttattggga aacaggtttt 2100

ctccctcttg ctttattgag gtataaccaa caaagtctta atctacttac agtgtgatgc 2160

tttgagaact gttatattgt ggttgtatcc acttagtgta tccctcatcc ctggtatccc 2220

caccctcttc cttagctgta ctgagaacat ccaagaccta cctggagtag gtgctaggca 2280

cacagtatgg attttgatga caacttgaat gccattacct agtaaagcaa ggtatt 2336

<210> 3

<211> 2961

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

cagatcctct tctgagatga gtttttgttc atggcgaccg ggcttgggga gcctgtctac 60

ggactttcgg aagaggaggg agaatcccgt atcctcagag taaaagttgt ttctggaatt 120

gacctcgcca agaaggacat atttggagcc agcgacccat acgtgaagct gtccttgtat 180

gtagctgatg agaatagaga acttgctctg gttcaaacca agacaattaa aaagacgctg 240

aacccaaagt ggaatgaaga gttttatttt agggtaaatc catctaatca caggctccta 300

tttgaagtat ttgacgagaa cagattgaca cgagacgact tcctgggcca ggtggacgtg 360

cctcttagtc accttccgac agaagatcca accatggaga gaccctatac atttaaggat 420

tttctcctgc gacctagaag tcataaatct cgagtcaagg ggtttttgag gttgaaaatg 480

gcctacatgc cgaaaaacgg aggtcaggat gaagaaaaca gcgagcagag ggatgacatg 540

gagcatggat gggaagtggt tgactccaac gactcagctt cccagcacca ggaggagctc 600

ccccctcctc ccctgccgcc aggatgggaa gagaaagtgg acaatttagg ccgaacttac 660

tatgtcaacc acaacaacag gagtactcag tggcaccgac ccagcttgat ggatgtgtcc 720

tcggaatcag acaataacat caggcagatc aaccaggagg ctgcacaccg acgcttccgc 780

tctcggaggc acattagtga agatttggag cctgaggcct ctgaaggcgg tggagaaggc 840

cctgagcctt gggagaccat ttcagaggaa atgaacatgg caggagattc tctcagcctg 900

gctctgcccc caccgcctgc ctccccagtg tcccggacca gcccccagga gctgtcggaa 960

gaagtgagcc gaaggttgca gatcactccg gactccaacg gggaacagtt cagttctctg 1020

attcagagag agccctcgtc aaggcttcgg tcctgcagtg ttaccgacac ggttgctgag 1080

caagctcacc ttccaccgcc cagcacccca actaggcgag cccgttcgtc aactgtcacg 1140

ggtggtgagg aatccacgcc atcagtggcc tatgtacata ccacgccggg cctgccttca 1200

ggctgggaag aaagaaagga tgcaaaggga cgcacatact atgtcaatca taacaatcga 1260

accacaactt ggactcggcc aatcatgcag cttgcagaag acggtgcctc cggatcagcc 1320

acaaacagta acaaccacct agtcgaaccc cagatccgcc ggccccgtag cctcagctcg 1380

ccaacagtaa ctttatctgc cccactggag ggtgccaagg attcacccat ccgccgtgcc 1440

gtgaaagata ccctttccaa tccacagtcc cctcagccat caccttacaa ctcccccaaa 1500

ccacaacaca aagtcacaca gagcttcctg ccaccaggct gggagatgag gatagccccc 1560

aacggccgac ccttcttcat tgaccataac acaaagacta caacctggga agatccacgg 1620

ttgaaatttc cagtacacat gcggtcaaaa gcatctttaa accccaatga cctgggccct 1680

cttcctcctg gctgggaaga gaggatccac ttggatggcc gcacgtttta cattgaccat 1740

aatagtaaaa ttacccagtg ggaagatcca agactacaga acccagccat cactggtccg 1800

gctgttccgt actccagaga gtttaagcag aaatacgact actttaggaa gaaattaaag 1860

aagcctgctg atattccaaa caggtttgaa atgaaacttc acagaaacaa catatttgaa 1920

gagtcctatc ggaggatcat gtctgtaaag agacctgacg tcctaaaggc taggctgtgg 1980

attgagtttg aatcagagaa aggcctggac tatgggggcg tggccagaga atggttcttc 2040

ttactgtcca aagagatgtt taacccctac tatggcctct tcgagtactc tgccacggac 2100

aactacacac ttcagatcaa tcccaactca ggcctctgta atgaagacca tttgtcctat 2160

ttcaccttca ttggaagagt tgctggccta gcggtgtttc atgggaaact cttagatgga 2220

ttcttcattc gaccattcta caagatgatg ctggggaagc agataacgct gaacgacatg 2280

gagtccgtgg acagcgagta ctacaactct ttgaagtgga tcttagaaaa cgaccccacg 2340

gaacttgacc tcatgttctg catagacgaa gagaactttg ggcagacata ccaagtggat 2400

ctgaagccca acgggtcaga aataatggta accaatgaga acaaacgaga atacattgac 2460

ttagtcatcc agtggagatt tgtgaacagg gtccagaagc aaatgaatgc cttcttggag 2520

ggatttacag aacttcttcc aatcgacttg attaaaattt ttgatgaaaa tgagctggag 2580

ttgctgatgt gcggccttgg tgatgtcgac gtgaacgact ggagacagca ctctatttac 2640

aagaacggct actgccccaa ccaccctgtc atccagtggt tctggaaggc cgtgctcctg 2700

atggatgctg agaagcgcat ccggttacta cagtttgtca caggcacctc cagagtaccc 2760

atgaatggat ttgccgaact ctatggttcc aatggtcctc agctgtttac aatagagcaa 2820

tggggcagtc ccgaaaaact acccagagct catacatgct ttaatcgcct tgatttacct 2880

ccatatgaaa cctttgaaga tttacgggag aaacttctca tggctgtgga aaacgctcaa 2940

ggcttcgaag gtgtggatta a 2961

<210> 4

<211> 2193

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

catggtcgag gtgagcccca cgttctgctt cactctcccc atctcccccc cctccccacc 60

cccaattttg tatttattta ttttttaatt attttgtgca gcgatggggg cggggggggg 120

ggggggggcg cgccgggggg gggggggggg gggggggggg gggggggggg gcggagaggt 180

gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc cttttatggc gaggcggcgg 240

cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg gagtcgctgc gcgctgcctt 300

cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc cccggctctg actgaccgcg 360

ttactcccac aggtgagcgg gcgggacggc ccttctcctc cgggctgtaa ttagcgcttg 420

gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa gccttgaggg gctccgggag 480

ggccctttgt gcggggggag cggctcgggg ctgtccgcgg ggggacggct gccttcgggg 540

gggacggggc agggcggggt tcggcttctg gcgtgtgacc ggcggctcta gagcctctgc 600

taaccatgtt catgccttct tctttttcct acagctcctg ggcaacgtgc tggttattgt 660

gctgtctcat cattttggca aagaattgga tccgccacca tggtgagcaa gggcgaggag 720

ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag 780

ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc 840

atttgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac 900

ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc 960

gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac 1020

aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag 1080

ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac 1140

agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag 1200

atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc 1260

cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc 1320

ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc 1380

gccgggatca ctctcggcat ggacgagctg tacaagtaag aattcgatat caagcttatc 1440

gataatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt 1500

gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc 1560

cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag 1620

ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc 1680

actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc 1740

cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg 1800

ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg 1860

ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc 1920

ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt 1980

cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcatcgatac 2040

cgtcgacccg ggcggccgct tcgagcagac atgataagat acattgatga gtttggacaa 2100

accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct 2160

ttatttgtaa ccattataag ctgcaataaa caa 2193

<210> 5

<211> 1604

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atggccaaag gagaaggcgc cgagagcggt tccgcggcgg ggctgctccc caccagcatc 60

ctccaagcca gtgaacggcc ggtccaggtg aagaaggaac caaaaaagaa gcagcaactg 120

tccatttgca acaagctttg ctatgcagtt ggaggggccc cgtaccagtt gaccggctgc 180

gcactgggat tcttcctgca gatctaccta ttggatgtgg ctaaggtgga accacttcct 240

gcttccatta tcctttttgt gggccgagcc tgggatgcct tcactgaccc tctggtgggc 300

ttctgcatta gcaagtcctc ctggacccgc ctgggccgcc tcatgccctg gatcatcttc 360

tccactcccc tggccatcat tgcttacttc ctcatctggt ttgtgcctga cttcccatca 420

gggacggaaa gttcacacgg cttcctttgg tacctgcttt tctattgcct ctttgagaca 480

ctggtcacgt gctttcatgt tccctactca gcgctcacca tgttcatcag cacggagcag 540

agtgagcgtg actcagccac ggcatacaga atgactgtgg aggtgctggg cacagtgata 600

ggcacagcga ttcaaggaca aattgtgggc caagccaagg caccttgtct ccaggaccag 660

aatggctctg tggtggtctc agaagttgcc aatcgcaccc agagtactgc ctccctcaaa 720

gacacgcaaa atgcttacct gctggcagca gggatcatcg cctccatcta cgtcctctgt 780

gccttcattc tgatcctagg cgtgcgggag cagagagaac tctacgagtc ccagcaggct 840

gagtcaatgc ccttctttca gggcctccgg ctggtcatgg gtcatggccc ctatgtcaag 900

ctcattgccg gcttcctttt tacctccctg gctttcatgc tggtggaggg taactttgcc 960

ttgttctgca cctatacctt ggacttccga aatgagttcc agaacctcct cctggccatc 1020

atgctctcgg ccacattcac catccctatc tggcagtggt tcctaacccg gtttggcaag 1080

aagacagctg tatacatcgg gatctcttct gcagttcctt ttctcatctt ggtggccctc 1140

atggagcgta atctaatcgt cacttacgtg gtggccgtag cagctggcgt cagtgtagca 1200

gctgccttcc tactaccatg gtccatgctg cctgacgtta tcgatgactt ccacctgaaa 1260

caccctcact cccctggcac cgagcccata ttcttctcct tctatgtctt cttcaccaag 1320

tttgcctctg gagtctcact gggtgtctct accctcagtc tcgactttgc caactaccag 1380

aggcagggat gctcccagcc agaacaggtc aagtttacac tgaagatgct ggtgaccatg 1440

gctcctatca tcctcatctt gctgggcctg ctgctcttca agctctaccc cattgatgag 1500

gagaagcggc gacagaataa gaaagctctg caggctctac gagaagaagc cagcagctca 1560

ggttgctcgg atacagactc cacagagctg gccagtattc tcag 1604

<210> 6

<211> 1692

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg 60

acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa 120

tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca 180

agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac 240

atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc 300

atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga 360

tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg 420

gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta 480

cggtgggagg tctatataag cagagctggt ttagtgaacc gtcagatcct gcagaagttg 540

gtcgtgaggc actgggcagg taagtatcaa ggttacaaga caggtttaag gagaccaata 600

gaaactgggc ttgtcgagac agagaagact cttgcgtttc tgataggcac ctattggtct 660

tactgacatc cactttgcct ttctctccac aggtgtccag gcggccgcca tggtgagcaa 720

gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa 780

cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac 840

cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac 900

cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt 960

cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga 1020

cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat 1080

cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta 1140

caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt 1200

gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca 1260

gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac 1320

ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt 1380

cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaaa gggcctattt 1440

cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat aattagaatt 1500

aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt 1560

cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta 1620

acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg atgaagctat 1680

tggcaagata tt 1692

<210> 7

<211> 976

<212> PRT

<213> mouse (Mus musculus)

<400> 7

Met Ala Thr Gly Leu Gly Glu Pro Val Tyr Gly Leu Ser Glu Glu Glu

1 5 10 15

Gly Glu Ser Arg Ile Leu Arg Val Lys Val Val Ser Gly Ile Asp Leu

20 25 30

Ala Lys Lys Asp Ile Phe Gly Ala Ser Asp Pro Tyr Val Lys Leu Ser

35 40 45

Leu Tyr Val Ala Asp Glu Asn Arg Glu Leu Ala Leu Val Gln Thr Lys

50 55 60

Thr Ile Lys Lys Thr Leu Asn Pro Lys Trp Asn Glu Glu Phe Tyr Phe

65 70 75 80

Arg Val Asn Pro Ser Asn His Arg Leu Leu Phe Glu Val Phe Asp Glu

85 90 95

Asn Arg Leu Thr Arg Asp Asp Phe Leu Gly Gln Val Asp Val Pro Leu

100 105 110

Ser His Leu Pro Thr Glu Asp Pro Thr Met Glu Arg Pro Tyr Thr Phe

115 120 125

Lys Asp Phe Leu Leu Arg Pro Arg Ser His Lys Ser Arg Val Lys Gly

130 135 140

Phe Leu Arg Leu Lys Met Ala Tyr Met Pro Lys Asn Gly Gly Gln Asp

145 150 155 160

Glu Glu Asn Ser Glu Gln Arg Asp Asp Met Glu His Gly Trp Glu Val

165 170 175

Val Asp Ser Asn Asp Ser Ala Ser Gln His Gln Glu Glu Leu Pro Pro

180 185 190

Pro Pro Leu Pro Pro Gly Trp Glu Glu Lys Val Asp Asn Leu Gly Arg

195 200 205

Thr Tyr Tyr Val Asn His Asn Asn Arg Ser Thr Gln Trp His Arg Pro

210 215 220

Ser Leu Met Asp Val Ser Ser Glu Ser Asp Asn Asn Ile Arg Gln Ile

225 230 235 240

Asn Gln Glu Ala Ala His Arg Arg Phe Arg Ser Arg Arg His Ile Ser

245 250 255

Glu Asp Leu Glu Pro Glu Ala Ser Glu Gly Gly Gly Glu Gly Pro Glu

260 265 270

Pro Trp Glu Thr Ile Ser Glu Glu Met Asn Met Ala Gly Asp Ser Leu

275 280 285

Ser Leu Ala Leu Pro Pro Pro Pro Ala Ser Pro Val Ser Arg Thr Ser

290 295 300

Pro Gln Glu Leu Ser Glu Glu Val Ser Arg Arg Leu Gln Ile Thr Pro

305 310 315 320

Asp Ser Asn Gly Glu Gln Phe Ser Ser Leu Ile Gln Arg Glu Pro Ser

325 330 335

Ser Arg Leu Arg Ser Cys Ser Val Thr Asp Thr Val Ala Glu Gln Ala

340 345 350

His Leu Pro Pro Pro Ser Thr Pro Thr Arg Arg Ala Arg Ser Ser Thr

355 360 365

Val Thr Gly Gly Glu Glu Ser Thr Pro Ser Val Ala Tyr Val His Thr

370 375 380

Thr Pro Gly Leu Pro Ser Gly Trp Glu Glu Arg Lys Asp Ala Lys Gly

385 390 395 400

Arg Thr Tyr Tyr Val Asn His Asn Asn Arg Thr Thr Thr Trp Thr Arg

405 410 415

Pro Ile Met Gln Leu Ala Glu Asp Gly Ala Ser Gly Ser Ala Thr Asn

420 425 430

Ser Asn Asn His Leu Val Glu Pro Gln Ile Arg Arg Pro Arg Ser Leu

435 440 445

Ser Ser Pro Thr Val Thr Leu Ser Ala Pro Leu Glu Gly Ala Lys Asp

450 455 460

Ser Pro Ile Arg Arg Ala Val Lys Asp Thr Leu Ser Asn Pro Gln Ser

465 470 475 480

Pro Gln Pro Ser Pro Tyr Asn Ser Pro Lys Pro Gln His Lys Val Thr

485 490 495

Gln Ser Phe Leu Pro Pro Gly Trp Glu Met Arg Ile Ala Pro Asn Gly

500 505 510

Arg Pro Phe Phe Ile Asp His Asn Thr Lys Thr Thr Thr Trp Glu Asp

515 520 525

Pro Arg Leu Lys Phe Pro Val His Met Arg Ser Lys Ala Ser Leu Asn

530 535 540

Pro Asn Asp Leu Gly Pro Leu Pro Pro Gly Trp Glu Glu Arg Ile His

545 550 555 560

Leu Asp Gly Arg Thr Phe Tyr Ile Asp His Asn Ser Lys Ile Thr Gln

565 570 575

Trp Glu Asp Pro Arg Leu Gln Asn Pro Ala Ile Thr Gly Pro Ala Val

580 585 590

Pro Tyr Ser Arg Glu Phe Lys Gln Lys Tyr Asp Tyr Phe Arg Lys Lys

595 600 605

Leu Lys Lys Pro Ala Asp Ile Pro Asn Arg Phe Glu Met Lys Leu His

610 615 620

Arg Asn Asn Ile Phe Glu Glu Ser Tyr Arg Arg Ile Met Ser Val Lys

625 630 635 640

Arg Pro Asp Val Leu Lys Ala Arg Leu Trp Ile Glu Phe Glu Ser Glu

645 650 655

Lys Gly Leu Asp Tyr Gly Gly Val Ala Arg Glu Trp Phe Phe Leu Leu

660 665 670

Ser Lys Glu Met Phe Asn Pro Tyr Tyr Gly Leu Phe Glu Tyr Ser Ala

675 680 685

Thr Asp Asn Tyr Thr Leu Gln Ile Asn Pro Asn Ser Gly Leu Cys Asn

690 695 700

Glu Asp His Leu Ser Tyr Phe Thr Phe Ile Gly Arg Val Ala Gly Leu

705 710 715 720

Ala Val Phe His Gly Lys Leu Leu Asp Gly Phe Phe Ile Arg Pro Phe

725 730 735

Tyr Lys Met Met Leu Gly Lys Gln Ile Thr Leu Asn Asp Met Glu Ser

740 745 750

Val Asp Ser Glu Tyr Tyr Asn Ser Leu Lys Trp Ile Leu Glu Asn Asp

755 760 765

Pro Thr Glu Leu Asp Leu Met Phe Cys Ile Asp Glu Glu Asn Phe Gly

770 775 780

Gln Thr Tyr Gln Val Asp Leu Lys Pro Asn Gly Ser Glu Ile Met Val

785 790 795 800

Thr Asn Glu Asn Lys Arg Glu Tyr Ile Asp Leu Val Ile Gln Trp Arg

805 810 815

Phe Val Asn Arg Val Gln Lys Gln Met Asn Ala Phe Leu Glu Gly Phe

820 825 830

Thr Glu Leu Leu Pro Ile Asp Leu Ile Lys Ile Phe Asp Glu Asn Glu

835 840 845

Leu Glu Leu Leu Met Cys Gly Leu Gly Asp Val Asp Val Asn Asp Trp

850 855 860

Arg Gln His Ser Ile Tyr Lys Asn Gly Tyr Cys Pro Asn His Pro Val

865 870 875 880

Ile Gln Trp Phe Trp Lys Ala Val Leu Leu Met Asp Ala Glu Lys Arg

885 890 895

Ile Arg Leu Leu Gln Phe Val Thr Gly Thr Ser Arg Val Pro Met Asn

900 905 910

Gly Phe Ala Glu Leu Tyr Gly Ser Asn Gly Pro Gln Leu Phe Thr Ile

915 920 925

Glu Gln Trp Gly Ser Pro Glu Lys Leu Pro Arg Ala His Thr Cys Phe

930 935 940

Asn Arg Leu Asp Leu Pro Pro Tyr Glu Thr Phe Glu Asp Leu Arg Glu

945 950 955 960

Lys Leu Leu Met Ala Val Glu Asn Ala Gln Gly Phe Glu Gly Val Asp

965 970 975

Claims (9)

1. The application is P1 or P2, and the P1 is the application of a non-human mammal which specifically knocks out PTEN gene in a cerebral vascular endothelial cell in the development of a product for regulating permeability of a blood brain barrier or a product for drug delivery;

the P2 is an application of a substance for improving or promoting the expression quantity of NEDD4-2 gene or/and the activity or content of NEDD4-2 protein in the brain vascular endothelial cells of non-human mammals in the development of products for regulating and controlling blood brain barrier permeability or products for drug delivery.

2. Use according to claim 1, characterized in that: the non-human mammal in P1 is obtained by specifically knocking out PTEN gene in brain vascular endothelial cells by Cre/loxP system;

the substance described in P2 is a recombinant vector expressing NEDD4-2 protein or a recombinant microorganism expressing NEDD4-2 protein.

3. Use according to claim 2, characterized in that: the specific knockout of the PTEN gene in cerebrovascular endothelial cells in P1 is a specific knockout of the fifth, sixth and seventh exons in the PTEN gene in non-human mammalian cerebrovascular endothelial cells;

the recombinant microorganism in P2 is a recombinant adeno-associated virus expressing NEDD4-2 protein.

4. A method of modulating blood brain barrier permeability in a non-human mammal, comprising: the method comprises modulating permeability of the blood brain barrier using the following method of a1) or a 2):

A1) reducing or inhibiting the expression level of PTEN gene or/and the activity or content of PTEN protein in the brain vascular endothelial cells of the non-human mammal;

A2) increase or promote the expression level of NEDD4-2 gene or/and the activity or content of NEDD4-2 protein in the brain vascular endothelial cells of the non-human mammal.

5. The method of claim 4, wherein: the reduction or inhibition of the expression quantity of the PTEN gene or/and the activity or content of the PTEN protein in the brain vascular endothelial cells of the non-human mammals is to specifically knock the PTEN gene in the brain vascular endothelial cells of the non-human mammals;

or, the improvement or promotion of the expression quantity of the NEDD4-2 gene or/and the activity or content of the NEDD4-2 protein in the cerebral vascular endothelial cells of the non-human mammal is realized by using a recombinant adeno-associated virus for expressing the NEDD4-2 protein, and the NEDD4-2 protein is a protein of which the amino acid sequence is SEQ ID No.7 in the sequence table.

6. The method of claim 5, wherein: specifically knocking out the PTEN gene in the brain vascular endothelial cells of the non-human mammals comprises specifically knocking out the PTEN gene in the brain vascular endothelial cells of the non-human mammals by adopting a Cre/loxP system;

or, the recombinant adeno-associated virus contains a coding gene of the NEDD4-2 protein; the nucleotide sequence of the coding chain of the coding gene of the NEDD4-2 protein is a DNA molecule shown in SEQ ID No.3 in a sequence table or 31 th-2961 th site of SEQ ID No.3 in the sequence table.

7. Use according to any of claims 1-3 and/or method according to any of claims 4-7, characterized in that: the regulation and control of the permeability of the blood brain barrier of the non-human mammal is to increase the permeability of the blood brain barrier of the non-human mammal.

8. Use according to any of claims 1-3 or 7 and/or method according to any of claims 4-7, characterized in that: the non-human mammal is selected from any one of mouse, rat, guinea pig, hamster, pig, dog, sheep, monkey, rabbit, cat, cow and horse.

9. A product for modulating blood brain barrier permeability and/or a product for the development of drug delivery as claimed in claim 1 and/or a recombinant adeno-associated virus according to any of claims 4 to 6.

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