CN107475260A - Suppress the function research and development and application of the BACE1 hypotypes that A β accumulate in brain - Google Patents
Suppress the function research and development and application of the BACE1 hypotypes that A β accumulate in brain Download PDFInfo
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Abstract
The invention belongs to the DNA recombinant techniques field in bioengineering, it is related to that the invention belongs to technical field of bioengineering, genetic recombination specifically based on BACE1 alternative splicing mechanism goes out a kind of BACE1 for the 47KD hypotypes that antagonism and A β formation inhibitory action are cut to APP pathology, and detection 47KD hypotypes BACE1 is tested to A β by ELISA1‑40With A β1‑42The facilitation of generation, and its corrective action to AD mouse memory impairments is verified by Behaviors survey, verify its corrective action formed to intracerebral A β finally by pathological experiment.47KD hypotypes BACE1 provides foundation and clue for the further investigation of AD molecular targets and AD medicament research and developments.
Description
Technical field
The invention belongs to the DNA recombinant techniques field in bioengineering, specifically based on BACE1 alternative splicing mechanism
Genetic recombination go out it is a kind of antagonism and A β cut to APP pathology form the BACE1 hypotypes of inhibitory action, and verify it to AD mouse
The corrective action that memory impairment and intracerebral A β are formed, and its for the further investigation of AD molecular targets and AD medicament research and developments provide according to
According to and clue.
Background technology
Alzheimer's disease (Alzheimer's disease AD) is the important diseases of aging society, and as threat
One of four big killers of elderly population health.Amyloid A β is continuously generated and accumulated in the brain, causes significant starch
Sample patch is formed and Secondary cases neurotrosis, is the important pathogenesis of AD.A β are by APP (amyloid protein precursor) molecular disease
Reason cutting is formed, and beta-secretase (BACE1) is the major rate-limiting enzyme of A β generations, and AD medicament research and developments and molecular mechanism grind
The core target spot studied carefully.
BACE1 is typically considered the protease for the 55KD being made up of 501 amino acid, and most research at present also will
It carries out AD medicament research and development as target spot, but ends in failure more, and this has prompted the complexity of BACE1 molecules.It is newest
Document report, BACE1 transcript can be produced by alternative splicing mechanism be separately encoded containing 501,476,457 and
The BACE1 isomers of 432 amino acid, it is separately encoded 55KD, 52KD, 49KD and 47KD BACE1 albumen.Type B I-476 compared with
A types I-501 has lacked 25 amino acid in the region that extron 4 encodes, and c-type I-457 is then lacked in the region of exon 3 coding
Lack 44 amino acid, and D types I-432 has then lacked all amino acid that Type B I-476 and c-type I-457 are lacked simultaneously.
Big intracerebral wide expression the most is A/ type I-501, and other expression quantity of three kinds of forms in the brain are very low, mainly in pancreas
It is expressed Deng having in tissue, so researcher has not focused on cutting function of other three kinds of forms to APP molecules.
The A types BACE1 of big intracerebral most wide expression protein structure domain includes N-terminal signal peptide and pre-peptide region, middle part
Maturation protein catalytic domain, and the C-side extension tail that the trans-membrane region being made up of 17 amino acid and 24 amino acid form【Figure
1】.BACE1 N-terminal and C-terminal respectively contains an aspartic acid active catalytic region, and the two asparagicacid residues are for BACE1
It is active most important, BACE1 contains four glycosylation sites, and glycosylation is BACE1 ripe necessary condition, is influenceed simultaneously
BACE1 activity.Compared with A types BACE1, D types BACE1 missing 146-214 amino acids, lacked Asn153, Asn172 this
Whether two glycosylation sites, the change of this structure can change its function, and this is worth us to further investigate.
The content of the invention
The present invention is alternative splicing and AD occurrence characteristics, treatment difficult point based on BACE1, and one is invented by bioengineering
Duan Teyi protein sequence and nucleotide sequence, it was demonstrated that it, which has, cuts antagonism and A β formation inhibitory action to APP pathology, and
Its corrective action formed to AD mouse memory impairment and intracerebral A β is verified by zoopery.
The present invention utilizes biotechnology, and DNA sequence dna corresponding to 432 amino acid polypeptides of genetic recombination one section arrives
PcDNA3.1 carrier for expression of eukaryon.After sequence analysis proves to recombinate successfully, this eukaryotic expression recombinant polypeptide is transfected into people's embryo
In nephrocyte, Western blotting proves the protein expression of polypeptide, realizes the restructuring of polypeptide.Then to the antagonism APP pathology of polypeptide
Cutting function is studied, and cytologic experiment shows that there is this polypeptide APP pathology cutting antagonism and A β to be formed and suppress to make
With.Then by animal internal injection, Behaviors survey and pathological experiment all prove this polypeptide have AD mouse memory impairment and
The corrective action that intracerebral A β are formed
The polypeptide recombinant expression plasmid of the present invention is to connect this polypeptide corresponding DNA sequence into pcDNA3.1 eukaryotic expressions to carry
Body, polypeptide N-terminal amino acid sequence are：
Its corresponding nucleotides sequence is classified as：
The first step, 47KD hypotype BACE1 construction of eukaryon expression plasmid for expressing and its detection of expression
It is that template enters performing PCR with pcDNA3.1-BACE1 (55KD).PCR primer is converted with DH5 α competence, and
The bacterium colony being converted to is cultivated in a small amount, then extracted the bacterium solution cultivated in a small amount in a small amount, finally by the plasmid of extraction
It is sequenced.
The polypeptide eukaryotic expression vector transfection 293T cells that will correctly connect, collect sample, Western blotting knot after 48 hours
Fruit demonstrates the expression of this polypeptide.
Second step：47KD hypotypes BACE1 Function detection
1. cytologic experiment
AD cell model SHSY5Y cell supernatant is used as sample, in cotransfection recombinant peptide eukaryon expression plasmid and
APP wild plasmids or APPSWEAfter mutant plasmids 48h, cell culture fluid is centrifuged, takes 1/100 as sample detection A β1-40
With A β1-42Concentration.ELISA results show that there is the BACE1 of 47KD hypotypes APP pathology cutting antagonism and A β to form suppression
Make and use.
2. mouse experiment in vivo
The 16 monthly age AD mouse Jing Guo genotype identification are selected to be grouped, equivalent to mankind severe AD patients, every group of difference
The plasmid of high-purity is injected intraperitoneally, twice a week, each every 20ug plasmid, 2 totally months., will after behaviouristics detection is carried out
Embedded section is fixed in mouse brain, and the formation of intracerebral senile plaque expelling and the change of relevant enzyme are detected by the method for SABC.OK
For the corrective action learned and there is the BACE1 of pathology results display 47KD hypotypes AD mouse memory impairment and intracerebral A β to be formed.
Brief description of the drawings：
Fig. 1 is BACE1 protein structure figures；
Fig. 2 is structure BACE1 mutant plasmid principle sketch；
Fig. 3 compares for 47KD hypotype BACE1 sequencing results；
Fig. 4 is two kinds of antibody schematic diagrams of BACE1；
Fig. 5 is BACE1 mutant plasmids expression checking principle；
Fig. 6 is the expression checking Westernblot analyses of BACE1 mutant plasmids；
Fig. 7 is that ELISA detects A β in AD cell model SHSY5Y cell culture fluids1-40Concentration；
Fig. 8 is that ELISA detects A β in AD cell model SHSY5Y cell culture fluids1-42Concentration；
Fig. 9 is the experimental design that intraperitoneal injection BACE1 is acted on AD mouse behaviors cognition；
Figure 10 is that AD mouse Y labyrinths enter arm accuracy statistical chart after plasmid is injected；
Figure 11 is AD mouse orientation navigation exemplary trajectory figure and 6 day incubation period broken line graph；
Figure 12 is that exemplary trajectory figure and virtual platform residence time number statistical chart are navigated by water in the space exploration of AD mouse；
Figure 13 is that three kinds of BACE1 antibody tests intraperitoneal injection BACE1 mutant plasmids are overexpressed situation in AD mouse brains；
Figure 14 is the dyeing of AD Mice brain tissues thioflavine and mathematical statistics.
Embodiment：
Embodiment 1：47KD hypotype BACE1 construction of eukaryon expression plasmid for expressing and its detection of expression
1st, 47KD hypotypes BACE1 construction of eukaryotic expression vector
BACE1 full length genes obtain from Genebank, and gene order number is NM 001207048.D types I-432 (47KD)
BACE1 lacks 146-214aa compared with A types I-501 (55KD) BACE1【Fig. 2】, with pcDNA3.1-BACE1 (55KD) for mould
Plate, sense primer 5'-ACCTGCTTTGTGGTGCTGGCTTCCCCCTCA-3', anti-sense primer 5'-
CAAAGCAGGTCGGTGCCCAGCTCCCCTTCC-3', enter performing PCR.PCR primer is converted with DH5 α competence, and will
The bacterium colony being converted to is cultivated in a small amount, is then extracted the bacterium solution cultivated in a small amount in a small amount with alkaline lysis, will finally be carried
The plasmid taken is sequenced, and is compared, and is mutated successfully【Fig. 3】.
2nd, the detection of expression of 47KD hypotypes BACE1 eukaryon expression plasmids
According to existing both ends specific antibody【Fig. 4】, antibody BACE1190-214aaCan detect 55KD, 49KD both
The BACE1 of form, antibody BACE1146-189aaThe BACE1 of both forms of 55KD, 52KD can be detected；And existing BACE1 resists
Body can detect the BACE1 of these four hypotypes of 55KD, 52KD, 49KD, 47KD.Can be with specific recognition according to these three antibody
Clip types, devise mutant plasmid expression confirmatory experiment【Fig. 5】.The HEK293T cells of high transfection efficiency are selected,
The eucaryon plasmid of BACE1 hypotypes is overexpressed in HEK293T cells, is detected with above-mentioned three kinds of antibody Western blot, as a result shown
Show that mutant plasmid is successfully constructed and can expressed【Fig. 6】.
Embodiment 2：47KD hypotypes BACE1 Function detection
47KD hypotypes BACE1 is to A β in 1.ELISA detection AD cell models1-40With A β1-42Generation suppress
AD cell model SHSY5Y cell supernatant is used as sample, in cotransfection BACE1 mutant plasmid and APP
Wild plasmid or APPSWEAfter mutant plasmids 48h, cell culture fluid is centrifuged, takes 1/100 to be used as sample detection 47KD hypotypes
BACE1, according to standard concentration and absorbance curve, calculates sample concentration to the dissection of APP molecules.With blank control group
Compare, be either overexpressed APP wild types and be still overexpressed APPSWEThe BACE1 of saltant type, only 47KD can make A β1-40It is dense
Degree is obvious to be reduced【Fig. 7】；For A β1-42Generation test experience in, also obtained identical result【Fig. 8】.
Hence, it can be determined that D types BACE1 (47KD) can significantly inhibit A β1-40With A β1-42Generation, inhibiting rate reaches
More than 80%.This prompts our 47KD BACE1 to be different from the promotion work that traditional BACE1 is formed to the cutting of APP pathology and A β
With it has the antagonism of APP pathology cutting and the inhibitory action of A β formation.
2nd, the effect that 47KD hypotypes BACE1 recognizes to the behavior of AD mouse is probed into Y labyrinths
The 16 monthly age AD mouse Jing Guo genotype identification are selected, equivalent to mankind severe AD patients, normal raising sets five
Group, every group of 6 experimental mouses, including control group (empty carrier group), A types BACE1 (55KD) group, Type B BACE1 (52KD) group, c-type
BACE1 (49KD) is organized and D types BACE1 (47KD) group.Every group of plasmid that high-purity is injected intraperitoneally respectively, it is twice a week, every every time
20ug plasmids, 2 totally months【Fig. 9】.
Y maze experiments are a kind of behaviouristics means for being used to detect mouse Spatial cognitive Abilities and short term memory capacity, its
It is simple to operate, it is widely used.Experimental result shows, empty vector control group and other three kinds of BACE1 hypotypes Y fans after plasmid is injected
Palace path cognition accuracy rate declines, the Y maze paths cognition accuracy rate for the trangenic mice that only D types BACE1 (47KD) is overexpressed
Slightly rise【Figure 10】, there is significant difference by statistical analysis, and this group of looking for food for mouse is better than with mobility
Other each groups.Experimental result explanation, which only has D types BACE1 (47KD) to be overexpressed, can strengthen trangenic mice APPswe/PS1dE9's
Learn short term memory capacity.
3rd, Morris water mazes probe into the effect that 47KD hypotypes BACE1 recognizes to the behavior of AD mouse
Morris water mazes are a kind of very universal laboratory facilities for practising memory mechanism research in AD researchs on cerebrology.
The AD mouse at 16 monthly ages are have selected, normal five groups of raising setting, every group of 6 experimental mouses, including blank group (empty carrier group), A types
BACE1 (55KD) group, Type B BACE1 (52KD) group, c-type BACE1 (49KD) groups and D types BACE1 (47KD) group.Every group of difference abdomen
Chamber injects the plasmid of high-purity, twice a week, each every 20ug plasmid；Detected often by Morris water maze laboratories after 8 weeks
The ability of learning and memory of group model mouse.
Morris water mazes are carried out in a diameter of 1.2 meters of round pool, are first placed in the injection plasmid mouse of 8 weeks and are put
Have to determine Burden-Swimming Ability of KM in the pond of visible platform, and repeatedly train to deepen position memory of the mouse to platform.With laggard
Row orientation navigation is tested, and is lasted 6 days, is daily respectively put into mouse in water towards pool wall from the fixation place of entry of 4 quadrants, and
Record it and search out the time used in hidden platform under water, be incubation period by the timing definition.Navigated according to each group mouse in positioning
The exemplary trajectory figure of row experiment understand the mouse of the BACE1 groups of 47KD hypotypes find the distance hiding to be walked in underwater platform compared with
It is short, and other three groups are difficult then to find platform；With the increase of test number of days, each group mouse finds the time needed for concealment platform
Overall is in shortening trend.In general trend, compared with blank control group, d types BACE1 (47KD) group mouse find concealment platform institute
The time (incubation period) needed significantly shortens【Figure 11】.
Being removed at the 7th day and platform is hidden in water, the position that mouse finds original platform in water carries out space exploration experiment,
Search time is identical and distance is in the case of, and the mouse of the BACE1 groups of 47KD hypotypes can have accurately to original platform position
Memory and identification, different from other group blindnesses find【Figure 12】.Analyze the long-term memory ability and spatial memory energy of mouse
The result of power finds, the mouse of 47KD hypotype BACE1 groups passes through the number of original platform position apparently higher than other each groups, explanation
The BACE1 of 47KD hypotypes can deepen long-term memory of the mouse to position of platform, i.e., the memory cognition damage to Alzheimer's disease
The restitution of wound is notable.Each group mouse is in the BACE1 groups that the activity time of the quadrant where original platform is equally 47KD hypotypes
Mouse apparently higher than other groups, memory of the mouse to the direction in space of platform can be deepened by illustrating the BACE1 of 47KD hypotypes, i.e.,
It is significantly improved effect to spatial cognition and position memory, and to the extensive of AD disease mices spatial cognition and position memory ability
Multiple effect is obvious.
4th, overexpression situations of the BACE1 of SABC checking intraperitoneal injection different subtype in AD mouse brains
It is fixed to embed its murine brain by the continuously intraperitoneal injection BACE1 mutant plasmids AD mouse of 8 weeks execution from 16 monthly ages
And with three kinds of BACE1 antibody mediated immunity groups.Dyed under same time identical conditions and picture shooting, as a result shown, with
Control group is compared, and the mouse brain hippocampus dyeing of four groups of overexpression BACE1 different subtype plasmids is deepened；Can detect 55KD,
Two kinds of hypotype BACE1 of 49KD from antibody processed BACE1190-214aaAs a result show, the BACE1 groups and 55KD of hippocampus 49KD hypotypes
The dyeing of the BACE1 groups of hypotype is substantially deeper than remaining each group；The self-control that the BACE1 of two kinds of hypotypes of 55KD, 52KD can be detected resists
Body BACE1146-189aaAs a result show, the dyeing of the BACE1 groups of hippocampus 52KD hypotypes and the BACE1 groups of 55KD hypotypes is substantially deep
It is consistent with the result of hippocampus in remaining each group, cerebral cortex area BACE1 expression【Figure 13】.Result above proves intraperitoneal injection
BACE1 different subtypes plasmid can in mouse brain tissue normal expression.
5th, influences of the 47KD hypotypes BACE1 to AD mouse brain tissue beta amyloid patches is probed into thioflavine S dyeing
The limbic system of brain includes the structures such as amygdaloid nucleus and hippocampus, and key effect is played to memory, its deutocerebrum sea
Ma Qu is related to specific learning and memory, is storage short-term memory key position, while be also mankind's learning and memory acousto-optic taste
Etc. the brain region of event, i.e. long-term memory region.Thioflavin S dyeing is mainly used in showing extracellular amyloid material, can not
The aβ protein of dissolubility deposits in brain tissue forms beta amyloid patch with the characteristic combined with thioflavin S, thus selects
The method that this method carries out tissue section strain analyzes continuous intraperitoneal injection BACE1 mutant plasmids from 16 monthly ages 2 months
The change of beta amyloid plaque deposition in AD Mice brain tissues, and subregion carries out qualitative observation and quantitative analysis, as a result finds
The BACE1 of 47KD hypotypes has the deposition for significantly lowering AD mouse brain hippocampus beta amyloid patches, to hippocampus week
Enclosing cortical area and off-lying sea horse end cortical area equally has obvious intervention effect【Figure 14】.By the big intracerebral β-starch of transgenic mice
Sample patch total number carries out quantitative analysis and mathematical statistics, as a result proves that only injection 47KD hypotypes BACE1 transgenosis is small
The big intracerebral beta amyloid patch of mouse all has obvious reduction in patch number compared with empty vectors group, illustrates the BACE1 of 47KD hypotypes,
There is to substantially reduce and correct formation and the deposition of AD mouse intracerebral beta amyloid patches.
Claims (8)
1. nucleotides sequence of the invention protection with the BACE1 hypotypes that antagonism and A β formation inhibitory action are cut to APP pathology
Row.
2. amino acid sequence of the invention protection with the BACE1 hypotypes that antagonism and A β formation inhibitory action are cut to APP pathology
Row.
3. invention protection 47KD hypotypes BACE1, which has, cuts antagonism and A β formation inhibitory action to APP pathology.
4. invention protection is with the BACE1 hypotypes to AD mouse memory impairment corrective actions.
5. invention protection 47KD hypotypes BACE1 has the corrective action to AD mouse memory impairments.
6. invention protection is with the BACE1 hypotypes that corrective action is formed to AD mouse intracerebral A β.
7. invention protection 47KD hypotypes BACE1 has the corrective action formed to AD mouse intracerebral A β.
8. invention is protected based on the AD medicament research and developments described in right 1,2,3,4,5,6,7 and AD diagnosis.
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| US20050074456A1 (en) * | 2002-12-04 | 2005-04-07 | Marcus Ballinger | Constructs for homogenously processed preparations of beta site APP-cleaving enzyme |
| EP1801232B1 (en) * | 2004-10-01 | 2012-03-14 | Takeda Pharmaceutical Company Limited | Method of screening transmembrane enzyme inhibitory substance |
| CN103228674A (en) * | 2010-11-10 | 2013-07-31 | 霍夫曼-拉罗奇有限公司 | Methods and compositions for neural disease immunotherapy |
| CN106084057A (en) * | 2016-06-13 | 2016-11-09 | 东北师范大学 | The preparation of BACE1 shearing-type high-titer antibody and application |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050074456A1 (en) * | 2002-12-04 | 2005-04-07 | Marcus Ballinger | Constructs for homogenously processed preparations of beta site APP-cleaving enzyme |
| EP1801232B1 (en) * | 2004-10-01 | 2012-03-14 | Takeda Pharmaceutical Company Limited | Method of screening transmembrane enzyme inhibitory substance |
| CN103228674A (en) * | 2010-11-10 | 2013-07-31 | 霍夫曼-拉罗奇有限公司 | Methods and compositions for neural disease immunotherapy |
| CN106084057A (en) * | 2016-06-13 | 2016-11-09 | 东北师范大学 | The preparation of BACE1 shearing-type high-titer antibody and application |
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