CN100335500C - Human amyloid precursor protein 639, coded sequence and uses thereof - Google Patents

Abstract

The present invention discloses an amino acid sequence and a coding sequence of a novel polypeptide-human amyloid protein precursor protein 639 (APP639 protein), an antibody of the polypeptide and applications of the polypeptide and the nucleotide in screening medicines for treating senile dementia, preparing kits for detecting liver development conditions, etc.

Description

Amyloid precursor protein 639, its encoding sequence and purposes

Technical field

The invention belongs to biological technical field, concrete aminoacid sequence that the present invention relates to a kind of new polypeptide-human amyloid precursor protein 639 (APP639 albumen) and encoding sequence thereof and they are in the application of aspects such as the medicine of screening treatment senile dementia and preparation liver growth detection kit.

Background technology

Senile dementia (Alzheimer ' s disease, AD) be the most serious in the world present nerve degenerative diseases.Three kinds of anomalous structures are arranged in AD patient's brain: senile plaque (senile plaques, SP), neurofibrillary tangles (neurofibrillary tangles, NFT) and the kind of starch sample electrodeposition substance cerebrovascular (amyloid-laden cerebral vessels).Senile plaque and the cerebrovascular main ingredient of kind of starch sample electrodeposition substance are the amyloid-beta that contains 39-43 amino-acid residue (a, β-amyloidprotein).A is that (amyloid precursor protein is APP) through proteolysis by amyloid precursor protein.At present, found that app gene can produce 8 kinds of different mRNA forms in transcription.

Senile dementia (AD, Alzheimer ' s disease) with in, in the elderly population common be losing one's memory closely related.The present aging trend of China's population is serious, and the AD morbidity is in continuous rising.According to investigations, dull-witted total prevalence rate is 5.22% in the over-65s population, the total all kinds of dementia patients 5,000,000 in the whole nation, and wherein patient AD accounts for 3,400,000.A large amount of evidences show that A β plays a part the center in AD falls ill initial evolution.

A is that (amyloid precursor protein, APP) through proteolysis, amyloid precursor protein hence obtains one's name by amyloid precursor protein.App gene is positioned at karyomit(e) No. 21, is that first is found and familial senile dementia (familial Alzheimer ' s disease, FAD) relevant gene.APP is a popularity distribution albumen, and respectively organizing in body of mammals all has expression.APP albumen is a typical I-type conformity membrane albumen, contain a signal peptide sequence, the outer aminoterminal zone of big born of the same parents, single strides the film district and a little intracellular region contains N end signal peptide, a big N end ectodomain (comprising the abundant zone of a Cys, negative electricity district and N-glycosylation district), single are striden the C end intracellular region territory of a film district and a weak point.App gene is positioned on No. 21 karyomit(e), and its transcription product is the mRNA of 3.2-3.4kb, and app gene is all expressed in many tissues, and it is expressed in kidney and brain is the highest.App gene has 18 exons, is respectively 1-13,13a and 14-18 exon.App gene can produce 8 kinds of different transcription products through different montage processing, and these products are all named with amino acid number that it was contained, comprising APP770, APP751, APP714, APP695, L-APPs.The known splice site of app gene mainly occurs in exon 7-8 and exons 1 is located for 5 liang.

APP695cDNA separates the total length app gene transcription product that obtains the earliest, its protein that contains 695 amino-acid residues of encoding.Compare with APP695mRNA, the many fragment-exon 7s that contain 168 Nucleotide of APP751mRNA, one of this exons coding and Kunitz serpin family (KPI) highly homologous contain the fragment of 56 amino-acid residues.APP770mRNA has more exon 8 than APP751mRNA, fragment that contains 19 amino-acid residues of this exons coding, and the sequence of these 19 amino-acid residues has 47% homology with the MRC OX-2 antigen of finding at neurone and thymocyte.APP714 manys exon 8 than APP695.L-APPs does not contain exon No. 15, and gains the name because of they mainly exist in the microglia in white corpuscle (Leukocytes) and brain.Corresponding montage also takes place at exon 7,8, and produces a series of montage products except not containing exons 15 in L-APPs.PP695, APP751 and APP770 are three kinds of modal app gene transcription products.APP770 and APP751 have the KPI structural domain, and they are mainly expressed in peripheral tissues and adult primate brain; APP695 then mainly expresses in neural system.Have experiment to show, compare with normal control group, the expression of APP695mRNA in AD patient's brain reduces, and the expression level of APP770mRNA raises.These results suggest, the different montage products and the regulation and control on transcriptional level thereof of app gene may be relevant with AD.And the different montages of app gene, the adjusting of expression are more important than its possible structural changes.

Therefore, this area presses for the montage product (albumen) of the new app gene relevant with AD of research and development.

Summary of the invention

The new montage product amyloid precursor protein 639 that purpose of the present invention just provides a kind of app gene relevant with AD with and nucleotide sequence.

Another object of the present invention provides the method and the described proteic purposes of producing this albumen and nucleotide sequence.

A first aspect of the present invention, a kind of isolating amyloid precursor protein 639 is provided, it is characterized in that it comprises: polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative with SEQ ID NO:1 aminoacid sequence.Preferred this polypeptide is the polypeptide with SEQ ID NO:1 aminoacid sequence.

A second aspect of the present invention provides a kind of isolating polynucleotide, it is characterized in that, it comprises a kind of nucleotide sequence that is selected from down group:

(a) coding is as the polynucleotide of polypeptide as described in claim 1 and 2;

(b) with polynucleotide (a) complementary polynucleotide.

The polypeptide of SEQ ID NO:1 aminoacid sequence shown in preferred this polynucleotide encoding has.

A third aspect of the present invention provides a kind of carrier, and it contains the above-mentioned polynucleotide of the present invention.

A fourth aspect of the present invention provides a kind of genetically engineered host cell, and it contains the above-mentioned carrier of the present invention.Preferable, described host cell is an eukaryotic cell.Better, described host cell is a mammalian cell.Best, the cell that described host cell is behaved.

A kind of preparation method with active amyloid precursor protein 639 is characterized in that, this method comprises:

(a) being fit to express under amyloid precursor protein 639's the condition, cultivate the described host cell of claim 6;

(b) from culture, isolate and have the active polypeptide of amyloid precursor protein 639.

A sixth aspect of the present invention, provide a kind of can with the above-mentioned amyloid precursor protein 639's specificity of the present invention bonded antibody.Preferable, described antibody is monoclonal antibody.

A seventh aspect of the present invention provides a kind of test kit that detects the liver development condition, it is characterized in that it comprises the primer of specific amplification or transcript, perhaps with amyloid precursor protein 639's specificity bonded antibody.Preferably, detection be amyloid precursor protein 639's gene or transcript, and with normal amyloid precursor protein 639's nucleotide sequence comparing difference.

A eighth aspect of the present invention provides a kind of the present invention above-mentioned amyloid precursor protein 639's purposes, it is characterized in that, it is used to screen the medicine of treatment senile dementia, or preparation neurotrophy healthcare products.

Other aspects of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.

Description of drawings

The aminoacid sequence of Figure 1A PP639eDNA complete sequence and supposition.

The arrow indication is exon 1 and exon 3 connecting portions.What comprise in the square frame is A amyloid-beta aminoacid sequence.Poly (A) signal is sequence shown in the following horizontal line.

The app gene transcription product that montage takes place for Fig. 2 RT-PCR and sequencing analysis exon 2 places exists in people's embryonic tissue.

A:PCR primer and amplified fragments diagram.

B:RT-PCR analyzes the app gene transcription product of exon 2 places generation montage in people embryo brain and liver organization.

C: to the dna sequencing analysis of RT-PCR amplified production.The arrow indication be respectively exon 1 and exon 2 (on), exon 1 and exon 3 (descending) connection site.

Fig. 3 RT-PCR and the expression of Southern hybridization analysis APP639mRNA in the various tissues of people embryo

Fig. 4 RT-PCR analyzes the expression of APP639mRNA at grownup's pallium and liver organization.

A:RT-PCR analyzes APP639mRNA in the corticocerebral expression of grownup.

B:RT-PCR analyzes the expression of APP639mRNA at grownup's liver organization.

Fig. 5 Western blot analyzes the expression of APP639 fusion rotein in the HEK293 of transient transfection cell

Three of Fig. 6 people's app gene generation montage may the site.

Three of A:APP gene generation montage may site and APP770.

B: I site and the montage product thereof relevant with exon 7 and exon 8, comprising APP751, APP714 and APP695.

C: II site and the montage product L-APPs thereof relevant with exon 15, L-APPs is not except containing exons 15, corresponding montage also takes place at exon 7,8, and produce four respectively with the corresponding montage product of APP770, APP751, APP714 and APP695.

D: III site and the APP639 relevant with exon 2.

Embodiment

The inventor screens a new splicing form APP639 of people's app gene first through extensive and deep research.Sequencing result shows that new splice site is the 2nd exon, causes exons 1 directly to be connected with 3; APP639cDNA does not contain exon 2,7 and 8, and proteins encoded contains 639 amino-acid residues.RT-PCR and Southern hybridization analysis confirm that APP639mRNA exists in embryonic tissue.The contriver fails to detect APP639mRNA in grownup's pallium; Yet APP639mRNA has expression at grownup's liver.Western blot result shows that the proteic molecular weight of APP639 is approximately 110kD.

APP639 cDNA total length contains 3011 Nucleotide, and nucleotide sequence is arranged by 5 ' to 3 ' direction, comprises an open reading frame that 639 amino-acid residues are arranged in the sequence.Finished the present invention on this basis.

As used herein, term " amyloid precursor protein 639 " and " human amyloid amyloid protein precursor polypeptide 639 " are used interchangeably.

As used herein, " isolating " is meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification as polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.

As used herein, " isolating amyloid precursor protein 639 or polypeptide " is meant that the amyloid precursor protein 639 is substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can use the purified technology of protein purifying amyloid precursor protein 639 of standard.

Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide.Polypeptide of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology to produce from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell).The host used according to the recombinant production scheme, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated.

The present invention also comprises amyloid precursor protein 639's fragment, derivative and analogue.

Polynucleotide of the present invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can be identical with coding region sequence shown in Figure 1 or the varient of degeneracy.As used herein, " varient of degeneracy " is meant that in the present invention coding has the protein of SEQ ID NO:1, but with the differentiated nucleotide sequence of coding region sequence shown in Figure 1.

The invention still further relates to nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment " contains 15 Nucleotide at least, better is at least 30 Nucleotide, is more preferably at least 50 Nucleotide, preferably more than at least 100 Nucleotide.Nucleic acid fragment can be used for the amplification technique (as PCR) of nucleic acid to determine and/or to separate the polynucleotide of coding amyloid precursor protein 639.

Amyloid precursor protein 639's Nucleotide full length sequence or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, nucleotide sequence that can rat amyloid precursor protein 639 disclosed according to the present invention, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.

In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.

In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.

Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.

The extracellular can be expressed or be secreted into to recombinant polypeptide in the above methods in cell or on cytolemma.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, superly handle, the combination of super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.

Particularly, amyloid precursor protein 639's albumen of the present invention or polypeptide are of use in many ways.These purposes include, but is not limited to: the medicine that is used to screen the treatment senile dementia, or preparation neurotrophy healthcare products, antibody with the preparation amyloid precursor protein 639, can prepare the especially test kit of liver growth of sense organ, can be used as liver regeneration medical research model, and detect the proteic expression variation of APP639 in late period early in the senile dementia.The peptide molecule that can suppress or stimulate amyloid precursor protein 639's protein function that can be used for seeking therapeutic value with the reorganization amyloid precursor protein 639 protein screening peptide library of expressing.

On the other hand, the present invention also comprises complete monoclonal antibody or polyclonal antibody, but also comprises having immunocompetent antibody fragment, as Fab ' or (Fab) ₂Fragment; Heavy chain of antibody; Light chain of antibody; Or chimeric antibody, as have the murine antibody binding specificity but still keep antibody from people's antibody moiety.

Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, amyloid precursor protein 639's gene product of purifying or its have antigenic fragment, can be applied to animal (as rabbit, mouse etc.) to induce the generation of polyclonal antibody.Can use the reaction of multiple adjuvant enhancing immunity, comprising (but being not limited to) freund's adjuvant etc.Monoclonal antibody of the present invention can utilize hybridoma technology to prepare.The proteic antibody of anti-amyloid precursor protein 639 can be used in the immunohistochemistry technology, detects amyloid precursor protein 639 albumen in the biopsy specimen.The present invention's experiment has confirmed the molecular marked compound of the proteic expression level of detection APP639 that the amyloid precursor protein 639 can be used as.

Utilize albumen of the present invention,, can filter out with the amyloid precursor protein 639 interactional material takes place, as inhibitor, agonist or antagonist etc. by various conventional screening methods.

The present invention also provides a kind of pharmaceutical composition, and it contains amyloid precursor protein 639's albumen of the present invention, its coding nucleic acid and/or the antisense nucleic acid of safe and effective amount, and pharmaceutically acceptable carrier or vehicle.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example is prepared by ordinary method with the physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as tablet and capsule can be prepared by ordinary method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity, for example every day about 0.01 microgram/kg body weight-Yue 0.5 mg/kg body weight.In addition, polypeptide of the present invention also can use with the other treatment agent.If APP639 albumen is lower than the A β ability that APP695 produces, and its normal physiologic function does not change, so, can be by replace the method for APP695 with APP639, prevent or delay the AD illness.

In contriver's a specific embodiment, the contriver has found a kind of new montage product A PP639 of app gene, and it lacks exon 2,7 and 8.Utilize RT-PCR and Southern hybridizing method, the contriver finds that APP639 mRNA expresses in the many tissues of embryo, especially high expression level in the liver; In normal adult and patient's AD pallium, all do not investigate its existence, but in liver, still express.Utilize the Western hybridization technique, the APP639 molecular weight of albumen of cell heterogenous expression is about 110kD.

Muller etc. were at report in 1994, and they detect the mRNA of a kind of special shape of app gene in app gene mutant mice body, wherein lacked the 2nd exon.They have inserted one section and have contained neomycin resistance gene DNA module and a transcription termination sequence in the 2nd exon of the app gene of mouse, to reach the purpose of destroying the app gene function.RT-PCR result shows, in the brain and other tissue of the app gene sudden change homozygote mouse that they obtain, still there is the special rna transcription product of APP-, only detected APP mRNA expression level is than the low 5-10 of wild-type mice times, and this transcription product does not contain ruined the 2nd exon.The montage of low probability may take place in this results suggest, the 2nd exon of app gene in the mouse body.In this experiment, the contriver not only screens the APP639 cDNA that lacks the 2nd exon equally from people's tire brain cDNA library, and RT-PCR and Southern hybridization analysis have confirmed that also APP639mRNA exists in tissue.Thereby people's such as contriver and Muller experimental result shows that app gene exists really in the mechanism of the 2nd exon generation montage in mammalian body.

People's app gene is positioned on No. 21 karyomit(e), contains 18 exons altogether.The montage of app gene on transcriptional level that studies show that early mainly occurs in two sites.A site is exon 7,8 places, optionally montage produces four kinds of different forms of transcribing: the APP770 (Fig. 6 A) that comprises two exons, the APP751 that only contains the 7th exon only contains the APP714 of the 8th exon and two APP695 (Fig. 6 B) that exon does not all have.Another site relevant with the 15th exon (Fig. 6 C), the APPmRNA that does not contain exons 15 also has four kinds of forms, corresponds respectively to aforementioned four kinds of products that montage takes place at the 7th, 8 exon places.Because they are mainly expressed, thereby be named as L-APPs (Fig. 6 C) in white corpuscle (Leukocyte) and brain microglia.The contriver has found the new montage product A PP639 of of people's app gene first, and APP639 does not also have exon 2 except not containing exon 7 and 8.There is 3rd the possible splice site (Fig. 6 D) relevant with the 2nd exon in contriver's experiment confirm app gene at transcriptional level.

The contriver is cloned into the APP639 (Fig. 1) that lacks the 2nd exon from people's tire brain cDNA library, the result of RT-PCR and Southern hybridization has confirmed that there be (Fig. 3) in this splicing form in tire brain and other tissue.Yet, invent the expression (Fig. 4) of failing to detect APP639mRNA per capita non-dull-witted the elderly and AD patient's pallium.Have two reasons, once being that the APP splicing form that lacks the 2nd exon itself is not expressed in grownup's cerebral tissue; Another may be, the expression of this splicing form in grownup's cerebral tissue is very low, and to obtain tissue be a tediously long process, and RNA also can natural degradation in organizing the prolonged preservation process, crosses when low when rna content, is difficult to detect with conventional means.The result of RT-PCR and Southern hybridization shows that also in embryonic stage, APP639 is higher than cerebral tissue (Fig. 3) in the expression of liver.Utilize the RT-PCR method, the contriver has investigated the expression of APP639 at grownup's cerebral tissue and liver, although find in pallium, can not detect the existence of APP639 mRNA, and, the expression (Fig. 4) of APP639 is arranged in liver organization.Above experimental evidence prompting, APP639 may have certain function in liver.

According to homology relatively, APP albumen n end zone is conservative relatively, and traditional view thinks that the APP albumen n end has conservative functional area.Found that the APP albumen n end has a heparin land, can promote the growth of neural axon, regulated the generation of nerve synapse.The APP albumen n end is rich in halfcystine, can form disulfide linkage, guarantees the three-dimensional arrangement of APP albumen n end, and the N end structure is similar to rich halfcystine class somatomedin, and this conforms to the function that the APP albumen n end of external discovery has neurotrophic factor.The crystallology method shows that the APP albumen n end except that the heparin land, also has a conservative hydrophobic region, and the existence in these two zones may hold known neurotrophic function closely related with N.APP639 albumen lacks N and holds the 20th to 75 amino acids, but these 56 amino acid whose disappearances may be to not influencing N end functional area, because from aminoacid sequence, main functional domain heparin land and hydrophobic domain are not subjected to very big influence.So the contriver infers, other protein similars such as APP639 albumen n end neurotrophic function and APP695.But the disappearance of halfcystine may cause the change of N end structure, thereby influences its function.The contriver laboratory infers that the disappearance of exon 2 may influence the secretion of A β, is screening the APP639 stable expression cell strain at present.The contriver believes, to the further research of APP639 protein function, helps the contriver to illustrate the function of APP protein family.The discovery of this new splicing form of APP639 helps function and metabolism that the contriver understands app gene.

But regrettably, the contriver is not at embryo's brain, and liver detects the proteic expression of endogenic APP639 in kidney and patient AD and the normal adult brain.This is likely because APP639 albumen expression amount in vivo is very low.In a word, the APP639cDNA according to the contriver clone can express the APP639 albumen that lacks exon 2 accordingly, and can be proved by different APP antibody recognition.

The contriver has found a kind of new montage product A PP639 of app gene, and it lacks exon 2,7 and 8.Utilize RT-PCR and Southern hybridizing method, the contriver finds that APP639mRNA expresses in the many tissues of embryo, especially high expression level in the liver.In normal adult and patient's AD pallium, all do not investigate its existence, but in liver, still express.Utilize the Western hybridization technique, the APP639 molecular weight of albumen of cell heterogenous expression is about 110kD.In a word, this new splicing form of APP639 exists really in vivo.

Getting in touch of the 460bp that obtains for further conclusive evidence amplification and fragment about 300bp and app gene transcription product, the PCR product is purified, is cloned into the pGEM-T-easy carrier and (available from Promega company, USA), checks order.Sequencing result shows, corresponding to containing app gene the 2nd exon over against fragment according to about 460bp of APP695, and exons 1 be connected with exon 2 (Fig. 2 C); Corresponding to over against not containing the 2nd exon, make exons 1 directly link to each other with exon 3 (Fig. 2 C) according to the fragment about the 300bp of APP639.App gene genome sequence analysis revealed, two kinds of top montage processes are all finished by conservative rna editing GT-AG mechanism.

And, because exons 1 and exon 2 contain 57 and 168 Nucleotide respectively, just in time all be 3 integral multiple, so the disappearance of exon 2 can't change the proteic amino-acid sequence of corresponding APP of transcription product coding; The contriver infers that this may can not influence this APP albumen yet and exercise its normal function thus.Above evidence shows that the form of transcribing that this app gene of APP639 is new is present in human fetus's tissue really.

The international think-tank of Holland (Netherlands Brain Bank, NBB) the dead back sample that provides the europathology feature that derives from good clinography to determine case.Dementia patients is determined by clinical analysis; The clinical diagnosis of " possible AD patient " based on family, medical inspection and lab investigation, and is separated with other dull-witted illness.Clinical diagnosis is carried out with reference to the NINCDS-ADRDA standard, and the severe dementia patients is assessed with reference to GlobalDeterioration Scale.The old normal control group membership of non-dementia is not all having nerve and mental illness on family's history and on characterizing.

Be defined in the upward gene relevant with AD of heredity because app gene is first, the sudden change of app gene can cause familial inheritance AD.In addition, the expression variation of different mRNA montage products (APP695, APP751 and APP770) in brain that has experiment to show that app gene takes place at exon 7,8 places to produce after the montage also may be relevant with AD.These study prompting, and the different splicing forms of app gene and the regulation and control of transcriptional level thereof may produce relevant with AD.This impels the contriver to go to investigate the expression of APP639mRNA in AD patient and the dull-witted elderly brain tissue of non-.

In a specific embodiment, the contriver obtains 17 pallium samples altogether from the international think-tank of Holland, 8 sample sources is wherein arranged in AD, and 9 derive from non-dull-witted old contrast.The contriver uses primer that P3/P5 is carried out pcr amplification, can only obtain the product corresponding with APP695 in all samples, but fails to detect the amplified band that is consistent with the APP639 that lacks exon 2.Fig. 4 A has only shown the pcr analysis result of sample segment, and the pallium sample source of swimming lane 4-8 is in 5 non-dull-witted old contrasts; The sample source of swimming lane 9-13 is in 5 AD patients.As shown by arrows, in sample, only can increase and obtain the band corresponding, and not have the product (Fig. 4 A) corresponding with APP639 with APP695.

As shown in Figure 5, the proteic molecular weight of APP639-myc is slightly less than APP695-myc albumen, greatly about about 120kD.22C11 can not discern APP639-myc albumen, and 6E10, myc antibody all can be discerned APP695-myc and APP639-myc albumen.Because its C end contains the myc tumor-necrosis factor glycoproteins of about 60 amino-acid residues, so the proteic molecular weight of APP639 is approximately 110kD.

Because APP639mRNA is higher in the expression of fetal liver, thereby the contriver has investigated its expression at grownup's liver organization in another specific embodiment.Shown in Fig. 2-7B, utilize primer that P3/P5 is carried out pcr amplification, the contriver obtains two corresponding with APP695 and APP639 respectively amplified bands (indication of Fig. 4 arrow) in an adult liver organization.This mRNA that shows APP639 has expression in grownup's liver organization.The APP639 distribution and expression has the specificity of growing period and tissue, therefore can be used as especially liver growth detection kit of organ, can also be as liver regeneration medical research model.

The proteic N end of APP639 three-dimensional conformation changes, and may influence generation and the secretion of A β.APP albumen also may be as the novel drugs target spot of senile dementia.APP639 albumen can be used as the early stage disease severed finger mark of senile dementia, utilizes the APP639 specific antibody, detects APP639 protein expression variation in late period early in the senile dementia.Secretor type APP639 albumen segment may present the neontology function except that trophism, can be used as novel neurotrophy healthcare products.The discovery of the new splicing form of APP639 helps the contriver and studies the regulatory mechanism of different montages to the generation development of senile dementia.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1cDNA library screening and Southern are hybridized the preparation of used probe template

1) preparation of cDNA library screening probe template (Probe 1)

A) use TRIZOL reagent, from SK-N-SH cell (available from the Shanghai Inst. of Life Science, CAS cell bank), extract total RNA.

B) reverse transcription.

C) be template with the RT-PCR product, use primer that P1/P2 is carried out pcr amplification.

D) electrophoresis reclaims the purifying pcr amplification product, and the clone advances the pGEM-T-easy carrier, and enzyme is cut, order-checking.

E) Xho I and Sac I enzyme are cut, and the 682bp fragment that electrophoresis recovery purifying obtains promptly is Probe1.

2) Southern is hybridized the preparation of used probe template (Probe 2 and Probe 3)

a)Probe 2

The APP695cDNA that obtains with the sieve storehouse is a template, uses primer P3/P4 to carry out pcr amplification, and resulting 459bp fragment is Probe 2.

b)Probe 3

Cut the plasmid that contains APP695cDNA with Kpn I and Acc I enzyme, electrophoresis, the 167bp fragment that the recovery purifying obtains is Probe 3.

The acquisition of embodiment 2APP639cDNA

The contriver through two-wheeled hybridization, obtains 17 positive colonies with Probe 1 screening people tire brain cDNA library (available from GIBCO company) altogether.Utilizing primer P1/P6 (table one) to carry out pcr analysis and enzyme cuts and identifies these clones.Fig. 2-2 has shown the PCR result of part positive colony.In 17 positive colonies, there are 14 clones to contain the encoding sequence of app gene, one of them is cloned corresponding to APP770, and a clone is corresponding to APP751, and all the other 12 clones belong to the APP695 type.The contriver has selected several clones corresponding to APP695 and has checked order (by the order-checking of basic Kanggong department), found that Clone I3 except not containing the 7th, 8 exons, also lacks the 2nd exon, causes the 1st directly to be connected with the 3rd exon.Complete sequence determination shows that Clone I3 total length is 3011bp, reads frame and contains 1920bp, infers that encoded protein contains 639 amino acid.Therefore, the contriver is with Clone I3 called after APP639 (Fig. 1).

Table one PCR primer

Primer	Region	Direction	Sequence
				P1 P2 P3 P4 P5 P6	Exon	6 Exon 14 Exon 1 Exon 4 Exon 6 Exon 11	Sense Antisense Sense Antisense Antisense Antisense	5’-GAAGAGGCTGAGGAACCCTACG-3’ 5’-TCCATTCACGGGAAGGAGCTCC-3’ 5’-GTTTGGCACTGCTCCTGCTG-3’ 5’-TCTCTTTGGCGACGGTGTGC-3’ 5’-CACCATCCTCATCGTCCTCG-3’ 5’-TGAGCATGGCTTCCACTCTGGC-3’

The acquisition and the preservation of embodiment 3 tissues

Tissue-derived in 2 aborted fetuses (being provided by preclinical medicine institute of Fudan University), 8 have the sporadic AD patient of obvious pathological characters and 9 old normal controls of non-dementia.From the tissue of 23 months aborted fetuses, promptly obtained with interior at postabortal 3 hours, the tissue branch of collection is packed into to drop into immediately behind the clean 1.5m1 centrifuge tube and is filled in the container of liquid nitrogen, places-80 ℃ of environment to preserve then.From the cerebral tissue of the dull-witted old normal control group of AD patient and non-by Dutch international think-tank (Netherlands Brain Bank, NBB) necrotomy system acquisition fast.In case collect, cerebral tissue is through micrography, and the operation according to standard immediately separates.Tissue sample after the quick-frozen, is stored in-80 ℃ in liquid nitrogen.Grownup's liver organization cDNA is that the Zhao Mujun researcher of Institute Of Biochemistry And Cell Biology, Shanghai Institutes For Biological Sciences, Chinese Academy Of Sciences is so kind as to give.

Embodiment 4RT-PCR and the expression of Southern hybridization analysis APP 639mRNA in human embryo's different tissues

The different tissues that separates embodiment 3 described miscarriage embryos, extract RNA respectively, utilize primer P3/P5 to carry out RT-PCR (Fig. 3 A), carry out Southern hybridization with embodiment 1 described probe 2, probe 3 with pcr amplification product, analyze the expression of APP mRNA in tissues such as embryo and brain, kidney, liver, muscle, heart, pancreas, lung and small intestine.With APP639cDNA and APP695cDNA respectively as over against photograph; Distilled water carries out Southern hybridization as negative contrast.APP639cDNA among Fig. 3 (swimming lane 1), APP695cDNA (swimming lane 2), distilled water (swimming lane 3), total RNA derives from brain (swimming lane 4), kidney (swimming lane 5), spleen (swimming lane 6), lung (swimming lane 7), heart (swimming lane 8), muscle (swimming lane 9), liver (swimming lane 10), small intestine (swimming lane 11) respectively.

Fig. 3 B shows the expression of APP639mRNA in the various tissues of people embryo.These the institute in a organized way in, all the amplification obtain two fragments of molecular weight about 744bp and 576bp, they correspond respectively to APP695 and APP639.The fragment of 744bp should contain the 2nd exon, and the 576bp fragment does not then contain the 2nd exon.The Southern hybridization of Fig. 3 C for using Probe 2 to carry out as probe.Probe 2 comprises the sequence of exon 2 and both sides thereof, all can hybridize with two amplified fragments.The Southern hybridization of Fig. 3 D for using Probe 3 to carry out as probe.Probe 3 only contains the sequence of exon 2, can only hybridize with the amplified fragments that contains exon 2, thereby can only discern the big fragment of 744bp.The contriver uses Probe 2 and Probe 3 to carry out Southern hybridization.As shown in Figure 3A, Probe 2 contains the 2nd complete exon and the contiguous sequence of both sides thereof, corresponding to APP695cDNA the 158th to 616bp between nucleotide sequence; Thereby it can be hybridized with the adjacent domain outside exon 2 and the exon 2.And Probe 3 only contains the sequence of the 2nd exon, thereby it can only be hybridized with the nucleic acid fragment that contains the exon 2 sequence.This shows no matter two fragments that amplification obtains among Fig. 3 B comprise or do not comprise exon 2, can both hybridize with Probe 2; And the pcr amplified fragment that only contains the 2nd exon can be by Probe 3 marks.The result of Southern hybridization has confirmed contriver's supposition.With APP695 and APP639cDNA is over against photograph, and two pcr amplification product fragments in each swimming lane can both be by Probe 2 identifications (Fig. 3 C); Yet Probe 3 but can only the bigger amplified band corresponding with APP695 (Fig. 3 D) of tagged molecule amount.Above RT-PCR and Southern hybridization analysis show that APP639mRNA all has expression in everyone embryonic tissue that the contriver detected, and it is expressed in liver and muscle is abundanter, lower in other tissue (Fig. 3 C).The contriver uses primer that P3/P4 is repeated above experiment, has obtained identical result.

All can obtain corresponding two amplified fragments in people embryo brain and liver organization, molecular weight is approximately the fragment of 300bp corresponding to APP639, and the fragment about 460bp is corresponding to APP695 (Fig. 2-4A).And the segmental expression of 460bp is higher than the 300bp fragment in the tire brain, and then just in time opposite in the tire liver, the segmental expression of 300bp is higher than 460bp fragment (Fig. 2).

Getting in touch of the 460bp that obtains for further conclusive evidence amplification and fragment about 300bp and app gene transcription product, the PCR product is purified, is cloned into the pGEM-T-easy carrier and (available from Promega company, USA), checks order.Sequencing result shows, corresponding to containing app gene the 2nd exon over against fragment according to about 460bp of APP695, and exons 1 be connected with exon 2 (Fig. 2 C); Corresponding to over against not containing the 2nd exon, make exons 1 directly link to each other with exon 3 (Fig. 2 C) according to the fragment about the 300bp of APP639.

Embodiment 5RT-PCR analyzes the expression of APP639mRNA at grownup's pallium and liver organization.From embodiment 3 described pallium, extract RNA, utilize primer P3/P5 to carry out RT-PCR, carry out DNA polyacrylamide gel electrophoresis (concentration is 5%) and silver and dye.APP639cDNA among Fig. 4 A (swimming lane 1) and APP695cDNA (swimming lane 2) are respectively as over against photograph; Distilled water (swimming lane 3) is as negative contrast.The contriver (derives from AD patient: n=9 in all pallium samples that obtain; Derive from non-dull-witted old contrast: n=10) all fail to detect the app gene transcription product that lacks exon 2.The pcr amplification result who has only shown sample segment among the figure, the sample source of swimming lane 4-8 is in 5 non-dull-witted old contrasts; The sample source of swimming lane 9-13 is in 5 AD patients; Shown in arrow among the figure, in all brain cortical tissues, only can increase obtains the band corresponding with APP695.Fig. 4 A shows that APP639mRNA is in the corticocerebral expression of grownup.

The contriver is in an only liver sample reverse transcription product (Institute Of Biochemistry And Cell Biology, Shanghai Institutes For Biological Sciences, Chinese Academy Of Sciences gives), and amplification has obtained corresponding with APP695 and APP639 respectively band (shown in the arrow).Fig. 4 B: show the expression of APP639mRNA at grownup's liver organization.

Embodiment 6myc-APP639/pcDNA3 Construction of eukaryotic

1) with APP639cDNA is template, uses 5 ' primer (5 '-TTT TCT AGAGAT GCT GCC CGGTTT G-3 ') and 3 ' primer (5 '-GAG TCT AGACTA GTT CTG CAT CTG C-3 ') carries out pcr amplification.(in primer, import The site of Xba I)

2) purifying pcr amplified fragment uses Xba I enzyme to cut 37 ℃＞4 hours; Also use Xba I enzyme to cut to carrier myc/pcDNA3 (purchasing company) simultaneously in American I nvitrogen, 37 ℃ 2 hours, CIP handled 1 hour.

3) electrophoresis, purifying reclaim and insert fragment and carrier segments, quantitatively.

4) connect, 16 ℃, spend the night.

5) electric transform bacteria is coated with the AP plate, 37 ℃ of overnight incubation.

6) choose the clone, enzyme is cut evaluation.

The expression of embodiment 7APP639 fusion rotein in the HEK293 cell

From the APP639cDNA complete sequence that obtains, the contriver infers that its proteins encoded contains 639 amino-acid residues.In order further to confirm this cDNA sequence corresponding proteins matter of encoding really, the contriver clones into eukaryotic expression vector myc/pcDNA3 with APP639cDNA, and APP639 respectively transient transfection go into the HEK293 cell (blank of empty carrier, among Fig. 51,4,7 swimming lanes, the HEK293 cell is available from the Shanghai Inst. of Life Science, CAS cell bank), the HEK293 cell of APP695-myc/pcDNA3 is (among Fig. 52,5,8 swimming lanes) and the HEK293 cell of APP639-myc/pcDNA3 (among Fig. 53,6,9 swimming lanes), utilize the means of WesternBlot to detect the proteic expression of APP639.Utilize myc antibody (purchasing company), successfully detect APP695-myc fusion rotein (Fig. 5 swimming lane 2 is shown in the round dot) and APP639-myc fusion rotein (Fig. 5 swimming lane 3 is shown in the rhombus), and do not detect respective strap at control group in American I nvitrogen.(purchase company by APP antibody 22C11 simultaneously in German Roche, the proteic 66-81 amino acids of identification APP695, identification APP albumen n end 66-81 amino acids) and 6E10 (purchase Signet company in the U.S., identification A β 1-17 amino acids is corresponding to the proteic 597-613 amino acids of APP695) further confirmed associated protein.

22C11 can detect endogenous APP695 albumen (shown in the trilateral) and external source APP695-myc albumen, but can not detect APP639-myc albumen.And 6E10 is an A β 1-17 amino acids owing to what discern, so not only can discern endogenous APP695 albumen and external source APP695-myc albumen, and can detect APP639-myc albumen, this has further pointed out the APP639-myc protein band, and explanation APP639-myc albumen lacks exon 2 really.As Fig. 5, the proteic molecular weight of APP639-myc is slightly less than APP695-myc albumen, greatly about about 120kD.Because its C end contains the myc tumor-necrosis factor glycoproteins of about 60 amino-acid residues, so the contriver infers that the proteic molecular weight of APP639 is approximately 110kD.Bibliographical information, the proteic molecular weight of APP does not wait at 90kD-120kD.Contriver's result meets with it.

Embodiment 8 analyzes the disappearance of exon 2 to the proteometabolic influence of APP

Method as embodiment 6 makes up APP639/pcDNA3, and the APP695/pcDNA3 carrier is set up APP639, and the APP695 stable expression cell strain is set up A β detection model, and the disappearance of analyzing exon 2 is analyzed the variation that A β generates to the proteometabolic influence of APP.

Embodiment 9 investigates growth and the function effect of APP639 to each organ of animal especially liver

Cross expression APP639 in animal embryo, investigate growth and the function effect of the existence of APP639 each organ of animal especially liver; Set up APP639knock out animal model, investigate growth and the function effect of the inhibition of APP639, as the phenotype variation of liver each organ of animal especially liver.

Embodiment 10 observes differentiation influence and the possible aixs cylinder cynapse growth effect of APP639 albumen to neurocyte

Cultivate the APP639 cell, the collection condition nutrient solution is added in the neurocyte training liquid, observes differentiation influence and possible aixs cylinder cynapse growth effect to neurocyte.

SEQUENCE LISTING

＜110〉Shanghai Inst. of Life Science, CAS

＜120〉amyloid precursor protein 639, its encoding sequence and purposes

<130>030626

<160>1

<170>PatentIn version 3.1

<210>1

<211>639

<212>PRT

＜213〉amyloid precursor protein 639

<400>1

Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg

1 5 10 15

Ala Leu Glu Val Tyr Pro Glu Leu Gln Ile Thr Asn Val Val Glu Ala

20 25 30

Asn Gln Pro Val Thr Ile Gln Asn Trp Cys Lys Arg Gly Arg Lys Gln

35 40 45

Cys Lys Thr His Pro His Phe Val Ile Pro Tyr Arg Cys Leu Val Gly

50 55 60

Glu Phe Val Ser Asp Ala Leu Leu Val Pro Asp Lys Cys Lys Phe Leu

65 70 75 80

His Gln Glu Arg Met Asp Val Cys Glu Thr His Leu His Trp His Thr

85 90 95

Val Ala Lys Glu Thr Cys Ser Glu Lys Ser Thr Asn Leu His Asp Tyr

100 105 110

Gly Met Leu Leu Pro Cys Gly Ile Asp Lys Phe Arg Gly Val Glu Phe

115 120 125

Val Cys Cys Pro Leu Ala Glu Glu Ser Asp Asn Val Asp Ser Ala Asp

130 135 140

Ala Glu Glu Asp Asp Ser Asp Val Trp Trp Gly Gly Ala Asp Thr Asp

145 150 155 160

Tyr Ala Asp Gly Ser Glu Asp Lys Val Val Glu Val Ala Glu Glu Glu

165 170 175

Glu Val Ala Glu Val Glu Glu Glu Glu Ala Asp Asp Asp Glu Asp Asp

180 185 190

Glu Asp Gly Asp Glu Val Glu Glu Glu Ala Glu Glu Pro Tyr Glu Glu

195 200 205

Ala Thr Glu Arg Thr Thr Ser Ile Ala Thr Thr Thr Thr Thr Thr Thr

210 215 220

Glu Ser Val Glu Glu Val Val Arg Val Pro Thr Thr Ala Ala Ser Thr

225 230 235 240

Pro Asp Ala Val Asp Lys Tyr Leu Glu Thr Pro Gly Asp Glu Asn Glu

245 250 255

His Ala His Phe Gln Lys Ala Lys Glu Arg Leu Glu Ala Lys His Arg

260 265 270

Glu Arg Met Ser Gln Val Met Arg Glu Trp Glu Glu Ala Glu Arg Gln

275 280 285

Ala Lys Asn Leu Pro Lys Ala Asp Lys Lys Ala Val Ile Gln His Phe

290 295 300

Gln Glu Lys Val Glu Ser Leu Glu Gln Glu Ala Ala Asn Glu Arg Gln

305 310 315 320

Gln Leu Val Glu Thr His Met Ala Arg Val Glu Ala Met Leu Asn Asp

325 330 335

Arg Arg Arg Leu Ala Leu Glu Asn Tyr Ile Thr Ala Leu Gln Ala Val

340 345 350

Pro Pro Arg Pro Arg His Val Phe Asn Met Leu Lys Lys Tyr Val Arg

355 360 365

Ala Glu Gln Lys Asp Arg Gln His Thr Leu Lys His Phe Glu His Val

370 375 380

Arg Met Val Asp Pro Lys Lys Ala Ala Gln Ile Arg Ser Gln Val Met

385 390 395 400

Thr His Leu Arg Val Ile Tyr Glu Arg Met Asn Gln Ser Leu Ser Leu

405 410 415

Leu Tyr Asn Val Pro Ala Val Ala Glu Glu Ile Gln Asp Glu Val Asp

420 425 430

Glu Leu Leu Gln Lys Glu Gln Asn Tyr Ser Asp Asp Val Leu Ala Asn

435 440 445

Met Ile Ser Glu Pro Arg Ile Ser Tyr Gly Asn Asp Ala Leu Met Pro

450 455 460

Ser Leu Thr Glu Thr Lys Thr Thr Val Glu Leu Leu Pro Val Asn Gly

465 470 475 480

Glu Phe Ser Leu Asp Asp Leu Gln Pro Trp His Ser Phe Gly Ala Asp

485 490 495

Ser Val Pro Ala Asn Thr Glu Asn Glu Val Glu Pro Val Asp Ala Arg

500 505 510

Pro Ala Ala Asp Arg Gly Leu Thr Thr Arg Pro Gly Ser Gly Leu Thr

515 520 525

Asn Ile Lys Thr Glu Glu Ile Ser Glu Val Lys Met Asp Ala Glu Phe

530 535 540

Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu Val Phe Phe

545 550 555 560

Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val

565 570 575

Gly Gly Val Val Ile Ala Thr Val Ile Val Ile Thr Leu Val Met Leu

580 585 590

Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly Val Val Glu Val Asp

595 600 605

Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys Met Gln Gln Asn

610 615 620

Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gln Met Gln Asn

625 630 635

Claims (7)

1. an isolating amyloid precursor protein 639 is characterized in that, is the polypeptide of SEQ ID NO:1 aminoacid sequence.

2. isolating polynucleotide is characterized in that, for being selected from down a kind of nucleotide sequence of group:

(a) polynucleotide of polypeptide according to claim 1 of encoding;

(b) with the complete complementary polynucleotide of polynucleotide (a).

3. polynucleotide as claimed in claim 2 is characterized in that, this polynucleotide encoding aminoacid sequence is the polypeptide of SEQ ID NO:1.

4. a carrier is characterized in that, it contains the described polynucleotide of claim 2.

5. a genetically engineered host cell is characterized in that, it contains the described carrier of claim 4.

6. the preparation method with active amyloid precursor protein 639 is characterized in that, this method comprises:

(a) being fit to express under amyloid precursor protein 639's the condition, cultivate the described host cell of claim 6;

(b) from culture, isolate and have the active polypeptide of amyloid precursor protein 639.

7. energy and the described amyloid precursor protein 639's specificity of claim 1 bonded antibody.

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