CN1873005A

CN1873005A - Conjugated protein Tau of microtubule of earthworm, its coding gene, and application

Info

Publication number: CN1873005A
Application number: CN 200510011850
Authority: CN
Inventors: 赫荣乔; 赵静; 王东亮; 王兴胜; 赫坚
Original assignee: Institute of Biophysics of CAS
Current assignee: Institute of Biophysics of CAS
Priority date: 2005-06-02
Filing date: 2005-06-02
Publication date: 2006-12-06

Abstract

This invention provides E-Tau protein expressed by E-tau gene produced from Lumbricus terrestris total RNA by RT-PCR, recombinant vector containing E-tau gene and its subclones, recombinant microorganisms, eukaryotic expression vector containing E-tau gene and relevant transfected cell lines. E-tau gene is a new type of microtubule-associated gene, which is closely related to Alzheimer's disease. E-Tau protein can accelerate microtubule assembly and stabilize microtubule net. This invention also relates to the application of E-tau gene in neurodegenerative disease treatment.

Description

Conjugated protein Tau of microtubule of earthworm, its encoding gene and application thereof

Technical field

The invention belongs to the genetically engineered field, specifically, the gene Etau of the microtubule bindin of a kind of earthworm that the present invention relates to encode.The invention still further relates to the protein function of this genes encoding, the recombinant vectors that contains the said gene sequence and subclone and recombinant microorganism, and be by carrier for expression of eukaryon and the cells transfected that these subclones acquire.In addition, the invention still further relates to this gene in neurone microtubule assembling effect and clinical medicine in to the application of nerve degenerative diseases research.

Background technology

1975, Weingarten etc. have found that a kind of and microtubular protein has the protein of high-affinity, ranges microtubule bindin (microtubule associated protein with it in the molecular structure process of research neurone microtubule system, MAP), the neural tau of called after.This protein has the effect that promotes microtubule assembling and stabilize microtubules network, grows relevant with signal transduction, matter transportation and neurocyte.Studies show that of nearly more than ten years, neural tau and nerve degenerative diseases, particularly with presenile dementia (Alzheimer ' s disease, pathologic process AD) is closely related.The unusual super phosphorylation of Tau is the main component of neurofibrillary tangles (NFTs), and NFTs is a kind of important pathological feature of nerve degenerative diseases such as AD.1998, Hutton etc. have found that (frontotemporaldementia with parkinsonsm linked to chromosome 17, cause of disease FTDP-17) is that sudden change has taken place the tau gene on this karyomit(e) to sample volume temporal lobe dementia to the relevant Parkinson's disease of No. 17 karyomit(e) (Parkinson ' s PD).Studies show that recently this protein also is present in the neurocyte nuclear, can interact with DNA, has and stablizes double-stranded effect; Can also with some protein interaction with critical function, show the function of similar molecular chaperones.Because the critical role of tau in the research of nerve degenerative diseases pathomechanisms such as AD makes it become one of member who attracts most attention in the microtubule bindin family.

Since tau at first is extracted out from the adult cerebral tissue, people just know that it exists with several forms (isomer), found afterwards that this was the result (Matus, 1988) of developmental regulation.Molecular cloning research has now found that and has six kinds of Protein tau matter isomer in the adult mammalian nervous system at least, and they derive from same gene, different montages after mRNA transcribes and obtaining.

The Tau gene is positioned on No. 17 karyomit(e), and it is high conservative between kind of system.Up to now, clone (Lee etc., 1988 of tau from mouse, rat, ox and the mankind's expression library, have been obtained; Goedert etc., 1988; Goedert etc., 1989a; Himmler, 1989a; Himmler etc., 1989b; Mori etc., 1989; Kosik etc., 1989; Kanai etc., 1989; Goedert etc., 1989b).At present the isomer of six kinds of human tau that are separated with the full length cDNA clone form is made up of 352-441 amino acid respectively, and the existence that difference mainly is three insertion sequences is (Goedert etc., 1988 whether; Goedert etc., 1989a; Goedert etc., 1989b; Goedert and Jakes, 1990).

The illness relevant with Protein tau matter deposition has more than and is limited to alzheimer's disease, also can observe in many central nervous system diseases, such as paralysis (Joachim etc., 1987) on Pick's disease (Pick's disease) and the carrying out property nuclear.In addition, find that also tau is the integral part of pairing spiral sample fiber in most of aged Down's syndrome patient's brain.This prompting is for multiple nerve cell damage, and tau may be affected in a kind of non-relatively narrow spectrum mode.In other words, the different modifying of tau, each may cause the formation of similar neurofibrillary tangles at specific disease.

Tau is that Alzheimer disease pathologic is learned one of key molecule in the research, and relevant (Grundke-Iqbal etc., 1986a with other several nerve degenerative diseases; Grundke-Iqbal etc., 1986b; Iqbal etc., 1986).Statistical figure show that all pathology relevant with tau have occupied 75% of dementia.In these neural diseases, familial inheritance volume temporo page or leaf dementia has caused widely to be paid close attention to.The patient of this kind familial idiocy, some missense mutation (Poorkaj etc., 1998 have taken place in its tau gene; Hutton etc., 1998; What Spillantini etc., 1998), the single gene mutation that shows Protein tau matter caused its accumulation and processing can directly cause dull-witted the generation unusually.In order to understand fully the molecular mechanism of alzheimer's disease and other pathological processes relevant with Protein tau matter, the function of understanding Protein tau matter each side is very necessary.

Tau is found with a kind of important brain microtubule bindin at first, and it can promote the assembling of microtubule and the stable latter's structure (Weingarten etc., 1975).The nineties, several laboratories have reported that respectively tau is present in (Loomis etc., 1990 in the nucleus of various kinds of cell strain system; Davisand Johnson, 1999), people such as Greenwood find to examine tau has direct or indirect combining (Greenwood and Johnson, 1995) with DNA, therefore infers that Protein tau matter except that as the microtubule bindin, also has other function.The function of understanding fully tau will help to illustrate the molecular mechanism of the pathological process of alzheimer's disease and other and Protein tau qualitative correlation undoubtedly.

Up to the present, although in many animal species, found Tau protein and its function has been done big quantity research, never see and in earthworm, find the proteinic report of Tau.Conservative property according to fruit bat, rat, mouse and human Tau structure, the inventor is from total RNA of earthworm body portion and neuroganglion part, find a new Tau gene by RT-PCR, called after Etau, and its restricted zymogram, sequence and expressed protein are analyzed, and obtained polyclonal antibody.In addition, the inventor has also studied the expression pattern of this gene in eukaryotic cell, and it is relevant with signal transduction, matter transportation and neurocyte growth to find that this gene and the assembling of microtubule reach.

Summary of the invention

The invention provides protein E-Tau, the recombinant vectors that contains the said gene sequence and the subclone and the recombinant microorganism of a kind of gene E-tau that from the total RNA of earthworm, obtains and this genetic expression by RT-PCR, and by this gene constructed carrier for expression of eukaryon, transfectional cell series.This gene belongs to the gene of a kind of new microtubule-associated proteins Tau, and its expression product belongs to a kind of new protein.The effect that neural Tau protein has assembling of promotion microtubule and stabilize microtubules network is understood in this area part, grows relevant with signal transduction, matter transportation and neurocyte; Neural tau and nerve degenerative diseases, particularly with presenile dementia (Alzheimer ' s disease, pathologic process AD) is closely related.The invention still further relates to the application of protein E-Tau in the medicine of preparation treatment nerve degenerative diseases of this genetic expression, this nerve degenerative diseases is presenile dementia particularly.

Gene Etau provided by the invention has the polynucleotide sequence shown in SEQ ID NO.1.On the other hand, the disappearance of the one or more bases in this gene, insertion or substitute produces and has the varient that has an identical function with sequence shown in the SEQ ID NO.1 and also should be included within the content of the present invention.Therefore, the present invention should comprise the varient with polynucleotide of sequence shown in the SEQ ID NO.1, and the homology of sequence is at least 80% shown in the sequence of these varients and the SEQ ID NO.1, preferably is at least 90%.

So the present invention also provides the fragment of the varient of this gene.

On the other hand, the invention provides and contain the segmental recombinant vectors of this gene and varient or its thereof.The carrier of selecting can be brought into play function usually in the particular host cell of using, that is to say, carrier and host cell compatibility, thus the amplification that can realize gene with (or) expression of gene.In one embodiment of the invention, above-mentioned carrier is pET28a (NOVAGEN), insertion be Etau or its fragment, inserting the site can be Nco I and Xho I or EcoR I and BamH I etc.

Be used to realize preferred vector of the present invention be can be compatible with bacterium, yeast and mammalian host cell those carriers.Wherein especially comprise pCRII, pCR3 and pcDNA3.1 (Invitrogen, SanDiego, CA), pB SII (Stratagene, La Jolla, CA), pET 15 (Novagen, Madison, WI), pGEX (Pharmacia Biotech, Piscataway, NJ), pEGFP-N1 (Clontech, PaloAlto, CA), pETL (BlueBacII, Invitrogen), pDSR-α (PCT Pub.No.WO90/14363) and pFastBacDual (Gibco-BRL, Grand Island, NY).

On the other hand, the present invention also provides the recombinant microorganism that contains above-mentioned recombinant vectors.When vector construction is finished, be about to polynucleotide of the present invention and be inserted into after the suitable position of carrier, just the gained carrier can be imported in the appropriate host cell, be used for polynucleotide of the present invention amplification and (or) expression.Can carrier be transformed in the selected host cell with method well known in the art, these methods comprise, for example, and infection protocol, infection method, Calcium Chloride Method, electroporation, microinjection, lipofection, DEAE-dextran method, or known additive method.The method of selecting can have part to change according to the type of used host cell.These methods and other appropriate means are known by the person skilled in art, and " molecular cloning laboratory manual " (Cold Spring Harbor Laboratories people such as Sambrook, 1989) and in " molecular biology basic skills " (Elsevier, 1986) of people such as Davis all describe to some extent.

The amplification of polynucleotide of the present invention and (or) express can prokaryotic host cell, yeast host cell, insect host cell (rhabdovirus system) and (or) carry out in the eukaryotic host cell.Being chosen in of expression host cell depends on whether will carry out posttranslational modification (as glycosylation and/or phosphorylation) to target protein matter in a way.If desired, then preferably yeast host cell, insect host cell or mammalian host cell.The summary of relevant expression vector can be with reference to Meth.Enz., Vol.185 (D.V.Goeddel, ed., Academic Press 1990).

On the other hand, the present invention also provides a kind of method, in order to obtain marking protein of the present invention.This method comprises cultivates host cell with synthetic this protein, collects then and the cracking host cell, optionally reclaims product with method well known in the art again.

On the other hand, the present invention also provides this gene and varient or its thereof segmental transfectional cell series.In embodiments of the invention, the Etau that uses GFP to merge comes transfecting eukaryotic cells, and obtains the clone of stable transfection.This clone for example comprises HEK 293 (ATCC CRL-1573), HeLa (ATCC CCL-2), NIH 3T3 (ATCC CRL-1658), BNL CL.2 (ATCC TIB-73), HepG2 (ATCC HB-8065), COS7 (ATCC CRL-1651), CHO (ATCCCRL-9618), SH-SY5Y (ATCC CRL-2266), IMR 32 (ATCC CCL-127), MRC5 (ATCC CCL-171), MCF7 (ATCC HTB-22), 562 (ATCC CCL-243), SKOV-3 (ATCC HTB-77), HUV-EC (ATCC CRL-1730) (primary cell) and C6 eukaryotic cell lines (available from ATCC) such as (ATCC CCL-107).

According to this area routine techniques method, also obtain the polyclonal antibody of Etau in addition, the Etau clone of these marks GFP and polyclonal antibody can be used in situ hybridization studying the expression pattern of gene Etau of the present invention, but are not limited to this.

Because neural tau and nerve degenerative diseases, particularly with presenile dementia (Alzheimer ' sdisease, pathologic process AD) is closely related.The invention still further relates to the application of protein E-Tau in the medicine of preparation treatment nerve degenerative diseases, this nerve degenerative diseases is presenile dementia particularly.

Description of drawings

Fig. 1: the sepharose figure of the total RNA of expression earthworm.

Fig. 2: the sepharose figure of expression RT-PCR of the present invention.The M:DNA mark; 1:EtauDNA wherein is gene of the present invention in the square frame.

Fig. 3: the restriction enzyme digestion sepharose figure of expression polynucleotide of the present invention.The M:DNA mark; 1: recombinant vectors; 2: empty carrier; 3: the double digestion recombinant vectors; 4:Etau DNA.

Fig. 4: represent that gene of the present invention cuts sepharose figure through the enzyme of construction of eukaryotic expression vector Etau-GFP.The M:DNA mark; 1: recombinant vectors; 2: empty carrier; 3: the double digestion recombinant vectors; 4:Etau DNA.

Fig. 5: the expression of gene product of the present invention that detects purifying by SDS-PAGE.M: standard molecular weight; 1: the protein of the present invention of purifying, its molecular weight is about 46KDa.

Fig. 6: represent that gene of the present invention becomes the expression pattern of Etau-GFP in cell through construction of eukaryotic expression vector.

Fig. 7: the western blot figure that represents the polyclonal antibody of gene gained of the present invention.M: standard molecular weight; 1: the proteinic immune band of the present invention of purifying, its molecular weight is about 46KDa.

Fig. 8: the protein E-Tau and the interactional ultraviolet spectrogram of microtubule of expression genetic expression of the present invention.In microtubular protein (the 10 μ M) solution of 37 ℃ of insulations, add E-Tau (2 μ M), detect the variation (light absorption value of microtubule assembling back solution will become big) of solution at the 350nm light absorption value with Hitachi's spectrophotometer.Curve 1: have only microtubular protein in the solution; Curve 2: contain microtubular protein and E-Tau protein in the solution simultaneously.

The restriction map of Fig. 9: Etau determine and the structure of various subclones.

The contrast of Figure 10: Etau and human tau (Htau) protein-active (in microtubular protein (the 10 μ M) solution of 37 ℃ of insulations, add E-Tau or Htau (2 μ M), detect the variation of solution) at the 350nm light absorption value with Hitachi's spectrophotometer.

Figure 11: the expression product that detects the Etau subclone of purifying by SDS-PAGE.M: standard protein molecular weight, B: subclone B, E: subclone E.

Figure 12: the biological activity determination of subclone B, E protein expressioning product (in microtubular protein (the 10 μ M) solution of 37 ℃ of insulations, add B or E (2 μ M), detect the variation of solution) at the 350nm light absorption value with Hitachi's spectrophotometer.

Embodiment

Below in conjunction with preferred embodiment the present invention is done detailed explanation, but and do not mean that content constraints of the present invention.

Embodiment 1: the preparation of the total RNA of earthworm

Step 1: the processing and the preparation of experiment utensil

1) plastics: (comprising rifle head, EP pipe, homogenate pipe etc.)

Earlier DEPC (diethylpyrocarbonate) water is poured into from volumetric flask in the Porcelain Jar, plastics are soaked wherein one by one, wherein the lancet head needs suction pipe to squeeze into DEPC water, spend the night, high pressure is dried standby more then, before the experiment with the first-class suction nozzle platform of putting into of rifle, high pressure once (EP pipe) again;

2) glasswork: bubble acid is spent the night, and rinses well, covers tinfoil and dries standby (DEPC bubble) (clean bubble 1 ‰ DEPC of back elder generation and spend the night, dry);

3) homogenizer: after (comprising scissors, tweezers) clean earlier, high pressure (not needing to steep DEPC) again;

4) getting the scissors time spent of organizing usefulness bakes on fire.

Step 2: earthworm specimen preparation

1) at first is ready for aluminium foil or the freezing preservation pipe of packing sample, and writes sample number exactly at aluminium foil or frozen pipe appearance many places with permanent pen.

2) accurately excise the required tissue of earthworm (Eisenia foetida, Esiena fetida) after, reject the types of organization that non-institute such as reticular tissue and fatty tissue needs immediately on ice.

3) rapid rinsing sample in the PBS that does not contain RNase is to remove bloodstain and dirt.

4) load the parcel tissue with ready aluminium foil or freezing preservation pipe, drop into cooled with liquid nitrogen rapidly.

Step 3: the milling and homogenate of tissue samples

1) sample that very low temperature is preserved is removed sample sack, after weighing on the electronic balance, is transferred in the stone roller alms bowl with the liquid nitrogen precooling, with the pestle tissue of milling, constantly adds liquid nitrogen therebetween, and is Powdered until being milled into.

2) will be milled into pulverous sample, be transferred in the homogenate pipe that adds an amount of TRIzol reagent, the homogenate pipe will be placed ice bath, on the tissue homogenate pulverizer, carry out homogenate.Homogenate is more transparent and do not have particle and get final product.

3) carefully draw homogenate and change new centrifuge tube over to, place 5min at 15～30 ℃.

4) with centrifuge tube in 4 ℃, 12000rpm, 10min is centrifugal.

5) carefully draw supernatant liquor and change new centrifuge tube over to.

Step 4: the extracting of the total RNA of earthworm

1) in lysate, adds chloroform, cover tight centrifuge tube lid, with forced oscillation centrifuge tube (solution is fully emulsified, becomes milky white shape, no noted phase separation phenomena); Place 3min at 15～30 ℃.

2) in 4 ℃, 12000rpm, centrifugal 15min.

3) take out centrifuge tube carefully from whizzer, volume aspirated is about half supernatant of the initial add-on of TRIzol reagent to the centrifuge tube of another no RNA enzyme.

Step 5: the washing of the total RNA of earthworm and wash-out (use Qiagen RNA and extract test kit) are (QIAGEN)

1) in the centrifugal supernatant liquor that obtains of step 4, adding 70% long-pending ethanol of monoploid, puts upside down until mixing repeatedly.

2) careful absorption 700 μ L mixed solutions (comprising throw out wherein) are transferred to and are positioned in the RNease column spinner (spin column) that (provides in the test kit) in the 2mL collection tube, and 10, centrifugal 15 seconds of 000rpm discards filtered liquid and collection tube.

3) shift in RNease column spinner to the new 2mL collection tube.Draw 700 μ L BufferRW1 in the RNease column spinner, 10,000rpm is centrifugal, and 15sec washs, and discards filtered liquid and collection tube.

4) shift in RNease column spinner to the new 2mL collection tube.Draw 500 μ L BufferRPE to the RNease column spinner, 10,000rpm is centrifugal, and 15sec washs, and discards filtered liquid and collection tube.

5) the careful RNease column spinner of opening adds 500 μ L Buffer RPE, cover lid, 14, the centrifugal 3min of 000rpm.

6) shift in RNease column spinner to the new 2mL collection tube, 14, the centrifugal 1min of 000rpm.

7) shift the RNease column spinner in the 1.5mL centrifuge tube, add 30 μ L RNase-free water on RNease membrane surface, after room temperature is placed 1min, 10, the centrifugal 1min of 000rpm.

8) add 30 μ L RNase-free water, 10, the centrifugal 1min of 000rpm again.

Step 6: earthworm RNA detects

The total RNA of gained detects in 1% sepharose, as shown in Figure 1.

Precaution

All experimental procedures (comprising centrifugal) at room temperature (15～25 ℃) are carried out.

Embodiment 2 obtains Etau by RT-PCR

The following oligonucleotide sequence of chemosynthesis (worker is given birth in Shanghai):

Primer 1:5 ' ATGGAGCCCCGCCAGGAGTTCGAA 3 '

Primer 2: 5 ' CAAACCCTGCTTGGCCAGGGAGGC 3 '

Get the total RNA of earthworm among the embodiment 1, adopt RT-PCR one step kit (Qiagen Inc.), described to specifications method is operated.Utilize primer 1 and primer 2, use the Taq archaeal dna polymerase to carry out the PCR reaction, after the 95 ℃ of insulations 5 minutes, 50 ℃ of insulations 30 minutes, enter following circulation: 94 ℃ of sex change 1 minute, annealed 1 minute for 58 ℃, 72 ℃ were extended 1 minute, circulated 35 times, and 72 ℃ were extended 10 minutes, be cooled to 4 ℃, obtain the nucleotide sequence of Etau.In cloning vector pBluescript II KS-plasmid (NOVAGEN) EcoR I site, its sequence is shown in SEQ ID NO.1 with the dna clone of gained.

According to the characteristic of carrier pBluescript II KS, adopting T3 primer and T7 primer is primer, with T3 polysaccharase and T7 polysaccharase the positive and negative both direction of Etau is checked order (Shanghai life worker) the nucleotide sequence length overall 1053bp of this gene respectively.Its gene nucleotide sequence (being whole open reading-frame (ORF)s) the results are shown in SEQ ID NO.1, and the protein of being derived by this nucleotide sequence is 351 amino acid, and sequence is shown in SEQ ID NO.2.

The restriction map of embodiment 3:Etau determine and the structure of various subclones

Step 1: Etau clone of the present invention is carried out single endonuclease digestion, double digestion and multienzyme cut, and calculate each endonuclease bamhi size, and analyze its multiple clone site, draw out restriction map, see Fig. 3 by agarose gel electrophoresis.

Step 2: with Pst I Etau is carried out enzyme and cut, and reclaim this endonuclease bamhi by agarose gel electrophoresis, then with the pET28a carrier, (Pst I) cuts its enzyme with same enzyme, makes up by ligation at last and obtains subclone A and B; With Pfu I/Ace II Etau is carried out double digestion, make up and obtain subclone C; Use BlpI/Pfu I, Pfu I/Pst I, Pst I/Hind III, Pfu I/BsrFI, BsrF I/Xho I and Ace II/Xho I to the Etau double digestion in addition respectively, make up and obtain subclone D, E, F, G, H and I; Obtain 9 kinds of the subclones of Etau different fragments size altogether, but be not limited to this, see Fig. 9.

Embodiment 4: the structure of egfp and Etau fused protein (pEGFP-N1-Etau)

We use the carboxyl terminal that pEGFP-N1 carrier for expression of eukaryon (CLONTECH) is connected to the aminoterminal of GFP Etau.The Etau full length DNA is to use RT-PCR reaction amplification to obtain from the total RNA of earthworm.Upstream primer is:

5 ' CTCGAGATGGAGCCCCGCCAGGAGTTCGAA 3 ' has added Xho I site before codon ATG; Downstream primer is: 5 '-GGATCCAACAAACCCTGCTTGGCCAGGGAGGC 3 ', introduced BamH I site before the codon ATG of GFP.Be to make up the Etau-GFP fusion gene, Etau DNA is cloned in the pEGFP-N1 carrier by Xho I and BamH I double digestion earlier, is built into pEGFP-N1-Etau and through sequence verification, as shown in Figure 4.

The expression and purification of embodiment 5:Etau full-length gene

Step 1: the structure of expression plasmid.Cut Etau with Nco I and Xho I enzyme, simultaneously used expression vector pET 28a is also carried out double digestion with these two kinds of enzymes, with the T4DNA ligase enzyme above-mentioned fragment is coupled together called after pET 28a-Etau then.Restriction enzyme mapping and dna sequencing result show that the structure of expression plasmid is accurate.

Step 2: recombinant protein expression and purifying.Use CaCl ₂Method transforms (Stratagene) bacterial strain competent cell of BL21 (DE3) with pET 28a-Etau, with 37 ℃ of liquid culture of LB substratum.Work as OD ₆₀₀=0.6 o'clock, add IPTG (final concentration 0.4mM) and begin to induce expression of gene of the present invention, continue to cultivate centrifugal collection thalline 3 hours.With phosphoric acid buffer (pH 7.3, aprotinin 1 μ g/ml, PMSF 100 μ g/ml) the suspension thalline that adds the protein enzyme inhibitors, per 1 liter of nutrient solution suspends ultrasonic degradation thalline in ice bath with the 50ml damping fluid.Subsequently, add TritonX-100 (final concentration 1%), stirred gently 30 minutes.Centrifuging and taking supernatant (4 ℃ 10000rpm), are used Ni ⁺⁺The affine absorption of-chelating Sepharose 4B post is removed heteroproteins with 50mM imidazoles wash-out.Collect 200mM imidazoles elution fraction then, dialysis, vacuum lyophilization tests with SDS-PAGE at last and verify that this sample, result show its molecular weight and expection consistent (being about 46KDa), and its purity is seen Fig. 5 more than 90%.

Embodiment 5: the expression pattern of transfecting eukaryotic cells

Select HEK 293, HeLa, COS-7, CHO, eukaryotic cell lines such as 5HSY-5Y carry out transfection.

Step 1: prepare transfectional cell (using the transfection reagent lipofectamine2000 of Inventrogen)

Transfection the day before yesterday, trypsin digestion cell and counting, the cell bed board contains serum at 500 μ L/2mL, does not contain (every hole 0.5-2 * 10 in the substratum (LB substratum) of antibiotic normal growth ⁵/ 3 * 10 ⁵Individual cell).Make it can reach the fusion of 90-95% in transfection day.

Step 2: prepare dna/liposome complex

1), uses 50 μ L/250 μ L serum free mediums (as OPTI-MEMI, DMEM substratum) dilution, 0.8 μ g DNA/4.0 μ g DNA, mixing gently for every porocyte.

2) use before with Lipofectamine 2000 transfection reagents mixing gently, every porocyte dilutes 2 μ L/10 μ L Lipofectamine, 2000 transfection reagents with 50 μ L/250 μ L serum free mediums.Mixing gently, incubated at room 5 minutes.

3) Lipofectamine 2000 of the DNA of mixed diluting and dilution, this moment, cumulative volume was 100 μ L/500 μ L.Mixing gently, room temperature were placed 20 minutes so that DNA-Lipofectamine 2000 mixtures form.Solution may compare muddiness, but this does not influence transfection efficiency.

Step 3: transfectional cell

1), cleans twice with serum free medium with the old nutritive medium sucking-off in 24 orifice plates.Add the 0.5ml/2ml serum-free and join foster base.

2) dropwise add 100 μ L/500 μ L liposome/DNA mixtures (from culture hole on one side to the other side) in every hole, waggle culture plate, mixing gently before and after the edged of limit.

3) at 37 ℃, 5%CO ₂In hatched 24-48 hour.Need not to remove mixture or change substratum.Perhaps after 4-5 hour, change the incubation growth base and also can not reduce transfection activity.

4) add mixture after 24-72 hour in cell, fluoroscope is observed transfection efficiency down, and analysis of cells extract or carry out the original position cell dyeing detects every index.

Step 4: screening stably transfected cell line

1) the after-applied screening pressure of transfection 24h is used instead and is contained G418 (C ₂₀H ₄₀N ₄O ₁₀2H ₂SO ₄, no Chinese is a kind of microbiotic) culture medium culturing.After continuing to cultivate 14 days under the G418 screening concentration, choose mono-clonal, enlarged culturing, during changed liquid once in per 3 days.

2) cell is cultivated 18～24h after the transfection in non-selective nutrient solution, after making the foreign gene of transfection obtain expressing, trysinization, by 1: 15～1: 20 diluted passage, inoculation goes into to continue in the various selectivity nutrient solutions to cultivate, per 2～4 days replacing nutrient solutions once make resistance clone grown (as Fig. 6) in 2～3 weeks.

3) selected clone carries out enlarged culturing, carries out the hybridization analysis of DNA or RNA then, the detection of the new proteinic detection of synthetic and relevant every index.

Embodiment 5: the Etau product of application of purified is with preparation antibody

After purified recombinant expressed Etau product was further purified, directly soluble in water, the single protein that obtains like this can be with method immune animal known in the art with the preparation polyclonal antibody.After obtaining polyclonal antibody, detect it and tire and immunological characteristic.As Fig. 7.

Embodiment 6: the Etau product of purifying is applicable to molecular neurobiology

The product Etau of known this gene is that a kind of neural Tau protein has the effect (as Fig. 8) that starts microtubule assembling and stabilize microtubules network, grow relevant with signal transduction, matter transportation and neurocyte, neural tau and nerve degenerative diseases, particularly with presenile dementia (Alzheimer ' sdisease, pathologic process AD) is closely related.Therefore use this protein and can study itself and DNA and protein interactions, further to define the regulating and controlling effect in the Tau nerve degenerative diseases.Therefore, protein E-Tau of the present invention can also be used to prepare the medicine for the treatment of nerve degenerative diseases, and this nerve degenerative diseases is presenile dementia particularly.

The antibody of embodiment 7:Etau product is applicable to immunocytochemistry and immunohistochemistry

According to " molecular cloning laboratory manual " (Cold Spring Harbor Laboratories, 1989) method described in, the antibody of using Etau can carry out in situ hybridization to earthworm tissue and cell, thereby carry out time and position that Etau expression of gene pattern and Tau protein are carried out function, and expression intensity.

The bioactive evaluation of embodiment 8:Etau subclone product albumen

Expression method according to the Etau full-length gene, subclone fragment B, E among the embodiment 3 are connected among the expression vector pET 28a that corresponding enzyme cuts with the T4 ligase enzyme behind the double digestion of correspondence, then plasmid is changed over to the BL21 competent cell, expression and purification obtains corresponding proteins (seeing Figure 11), find subclone protein B, E, all have certain activity, promptly promote the assembling (seeing Figure 12) of tubulin.

Embodiment 9: the specific activity of earthworm Etau and people Htau

((tubulin extracts in the pig brain to add E-Tau or Htau (2 μ M) in microtubular protein (the 10 μ M) solution of 37 ℃ of insulations in the contrast of Etau and human tau (Htau) protein-active, extracting method reference (Robley C.1982), Htau albumen is will be connected to earlier according to document to obtain after people tau gene on the expression vector changes escherichia coli expression, purifying over to, express and purification process reference (Goedert and Jakes, 1990), detect the variation of solution) at the 350nm light absorption value with Hitachi's spectrophotometer.Experimental result is seen Figure 10.This shows that earthworm Etau has the activity of the promotion tubulin assembling higher than known people Htau.

Reference

1.Davis，P.K.and?Johnson，G.V.(1999)The?microtubule?binding?of?Tau?andhigh?molecular?weight?Tau?in?apoptotic?PC12?cells?is?impaired?because?ofaltered?phosphorylation.J.Biol.Chem.274，35686-35692

2.Goedert，M.，Wischik，C.M.，Crowther，R.A.，Walker，J.E.and?Klug，A.(1988)Cloning?and?sequencing?of?the?cDNA?encoding?a?core?protein?of?thepaired?helical?filament?of?Alzheimer?disease：identification?as?the?microtubule-associated?protein?tau.Proc.Natl.Acad.Sci.USA?85，4051-4055

3.Goedert，M.，Spillantini，M.G.，Potier，M.C.，Ulrich，J.，and?Crowther，R.A.(1989a)Cloning?and?sequencing?of?the?cDNA?encoding?an?isoform?ofmicrotubule-associated?protein?tau?containing?four?tandem?repeats：differentialexpression?of?tau?protein?mRNAs?in?human?brain.EMBO?J.8，393-399

4.Goedert，M.，Spillantini，M.G.，Jakes，R.，Rutherford，D.and?Crowther，R.A.(1989b)Multiple?isofoms?of?human?microtubule-associated?protein?tau：sequences?and?localization?in?neurofibrillary?tangles?of?Alzheimer′s?disease.Neuron?3，519-526.

5.Goedert，M.and?Jakes，R.(1990)Expression?of?separate?isoforms?of?humantau?protein：correlation?with?the?tau?pattern?in?brain?and?effects?on?tubulinpolymerization.EMBO?J.9，4225-4230

6.Greenwood，J.A.and?Johnson，G.V.W.(1995)Localization?and?in?situphosphorylation?state?of?nuclear?tau.Exp?Cell?Res?220，332-337.

7.Grundke-Iqbal，I.，Iqbal，K.，Quinlan，M.，Tung，Y.-C.，Zaidi，M.S.，andWisniewski，H.M.(1986a)Microtubule-associated?protein?tau.A?componentof?Alzheimer?paired?helical?filaments.J.Biol.Chem.261，6084-6089

8.Grundke-Iqbal，I.，Iqbal，K.，Tung，Y.-C.，Quinlan，M.，Wisniewski，H.M.，and?Binder，L.I.(1986b)Abnormal?phosphorylation?of?the?microtubule-associated?protein?tau(tau)in?Alzheimer?cytoskeletal?pathology.Proc.Natl.Acad.Sci.USA?83，4913-4917

9.Himmler，A.(1989a)Structure?of?the?bovine?tau?gene；alternatively?splicedtranscripts?generate?a?protein?family.Mol.Cell.Biol.9，1389-1396.

10.Himmler，A.，Drechsel，D.，Kirschner，M.W.and?Martin，D.W.Jr.(1989b)Tau?consists?of?a?set?of?proteins?with?repeated?C-terminal?microtubule-bindingdomains?and?variable?N-terminal?domains.Mol.Cell.Biol.9，1381-1388

11.Hutton，M.，Lendon，C.L.，Rizzu，P.，Baker，M.，Froelich，S.，Houlden，H.，Pickering-Brown，S.，Chakraverty，S.，Isaacs，A.，Grover，A.，Hackett，J.，Adamson，J.，Lincoln，S.，Dickson，D.，Davies，P.，Petersen，R.C.，Stevens，M.，de?Graaff，E.，Wauters，E.，van?Baren，J.，Hillebrand，M.，Joosse，M.，Kwon，J.M.，Nowotny，P.，Heutink，P.，et?al.(1998)Association?of?missense?and?5′-splice-site?mutations?in?tau?with?the?inherited?dementia?FTDP-17.Nature?393，702-705

12.Iqbal，K.，Grundke-Iqbal，I.，Zaidi，T.，Merz，P.A.，Wen，G.Y.，Shaikh，S.S.，Wisniewski，H.M.，Alafuzoff，I.，and?Winblad，B.(1986)Defective?brainmicrotubule?assembly?in?Alzheimer′s?disease.Lancet.2，421-426

13.Joachim，C.L.，Morris，J.H.，Kosik，K.S.and?Selkoe，D.J.(1987)Tauantisera?recognize?neurofibrillary?tangles?in?a?range?of?neurodegenerativedisorders.Ann.Neurol.22，514-520.

14.Kanai，Y.，Takemura?R，Oshima?T，Mori?H，Ihara?Y，Yanagisawa?M，MasakiT，Hirokawa?N.(1989)Expression?of?multiple?tau?isoforms?and?microtubulebundle?formation?in?fibroblasts?transfected?with?a?single?tau?cDNA.J.Cell?Biol.109，1173-1184.

15.Kosik，K.S.，Orecchio，L.D.，Bakalis，S.and?Neve，R.L.(1989)Developmentally?regulated?expression?of?specific?tau?sequences.Neuron?2，1389-1397

16.Lee，G.，Newman，S.T.，Gard，D.L.，Band，H.and?Panchamoorthy，G.(1998)Tau?interacts?with?src-family?non-receptor?tyrosine?kinases.J.Cell?Sci.111，3167-3177

17.Loomis，P.A.，Howard，T.H.，Castleberry，R.P.，and?Binder?LI(1990)Identification?of?nuclear?tau?isoforms?in?human?neuroblastoma?cells.Proc.Natl.Acad.Sci.USA?87，8422-8426

18.Matus，A.(1988)Microtubule-associated?proteins：their?potential?role?indetermining?neuronal?morphology.Annu.Rev.Neurosci.11，29-44.

19.Mori?H，Hamada?Y，Kawaguchi?M，Honda?T，Kondo?J，Ihara?Y.(1989)Adistinct?form?of?tau?is?selectively?incorporated?into?Alzheimer′s?paired?helicalfilaments.Biochem.Biophys.Res.Commun.159，1221-1226

20.Poorkaj，P.，Bird，T.D.，Wijsman，E.，Nemens，E.，Garruto，R.M.，Anderson，L.，Andreadis，A.，Wiederholt，W.C.，Raskind，M.，and?Schellenberg，G.D.(1998)TAU?as?a?susceptibility?gene?for?amyotropic?lateral?sclerosis-parkinsonism?dementia?complex?of?Guam.Ann.Neurol.43，815-825

21.Robley?C.，Williams?J.R.and?James?C.L.(1982)Preparation?of?tubulinfrom?brain.Methods?Enzymol.，85，376-85.

22.Spillantini，M.G.，Murrell，J.R.，Goedert，M.，Farlow，M.R.，Klug，A.，andGhetti，B.(1998)Mutation?in?the?tau?gene?in?familial?multiple?systemtauopathy?with?presenile?dementia.Proc.Natl.Acad.Sci.USA?95，7737-7741

23.Weingarten，M.D.，Lockwood，A.H.，Hwo，S.Y.，and?Kirschner，M.W.(1975)A?protein?factor?essential?for?microtubule?assembly.Proc.Natl.Acad.Sci.USA?72，1858-1862

Sequence table

＜110〉Institute of Biophysics, Academia Sinica

＜120〉conjugated protein Tau of microtubule of earthworm, its encoding gene and application thereof

<130>IB052846

<160>2

<170>PatentIn?version?3.1

<210>1

<211>1053

<212>DNA

＜213〉earthworm (Esiena fetida)

<400>1

atggagcccc?gccaggagtt?cgaagtgatg?gaagatcacg?ctgggacgta?cgggttgggg 60

gacaggaaag?atcagggggg?ctacaccatg?caccaagacc?aagagggtga?cacggacgct 120

ggcctgaaag?ctgaagaagc?aggcattgta?gacaccccca?gcctggaaga?cgaagctgct 180

ggtcacgtga?cccaagctcg?catggtcagt?aaaagcaaag?acgggactgg?aagcgatgac 240

aaaaaagcca?agggggctga?tggtaaaacg?aagatcgcca?caccgcgggg?agcagcccct 300

ccaggccaga?agggccaggc?caacgccacc?aggattccag?caaaaacccc?gcccgctcca 360

aagacaccac?ccagctctgg?tgaacctcca?aaatcagggg?atcgcagcgg?ctacagcagc 420

cccggctccc?caggcactcc?cggcagccgc?tcccgcaccc?cgtcccttcc?aaccccaccc 480

acccgggagc?ccaagaaggt?ggcagtggtc?cgtactccac?ccaagtcgcc?gtcttccgcc 540

aagagccgcc?tgcagacagc?ccccgtgccc?atgccagacc?tgaagaatgt?caagtccaag 600

atcggctcca?ctgagaacct?gaagcaccag?ccgggaggcg?ggaaggtgca?aatagtctac 660

aaaccagttg?acctgagcaa?ggtgacctcc?aagtgtggct?cattaggcaa?catccatcat 720

aaaccaggag?gtggccaggt?ggaagtaaaa?tctgagaagc?ttgacttcaa?ggacagagtc 780

cagtcgaaga?ttgggtccct?ggacaatatc?acccacgtcc?ctggcggagg?aaataaaaag 840

attgaaaccc?acaagctgac?cttccgcgag?aacgccaaag?ccaagacaga?ccacggggcg 900

gagatcgtgt?acaagtcgcc?agtggtgtct?ggggacacgt?ctccacggca?tctcagcaat 960

gtctcctcca?ccggcagcat?cgacatggta?gactcgcccc?agctcgccac?gctagctgac 1020

gaggtgtctg?cctccctggc?caagcagggt?ttg 1053

<210>2

<211>351

<212>PRT

＜213〉earthworm (Esiena fetida)

<400>2

Met?Glu?Pro?Arg?Gln?Glu?Phe?Glu?Val?Met?Glu?Asp?His?Ala?Gly?Thr

1 5 10 15

Tyr?Gly?Leu?Gly?Asp?Arg?Lys?Asp?Gln?Gly?Gly?Tyr?Thr?Met?His?Gln

20 25 30

Asp?Gln?Glu?Gly?Asp?Thr?Asp?Ala?Gly?Leu?Lys?Ala?Glu?Glu?Ala?Gly

35 40 45

Ile?Val?Asp?Thr?Pro?Ser?Leu?Glu?Asp?Glu?Ala?Ala?Gly?His?Val?Thr

50 55 60

Gln?Ala?Arg?Met?Val?Ser?Lys?Ser?Lys?Asp?Gly?Thr?Gly?Ser?Asp?Asp

65 70 75 80

Lys?Lys?Ala?Lys?Gly?Ala?Asp?Gly?Lys?Thr?Lys?Ile?Ala?Thr?Pro?Arg

85 90 95

Gly?Ala?Ala?Pro?Pro?Gly?Gln?Lys?Gly?Gln?Ala?Asn?Ala?Thr?Arg?Ile

100 105 110

Pro?Ala?Lys?Thr?Pro?Pro?Ala?Pro?Lys?Thr?Pro?Pro?Ser?Ser?Gly?Glu

115 120 125

Pro?Pro?Lys?Ser?Gly?Asp?Arg?Ser?Gly?Tyr?Ser?Ser?Pro?Gly?Ser?Pro

130 135 140

Gly?Thr?Pro?Gly?Ser?Arg?Ser?Arg?Thr?Pro?Ser?Leu?Pro?Thr?Pro?Pro

145 150 155 160

Thr?Arg?Glu?Pro?Lys?Lys?Val?Ala?Val?Val?Arg?Thr?Pro?Pro?Lys?Ser

165 170 175

Pro?Ser?Ser?Ala?Lys?Ser?Arg?Leu?Gln?Thr?Ala?Pro?Val?Pro?Met?Pro

180 185 190

Asp?Leu?Lys?Asn?Val?Lys?Ser?Lys?Ile?Gly?Ser?Thr?Glu?Asn?Leu?Lys

195 200 205

His?Gln?Pro?Gly?Gly?Gly?Lys?Val?Gln?Ile?Val?Tyr?Lys?Pro?Val?Asp

210 215 220

Leu?Ser?Lys?Val?Thr?Ser?Lys?Cys?Gly?Ser?Leu?Gly?Asn?Ile?His?His

225 230 235 240

Lys?Pro?Gly?Gly?Gly?Gln?Val?Glu?Val?Lys?Ser?Glu?Lys?Leu?Asp?Phe

245 250 255

Lys?Asp?Arg?Val?Gln?Ser?Lys?Ile?Gly?Ser?Leu?Asp?Asn?Ile?Thr?His

260 265 270

Val?Pro?Gly?Gly?Gly?Asn?Lys?Lys?Ile?Glu?Thr?His?Lys?Leu?Thr?Phe

275 280 285

Arg?Glu?Asn?Ala?Lys?Ala?Lys?Thr?Asp?His?Gly?Ala?Glu?Ile?Val?Tyr

290 295 300

Lys?Ser?Pro?Val?Val?Ser?Gly?Asp?Thr?Ser?Pro?Arg?His?Leu?Ser?Asn

305 310 315 320

Val?Ser?Ser?Thr?Gly?Ser?Ile?Asp?Met?Val?Asp?Ser?Pro?Gln?Leu?Ala

325 330 335

Thr?Leu?Ala?Asp?Glu?Val?Ser?Ala?Ser?Leu?Ala?Lys?Gln?Gly?Leu

340 345 350

Claims

1. polynucleotide, the homology of sequence is at least 80% shown in the sequence of these polynucleotide and the SEQ ID NO.1.

2. polynucleotide according to claim 1, wherein, the homology of sequence is at least 90% shown in the sequence of these polynucleotide and the SEQID NO.1.

3. polynucleotide according to claim 1, it has sequence shown in the SEQ ID NO.1.

4. protein, the homology of sequence is at least 80% shown in this proteinic aminoacid sequence and the SEQ ID NO.2.

5. protein according to claim 4, wherein, the homology of sequence is at least 90% shown in this proteinic aminoacid sequence and the SEQ ID NO.2.

6. protein according to claim 4, it has the aminoacid sequence shown in the SEQ ID NO.2.

7. recombinant vectors, this recombinant vectors is characterised in that and contains each described polynucleotide among the claim 1-3.

8. host cell that contains the described recombinant vectors of claim 7.

9. host cell according to claim 8, it is selected from prokaryotic cell prokaryocyte or eukaryotic cell.

10. the eukaryotic cell of each described polynucleotide transfection among the claim 1-3, this clone comprises: HEK 293, HeLa, NIH 3T3, BNL CL2, HepG2, COS-7, CHO, 5HSY-5Y, IMR 32, MRC5, MCF7,562, SKOV-3, IGROV-1, eukaryotic cell lines such as HUV-EC and C6.

11. a GFP fusion rotein is characterized in that containing the expressed protein of each described polynucleotide among the claim 1-3.

12. according to the application of each described polynucleotide among the claim 1-3 in the assembling of regulating cell microtubule.

13. according to any one the application of protein in the medicine of preparation treatment nerve degenerative diseases among the claim 4-6.

14. according to the application of claim 13, wherein said nerve degenerative diseases is a presenile dementia.

15. according to each described protein application in immunohistochemistry or Antibody Preparation in claim 4-6 and 11.