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

Abstract

The invention provides a gene E-tau obtained by RT-PCR from earthworm total RNA, a protein E-Tau expressed by the gene, a recombinant vector containing the above-mentioned gene sequence, its subclones and recombinant microorganisms, and a gene constructed from the gene E-tau. eukaryotic expression vectors and transfected cell lines. The gene belongs to a new microtubule-associated protein Tau gene, and its expression product belongs to a new protein. It has been partially understood in the field that neurotau is closely related to neurodegenerative diseases, especially the pathological process of Alzheimer's disease (AD); this protein can promote microtubule assembly and stabilize the microtubule network. The invention also relates to the application of the gene in the treatment of neurodegenerative diseases.

Description

Earthworm Microtubule Binding Protein Tau, Its Encoding Gene and Its Application

technical field

The invention belongs to the field of genetic engineering, in particular, the invention relates to a gene Etau encoding a microtubule binding protein of earthworms. The present invention also relates to the function of the protein encoded by the gene, the recombinant vector containing the above gene sequence and its subclones and recombinant microorganisms, as well as the eukaryotic expression vectors and transfected cell lines obtained from these subclones. In addition, the present invention also relates to the function of the gene in the assembly of neuron microtubules and its application in the study of neurodegenerative diseases in clinical medicine.

Background technique

In 1975, Weingarten et al. discovered a protein with high affinity to microtubule proteins in the process of studying the molecular structure of the neuron microtubule system, and classified it as a microtubule associated protein (MAP), named For neural tau. The protein can promote microtubule assembly and stabilize the microtubule network, and is related to signal transduction, material transportation and nerve cell development. Studies in the past ten years have shown that neuro tau is closely related to neurodegenerative diseases, especially the pathological process of Alzheimer's disease (AD). Abnormal hyperphosphorylation of Tau is a major component of neurofibrillary tangles (NFTs), which is an important pathological feature of neurodegenerative diseases such as AD. In 1998, Hutton et al. discovered that the cause of frontotemporal dementia with parkinsonsm linked to chromosome 17 (FTDP-17) was a mutation in the tau gene on the chromosome 17. . Recent studies have shown that this protein also exists in the nucleus of nerve cells, can interact with DNA, and has the function of stabilizing the double helix structure; it can also interact with some proteins with important functions, showing the function similar to molecular chaperones. Because tau plays an important role in the study of the pathological mechanism of neurodegenerative diseases such as AD, it has become one of the most watched members of the microtubule-binding protein family.

Since tau was first isolated from adult brain tissue, it has been known to exist in several forms (isomers), which were later found to be the result of developmental regulation (Matus, 1988). Molecular cloning studies have found that there are at least six tau protein isoforms in the nervous system of adult mammals, which are derived from the same gene and obtained by different splicing after mRNA transcription.

The Tau gene is located on chromosome 17 and is highly conserved among species. So far, tau clones have been obtained from mouse, rat, bovine and human expression libraries (Lee et al., 1988; Goedert et al., 1988; Goedert et al., 1989a; Himmler, 1989a; Himmler et al., 1989b; Mori et al., 1989; Kosik et al., 1989; Kanai et al., 1989; Goedert et al., 1989b). The six isoforms of human tau that have been isolated in the form of full-length cDNA clones are composed of 352-441 amino acids, and the difference mainly lies in the presence or absence of three insertion sequences (Goedert et al., 1988; Goedert et al., 1989a; Goedert et al., 1989b; Goedert and Jakes, 1990).

Conditions associated with tau protein deposition are not limited to Alzheimer's disease, but are also observed in many central nervous system disorders, such as Pick's disease (pericardial pseudocirrhosis) and progressive supranuclear palsy (Joachim et al. , 1987). In addition, tau was found to be a component of paired helical fibers in the brains of most elderly Down syndrome patients. This suggests that tau may be affected in a relatively non-specific manner for the damage of various nerve cells. In other words, different modifications of tau, each targeting a specific disease, may lead to similar neurofibrillary tangle formation.

Tau is one of the key molecules studied in the pathology of Alzheimer's disease and has been implicated in several other neurodegenerative diseases (Grundke-Iqbal et al., 1986a; Grundke-Iqbal et al., 1986b; Iqbal et al., 1986). Statistics show that all tau-related pathologies account for 75% of dementias. Among these neurological diseases, familial hereditary frontotemporal dementia has attracted widespread attention. Patients with this type of familial dementia have certain missense mutations in the tau gene (Poorkaj et al., 1998; Hutton et al., 1998; Spillantini et al., 1998), indicating that single-gene mutations of the tau protein can cause abnormalities in its accumulation and processing. directly lead to dementia. In order to understand the molecular mechanism of Alzheimer's disease and other pathological processes related to tau protein, it is necessary to understand the function of tau protein in various aspects.

Tau was originally discovered as an important brain microtubule-binding protein that promotes microtubule assembly and stabilizes the latter's structure (Weingarten et al., 1975). In the 1990s, several laboratories reported that tau exists in the nucleus of various cell lines (Loomis et al., 1990; Davis and Johnson, 1999), and Greenwood et al. found that nuclear tau binds directly or indirectly to DNA (Greenwood and Johnson, 1995), so it is speculated that tau protein has other functions besides being a microtubule-binding protein. Understanding the function of tau will undoubtedly help to elucidate the molecular mechanism of Alzheimer's disease and other pathological processes related to tau protein.

So far, although Tau protein has been found in many animal species and a lot of research has been done on its function, there has never been a report of Tau protein found in earthworms. According to the conservation of the Tau structure of fruit flies, rats, mice and humans, the inventors found a new Tau gene by RT-PCR from the total RNA of earthworm body parts and ganglion parts, named Etau, and The restriction zymogram, sequence and expressed protein were analyzed, and polyclonal antibody was obtained. In addition, the inventors also studied the expression pattern of the gene in eukaryotic cells, and found that the gene is related to the assembly of microtubules, signal transduction, material transportation and nerve cell development.

Contents of the invention

The present invention provides a gene E-tau obtained by RT-PCR from earthworm total RNA and the protein E-Tau expressed by the gene, a recombinant vector containing the above-mentioned gene sequence, its subcloning and recombinant microorganism, and a gene constructed from the gene E-tau eukaryotic expression vectors and transfected cell lines. The gene belongs to a new microtubule-associated protein Tau gene, and its expression product belongs to a new protein. It has been partially understood in the art that neuronal Tau protein has the function of promoting microtubule assembly and stabilizing microtubule network, and is related to signal transduction, material transportation and nerve cell development; neuronal tau is associated with neurodegenerative diseases, especially Alzheimer's (Alzheimer's disease, AD) is closely related to the pathological process. The invention also relates to the application of the protein E-Tau expressed by the gene in the preparation of medicines for treating neurodegenerative diseases, especially Alzheimer's disease.

The gene Etau provided by the invention has a polynucleotide sequence as shown in SEQ ID NO.1. On the other hand, the deletion, insertion or substitution of one or more bases in the gene should also be included in the content of the present invention. Therefore, the present invention should include variants of the polynucleotide having the sequence shown in SEQ ID NO.1, the sequence of these variants has at least 80% homology with the sequence shown in SEQ ID NO.1, preferably at least 90%.

Therefore, the present invention also provides fragments of variants of the gene.

On the other hand, the present invention provides recombinant vectors containing the gene and its variants or fragments thereof. The selected vector is usually capable of functioning in the particular host cell used, that is, the vector is compatible with the host cell, thereby enabling gene amplification and/or gene expression. In one embodiment of the present invention, the above-mentioned carrier is pET28a (NOVAGEN), and what is inserted is Etau or its fragment, and the insertion site can be Nco I and Xho I or EcoR I and BamH I etc.

Preferred vectors for practicing the present invention are those compatible with bacterial, yeast and mammalian host cells. These include, inter alia, pCRII, pCR3 and pcDNA3.1 (Invitrogen, San Diego, CA), pB SII (Stratagene, La Jolla, CA), pET 15 (Novagen, Madison, WI), pGEX (Pharmacia Biotech, Piscataway, NJ), pEGFP -N1 (Clontech, Palo Alto, CA), pETL (BlueBacII, Invitrogen), pDSR-alpha (PCT Pub. No. WO90/14363) and pFastBacDual (Gibco-BRL, Grand Island, NY).

On the other hand, the present invention also provides a recombinant microorganism containing the above-mentioned recombinant vector. After the construction of the vector is completed, that is, after the polynucleotide of the present invention is inserted into the appropriate part of the vector, the resulting vector can be introduced into a suitable host cell for amplification and (or) expression of the polynucleotide of the present invention. Vectors can be transformed into selected host cells by methods well known in the art, including, for example, transfection, infection, calcium chloride, electroporation, microinjection, lipofection, DEAE-dextran method, or other known methods. The method of choice will vary in part according to the type of host cell used. These methods, as well as other suitable methods, are well known to those skilled in the art and are described in Sambrook et al., "Molecular Cloning Laboratory Manual" (Cold Spring Harbor Laboratories, 1989) and Davis et al., "Essential Methods in Molecular Biology" ( Both are described in Elsevier, 1986).

Amplification and (or) expression of the polynucleotide of the present invention can be carried out in prokaryotic host cells, yeast host cells, insect host cells (baculovirus system) and (or) eukaryotic host cells. The choice of expression host cell depends to some extent on whether post-translational modifications (such as glycosylation and/or phosphorylation) are to be performed on the protein of interest. Yeast host cells, insect host cells or mammalian host cells are preferred, if desired. For a review of expression vectors, refer to Meth. Enz., Vol. 185 (D.V. Goeddel, ed., Academic Press 1990).

On the other hand, the present invention also provides a method for obtaining the expressed protein of the present invention. The method involves culturing host cells to synthesize the protein, harvesting and lysing the host cells, and selectively recovering the product by methods well known in the art.

On the other hand, the present invention also provides transfected cell lines of the gene and its variants or fragments thereof. In an embodiment of the present invention, GFP-fused Etau is used to transfect eukaryotic cells, and a stably transfected cell line is obtained. Such cell lines include, for example, 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(ATCC CRL-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 (ATCC CCL-107) and other eukaryotic cell lines (purchased from ATCC).

In addition, polyclonal antibodies to Etau can also be obtained according to conventional techniques in the art. These GFP-labeled Etau cell lines and polyclonal antibodies can be used for in situ hybridization to study the expression pattern of the gene Etau of the present invention, but are not limited thereto.

Because neuro tau is closely related to neurodegenerative diseases, especially the pathological process of Alzheimer's disease (AD). The present invention also relates to the application of protein E-Tau in the preparation of medicines for treating neurodegenerative diseases, especially Alzheimer's disease.

Description of drawings

Figure 1: Agarose gel representation of earthworm total RNA.

Figure 2: Agarose gel diagram showing RT-PCR of the present invention. M: DNA marker; 1: EtauDNA, in which the gene of the present invention is in the box.

Figure 3: Restricted agarose gel representation of polynucleotides of the present invention. M: DNA marker; 1: recombinant vector; 2: empty vector; 3: double-digested recombinant vector; 4: Etau DNA.

Figure 4: Represents the enzyme-digested agarose gel diagram of Etau-GFP constructed by the gene of the present invention through the eukaryotic expression vector. M: DNA marker; 1: recombinant vector; 2: empty vector; 3: double-digested recombinant vector; 4: Etau DNA.

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

Figure 6: It shows the expression pattern of Etau-GFP in cells constructed from the gene of the present invention through the eukaryotic expression vector.

Fig. 7: Western blot diagram showing the polyclonal antibody obtained from the gene of the present invention. M: Standard molecular weight; 1: The immune band of the purified protein of the present invention, its molecular weight is about 46KDa.

Fig. 8: The ultraviolet spectrogram showing the interaction between the protein E-Tau expressed by the gene of the present invention and the microtubules. Add E-Tau (2 μM) to the microtubule protein (10 μM) solution incubated at 37 ° C, and use a Hitachi spectrophotometer to detect the change in the absorbance of the solution at 350 nm (the absorbance of the solution will become larger after the assembly of the microtubules). Curve 1: only microtubule protein in the solution; curve 2: both microtubule protein and E-Tau protein in the solution.

Figure 9: Determination of the restriction enzyme map of Etau and construction of various subclones.

Figure 10: Comparison of Etau and human tau (Htau) protein activity (add E-Tau or Htau (2 μM) to the tubulin (10 μM) solution incubated at 37 ° C, and use a Hitachi spectrophotometer to detect the absorbance of the solution at 350 nm Variety).

Figure 11: Examination of expression products of purified Etau subclones by SDS-PAGE. M: standard protein molecular weight, B: subclone B, E: subclone E.

Figure 12: Determination of biological activity of subclones B and E protein expression products (add B or E (2 μM) to the tubulin (10 μM) solution incubated at 37 ° C, and use a Hitachi spectrophotometer to detect the change in the absorbance value of the solution at 350 nm ).

Detailed ways

The present invention will be described in detail below in conjunction with preferred embodiments, but it is not meant to limit the content of the present invention.

Example 1: Preparation of total RNA from earthworms

Step 1: Handling and preparation of laboratory equipment

1) Plastic products: (including pipette tips, EP tubes, homogenate tubes, etc.)

First pour DEPC (diethyl pyrocarbonate) water from the volumetric flask into the ceramic jar, and soak the plastic products in it one by one. The small gun tip needs to be injected with DEPC water through a straw, overnight, then high pressure, and then dried for later use. Put the tip of the gun into the tip table before putting it into the tip table, and then press it again (EP tube);

2) Glass products: Soak in acid overnight, rinse well, and dry with tin foil for later use (DEPC blisters) (after washing, first soak in 1‰ DEPC overnight, then dry);

3) Homogenizer: (including scissors, tweezers) wash first, then high pressure (no need to soak DEPC);

4) Roast the scissors used for tissue removal on the fire.

Step 2: Earthworm Sample Preparation

1) First prepare the aluminum foil or cryopreservation tube used to pack the sample, and write the sample number on the surface of the aluminum foil or cryopreservation tube with an oil-based marker.

2) After accurately excising the required tissues of earthworms (Esiena fetida), immediately remove connective tissue and adipose tissue and other non-research-required tissue types on ice.

3) Rapidly rinse the sample in RNase-free PBS to remove blood stains and dirt.

4) Load the wrapped tissue with the prepared aluminum foil or cryopreservation tube, and quickly put it into liquid nitrogen for cooling.

Step 3: Grinding and Homogenization of Tissue Samples

1) Remove the sample bag from the cryopreserved sample, weigh it on an electronic balance, transfer it to a mortar pre-cooled with liquid nitrogen, and grind the tissue with a pestle, adding liquid nitrogen continuously until it is ground into a powder .

2) Transfer the powdered sample to a homogenization tube to which an appropriate amount of TRIzol reagent has been added, place the homogenization tube in an ice bath, and perform homogenization on a tissue homogenizer. The homogenate should be transparent and free of particles.

3) Carefully draw the homogenate into a new centrifuge tube, and place it at 15-30°C for 5 minutes.

4) Centrifuge the centrifuge tube at 4° C., 12000 rpm, for 10 min.

5) Carefully pipette the supernatant into a new centrifuge tube.

Step 4: Extraction of earthworm total RNA

1) Add chloroform to the lysate, close the cap of the centrifuge tube tightly, vibrate the centrifuge tube vigorously (the solution is fully emulsified, becomes milky white, and has no phase separation); place at 15-30°C for 3 minutes.

2) Centrifuge at 12000 rpm for 15 minutes at 4°C.

3) Carefully take out the centrifuge tube from the centrifuge, and draw the supernatant with a volume about half of the initial volume of TRIzol reagent added to another RNase-free centrifuge tube.

Step 5: Washing and elution of earthworm total RNA (using Qiagen RNA extraction kit) (QIAGEN)

1) Add one volume of 70% ethanol to the supernatant obtained by centrifugation in step 4, and invert repeatedly until evenly mixed.

2) Carefully draw 700 μL of the mixture (including the precipitate) and transfer it to the RNease spin column (spin column) placed in a 2 mL collection tube (provided in the kit), centrifuge at 10,000 rpm for 15 seconds, discard the filtrate and collection tube.

3) Transfer the RNease spin column to a new 2mL collection tube. Pipette 700μL BufferRW1 into the RNease spin column, centrifuge at 10,000rpm for 15sec for washing, discard the filtrate and collection tube.

4) Transfer the RNease spin column to a new 2mL collection tube. Pipette 500μL BufferRPE onto the RNease spin column, centrifuge at 10,000rpm for 15sec for washing, discard the filtrate and collection tube.

5) Open the RNease spin column carefully, add 500μL Buffer RPE, close the lid, and centrifuge at 14,000rpm for 3min.

6) Transfer the RNease spin column to a new 2mL collection tube and centrifuge at 14,000rpm for 1min.

7) Transfer the RNease spin column to a 1.5mL centrifuge tube, add 30μL RNase-free water on the surface of the RNease membrane, leave it at room temperature for 1min, and centrifuge at 10,000rpm for 1min.

8) Add 30 μL of RNase-free water and centrifuge at 10,000 rpm for 1 min.

Step 6: Earthworm RNA detection

The resulting total RNA was detected in 1% agarose gel, as shown in Figure 1.

Precautions

All experimental procedures (including centrifugation) must be performed at room temperature (15-25°C).

       Example 2 Obtaining Etau by RT-PCR

The following oligonucleotide sequences were chemically synthesized (Shanghai Sangong):

Primer 1: 5'ATGGAGCCCCGCCAGGAGTTCGAA 3'

Primer 2: 5'CAAACCCTGCTTGGCCAGGGAGGC 3'

The earthworm total RNA in Example 1 was taken, and RT-PCR one step kit (Qiagen Inc.) was used to operate according to the method described in the instructions. Using primer 1 and primer 2, use Taq DNA polymerase to carry out PCR reaction, incubate at 95°C for 5 minutes, after incubating at 50°C for 30 minutes, enter the following cycle: denaturation at 94°C for 1 minute, annealing at 58°C for 1 minute, extension at 72°C for 1 minute , cycled 35 times, extended at 72°C for 10 minutes, and cooled to 4°C to obtain the nucleotide sequence of Etau. The resulting DNA is cloned at the EcoR I site of the cloning vector pBluescript II KS-plasmid (NOVAGEN), and its sequence is shown in SEQ ID NO.1.

According to the characteristics of the carrier pBluescript II KS, T3 primer and T7 primer were used as primers, and T3 polymerase and T7 polymerase were used to sequence the positive and negative directions of Etau respectively (Shanghai Sangong). The total length of the nucleic acid sequence of this gene is 1053bp. The gene nucleotide sequence (that is, the entire open reading frame) is shown in SEQ ID NO.1, and the protein deduced from the nucleotide sequence is 351 amino acids, and the sequence is shown in SEQ ID NO.2.

Example 3: Determination of the restriction enzyme map of Etau and construction of various subclones

Step 1: Carry out single-digestion, double-digestion and multiple-digestion on the Etau clone of the present invention, and calculate the size of each fragment by agarose gel electrophoresis, analyze its multiple cloning site, and draw a restriction enzyme map , see Figure 3.

Step 2: Etau was digested with Pst I, and the digested fragment was recovered by agarose gel electrophoresis, then digested with the same enzyme (Pst I) as the pET28a vector, and finally obtained by ligation reaction construction Clones A and B; Etau was double digested with Pfu I/Ace II to construct subclone C; in addition, BlpI/Pfu I, Pfu I/Pst I, Pst I/Hind III, Pfu I/BsrFI, BsrF were respectively used I/Xho I and Ace II/Xho I double digested Etau to construct subclones D, E, F, G, H and I; a total of 9 subclones of different fragment sizes of Etau were obtained, but not limited to this, See Figure 9.

Embodiment 4: Construction of green fluorescent protein and Etau fusion protein (pEGFP-N1-Etau)

We used pEGFP-N1 eukaryotic expression vector (CLONTECH) to connect the amino-terminal of GFP to the carboxyl-terminal of Etau. Etau full-length DNA was amplified from earthworm total RNA by RT-PCR reaction. The upstream primers are:

5'CTCGAGATGGAGCCCCGCCAGGAGTTCGAA 3', an Xho I site was added before the start codon ATG; the downstream primer was: 5'-GGATCCAACAAACCCTGCTTGGCCAGGGAGGC 3', a BamH I site was introduced before the start codon ATG of GFP. In order to construct the Etau-GFP fusion gene, the Etau DNA was first cloned into the pEGFP-N1 vector by Xho I and BamH I double enzyme digestion, and pEGFP-N1-Etau was constructed and verified by sequencing, as shown in Figure 4.

    Example 5: Expression and purification of the full-length Etau gene

Step 1: Construction of expression plasmid. Etau was digested with Nco I and Xho I, and the expression vector pET 28a used was also double digested with these two enzymes, and then the above fragments were connected with T4 DNA ligase, named pET 28a-Etau. The restriction map and DNA sequencing results showed that the construction of the expression plasmid was correct.

Step 2: Expression and purification of recombinant protein. Transform pET 28a-Etau into BL21(DE3) (Stratagene) strain competent cells by CaCl ₂ method, and culture in LB medium at 37°C. When OD ₆₀₀ =0.6, IPTG (final concentration: 0.4 mM) was added to induce the expression of the gene of the present invention, and the culture was continued for 3 hours, and the cells were collected by centrifugation. Suspend the cells with phosphate buffer (pH 7.3, aprotinin 1 μg/ml, PMSF 100 μg/ml) added with protease inhibitors, suspend with 50 ml of buffer per 1 liter of culture medium, and lyse the cells by ultrasonic in an ice bath. Subsequently, TritonX-100 (final concentration 1%) was added and stirred gently for 30 minutes. The supernatant was collected by centrifugation (4°C, 10000 rpm), affinity-adsorbed with Ni ⁺⁺ -chelating Sepharose 4B column, and eluted with 50 mM imidazole to remove impurity proteins. Then collect 200mM imidazole elution fraction, dialyze, vacuum freeze-dry, this sample is verified by SDS-PAGE experiment at last, the result shows that its molecular weight is consistent with expectation (about 46KDa), and its purity is more than 90%, see Fig. 5 .

    Example 5: Expression pattern of transfected eukaryotic cells

Choose HEK 293, HeLa, COS-7, CHO, 5HSY-5Y and other eukaryotic cell lines for transfection.

Step 1: Prepare transfected cells (using Inventrogen's transfection reagent lipofectamine2000)

The day before transfection, cells were trypsinized and counted, and the cells were plated in 500 μL/2 mL of serum-containing, normal growth medium (LB medium) without antibiotics (0.5-2×10 ⁵ /3×10 ⁵ per well cells). Make it reach 90-95% confluence on the day of transfection.

Step 2: Prepare DNA/liposome complexes

1) For each well of cells, dilute 0.8 μg DNA/4.0 μg DNA with 50 μL/250 μL serum-free medium (such as OPTI-MEMI, DMEM medium), and mix gently.

2) Gently mix the Lipofectamine 2000 transfection reagent before use, and dilute 2 μL/10 μL Lipofectamine 2000 transfection reagent with 50 μL/250 μL serum-free medium for each well of cells. Mix gently and incubate at room temperature for 5 minutes.

3) Mix the diluted DNA and the diluted Lipofectamine 2000, the total volume at this time is 100 μL/500 μL. Mix gently and let stand at room temperature for 20 minutes to allow the DNA-Lipofectamine 2000 complex to form. The solution may be cloudy, but this does not affect transfection efficiency.

Step 3: Transfect cells

1) Aspirate the old nutrient solution in the 24-well plate and wash it twice with serum-free medium. Add 0.5ml/2ml serum-free medium.

2) Add 100μL/500μL liposome/DNA mixture dropwise to each well (from one side of the culture well to the other side), shake the culture plate back and forth while adding, and mix gently.

3) Incubate at 37°C, 5% CO ₂ for 24-48 hours. There is no need to remove complexes or change media. Or changing the culture growth medium after 4-5 hours will not reduce the transfection activity.

4) After adding the complex to the cells for 24-72 hours, observe the transfection efficiency under a fluorescent microscope, analyze the cell extract or perform in situ cell staining, and detect various indicators.

Step 4: Screen for Stably Transfected Cell Lines

1) Apply screening pressure 24 hours after transfection, and culture in medium containing G418 (C ₂₀ H ₄₀ N ₄ O ₁₀ ·2H ₂ SO ₄ , no Chinese name, an antibiotic). After 14 days of continuous culture at the screening concentration of G418, pick out a single clone and expand the culture, during which the medium was changed every 3 days.

2) After transfection, the cells were cultured in non-selective medium for 18-24 hours to express the transfected exogenous gene, digested with trypsin, diluted and passaged at 1:15-1:20, and inoculated into various selective The culture was continued in the culture medium, and the culture medium was changed every 2 to 4 days within 2 to 3 weeks, so that the resistant clones could grow (as shown in Figure 6).

3) Select clones for expanded culture, and then carry out hybridization analysis of DNA or RNA, detection of newly synthesized proteins and detection of related indicators.

     Example 5: Application of purified Etau products to prepare antibodies

After the purified recombinantly expressed Etau product is further purified, it is directly dissolved in water, and the single protein thus obtained can be used to immunize animals with methods well known in the art to prepare polyclonal antibodies. After the polyclonal antibody was obtained, its titer and immune characteristics were tested. Figure 7.

   Example 6: The purified Etau product is suitable for molecular neurobiology

It is known that the product of this gene, Etau, is a neural Tau protein that can initiate microtubule assembly and stabilize the microtubule network (as shown in Figure 8), and is related to signal transduction, material transport, and nerve cell development. Neurotau is associated with neurodegeneration Diseases, especially closely related to the pathological process of Alzheimer's disease (AD). Therefore, the protein product can be used to study its interaction with DNA and protein, so as to further clarify the regulatory role of Tau in neurodegenerative diseases. Therefore, the protein E-Tau of the present invention can also be used to prepare drugs for treating neurodegenerative diseases, especially Alzheimer's disease.

Example 7: Antibodies to Etau products are suitable for immunocytochemistry and immunohistochemistry

According to the method described in the "Molecular Cloning Experiment Manual" (Cold Spring Harbor Laboratories, 1989), the Etau antibody can be used to perform in situ hybridization on earthworm tissues and cells, so as to determine the expression pattern of the Etau gene and the timing and timing of the function of the Tau protein. location, and expression intensity.

Example 8: Identification of biological activity of Etau subcloning product protein

According to the expression method of the full-length Etau gene, the subcloned fragments B and E in Example 3 were connected to the corresponding expression vector pET 28a with T4 ligase after corresponding double enzyme digestion, and then the plasmid was transferred into BL21 Competent cells were expressed and purified to obtain the corresponding proteins (see Figure 11), and it was found that the subcloned proteins B and E all had certain activities, namely promoting the assembly of tubulin (see Figure 12).

Example 9: Activity comparison between earthworm Etau and human Htau

Comparison of Etau and human tau (Htau) protein activity (add E-Tau or Htau (2 μM) to the tubulin (10 μM) solution incubated at 37 ° C (tubulin is extracted from pig brain, extraction method reference (Robley C.1982), Htau protein is obtained after the human tau gene connected to the expression vector is transferred into Escherichia coli to express and purify according to the literature. The meter detects the change of the absorbance value of the solution at 350nm). The experimental results are shown in Figure 10. It can be seen that the earthworm Etau has a higher activity of promoting tubulin assembly than the known human Htau.

references

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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-binding domains 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 neurodegenerative disorders. 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

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Sequence Listing

<110> Institute of Biophysics, Chinese Academy of Sciences

<120>Earthworm microtubule-binding protein Tau, its coding gene and its application

<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 aggggggctga tggtaaaacg aagatcgcca caccgcgggg agcagcccct 300

ccaggccaga agggccaggc caacgccacc aggattccag caaaaaccccc gcccgctcca 360

aagacaccac ccagctctgg tgaacctcca aaatcagggg atcgcagcgg ctacagcagc 420

cccggctccc caggcactcc cggcagccgc tcccgcaccc cgtcccttcc aacccccaccc 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 accacgtcc 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 (15)

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.

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