CN101258241A - Nucleic acid capable of binding to immunoglobulin G and use thereof - Google Patents

Abstract

A novel aptamer against IgG, use of the aptamer and others. More specifically, an aptamer capable of binding to the Fc segment of IgG (e.g., human IgG); a complex comprising the aptamer and a functional substance bound to the aptamer (e.g., an affinity substance, a labeling substance, an enzyme, a medicinal substance, a toxin, a drug delivery medium); a solid support having the aptamer or the complex immobilized thereto; a device for medical purposes comprising the solid support; a method for purification of an antibody, comprising holding an IgG antibody by adsorption on the solid support and eluting the adsorbed IgG antibody with an eluent; a process for producing a purified antibody, comprising preparing an IgG antibody and purifying the IgG antibody using the solid support; and others.

Description

With immunoglobulin G bonded nucleic acid and utilize method

Technical field

The present invention relates to immunoglobulin G (IgG) is had in conjunction with active nucleic acid.Purifying, mark, immobilization, modification that makes universal antibody by this nucleic acid etc. becomes possibility.

Background technology

IgG is one of main protein of serum, serves as the key player of identification foreign matter, guiding eliminating in immunity system.Apply flexibly this characteristic with its be applied to the curative of various diseases, the research in diagnosis medicine or the reagent is extensively carried out.One of them is in the antibody therapy to cancer, carrying out the exploitation of following aspect: utilize cytotoxicity (ADCC) that relies on antibody or the therapy that relies on the cytotoxicity (CDC) of complement, carry out the molecular targeted agents of hungry strategy (starvation tactics) with the acceptor of expressing in the antibodies specific blocking-up cancer cells etc., perhaps utilize guided missile (missle) therapy that is combined with the antibody of carcinostatic agent in the cancer cells specific antibody etc.Wherein, anti-HER2 acceptor peopleization monoclonal antibody is developed listing (trade(brand)name: Trastuzumab (Herceptin)) as the therapeutical agent of malignant tumours such as mammary cancer.In addition, utilize itself and antigen-specific bonded character, IgG to be used as and be determined as the cell of representative or the essential instrument of various biochemical experiments such as proteinic biological function explore, expression of gene screening with immunology.

IgG is that 2 H chains and 2 L chains are with disulfide linkage (S-S key) bonded Y font structure.Decompose if IgG is used as the papoid of protein decomposition enzyme, then can divide for Fc fragment of forming by constant region and the Fab fragment that contains antigen-binding site.In addition, IgG has subgroup to exist, and for human IgG four kinds of IgG1, IgG2, IgG3, IgG4 is arranged.

Antibody gets from serum or hybridoma (hybridoma) cell culture supernatant purifying with post for using antibody purification.Usually in the purifying of fs, use ProteinA as part.ProteinA is the protein of the molecular weight 42kDa of streptococcus aureus (Staphylococcus aureus) generation, and mortise is in the Fc zone of IgG.ProteinA price height, and the situation that can not obtain high purity antibody is arranged according to the difference of animal kind or subgroup, or the situation of antibody sex change is arranged under the purification condition that has used Protein A, seeking the novel separating agent that performance surpasses Protein A at present.

Antibody by fluorescent substance or enzyme labelling is used to various experiments such as immunohistochemical assay, tissue staining, ELISA, Western blotting, flow cytometry.For example in tissue staining, the antibody that can be combined with fluorescent substances such as FITC by use comes the proteinic tissue positioned of goal in research.In addition, in the mensuration of ELISA or Western blotting etc., an anti-material that detects with desire is reacted, then make and two anti-react of this anti-bonded, carry out the more mensuration of high sensitivity by this through mark.For example, in the ECL system of GE medical treatment (healthcare) company, use the antibody that is combined with horseradish peroxidase anti-, make the luminol,3-aminophthalic acid cyclic hydrazide oxy-luminescence, detect target substance by this by its katalysis as two.But making mark substance be incorporated into antibody by chemically modified wastes time and energy, and the sex change sometimes of different antibodies according to circumstances needs the new antibody labeling technology of exploitation.In addition, also expectation have more cheap and high sensitivity through marking two anti-.

As diagnosis chip, carrying out the exploitation of antibody chip at various diseases.One of its problem is: in the fixing means of antibody to substrate, make the exploitation of the method that the antigen-binding site of antibody arranges with the high reactivity state to high-density on the surface.In the fixation method of the fixation method of utilizing non-specific adsorption or utilization amino, antibody is arranged out of orderly and can not be obtained sufficient sensitivity.

As the molecular targeted curative for diseases such as cancer or rheumatosis, the development research of antibody is advanced rapidly, and existing so far about 20 kinds of antibody drugs are practical, and the clinical trial of about 300 kinds of candidate's antibody drugs is being implemented in the whole world in addition.At the beginning of the exploitation, use mouse monoclonal antibody as antibody drug, still, mouse antibodies is identified as foreign matter at human immune system, induces human anti-mouse antibody's generation, therefore can not fully improve result of treatment.Thereby the use gene recombination technology, developed constant zone with mouse antibodies be replaced as people's antibody constant zone chimera antibody maybe will except that the part the complementarity-determining region territory of mouse antibodies all displacement be the humanized antibodies of people's antibody.In addition, also developing the making method of the human monoclonal antibodies that has used the mouse (KM mouse) that produces people's antibody.

One of monoclonal antibody drug that uses in the antibody therapy makes its internalization (internalization) to target cell for by making carcinostatic agent or toxin be incorporated into the antibody of specific recognition cancer cells, and target cell is killed.Carcinostatic agent or toxin need to break away from from antibody after by internalization.Therefore, want to make antibody to be connected and contain proteolytic enzyme recognition site etc. in the base, the operation of implementing after by internalization to make carcinostatic agent or toxin to break away from and so on from antibody with carcinostatic agent or toxin bonded.For example the gemtuzumab ozogamicin (WAY-CMA 676 (Mylotarg)) that develops as the curative of acute myelogenous leukemia is for being incorporated into calicheamicin (calicheamicin) derivative the medicine of peopleization anti-CD 33 monoclonal antibody, therefore if WAY-CMA 676 is incorporated into CD33, internalization in cell, then the calicheamicin derivative takes place to dissociate, and cell is killed.Therefore, the design that antibody is connected base with carcinostatic agent or toxin bonded is very important, in order to draw higher drug effect, is carrying out the exploitation of novel connection base.

In recent years, RNA aptamers (aptamer) is gazed in the application of curative, diagnosis medicine, reagent, has several RNA aptamers to enter clinical stage or practicability stage.In December, 2004, what world's initiative had been admitted by the U.S. is the curative of the Ma Kugen (Macugen) of RNA aptamers medicine as senile macular degeneration SMD disease.The RNA aptamers is meant and target substance specificity bonded RNA such as protein, can use SELEX method (Fas lignand system of exponential enrichment is evolved (Systematic Evolution of Ligands by Exponential Enrichment)) preparation (Ellington etc., (1990) Nature, 346,818-822; Tuerk etc., (1990) Science, 249,505-510).The SELEX method is meant from about 10 ¹⁴The RNA pond (pool) with different IPs nucleotide sequence in screening and the method for target substance specificity bonded RNA.The stochastic sequence of the RNA that uses about for about 40 residues is clipped in the structure between the primer sequence.This RNA pond and target substance are merged, use only recovery and target substance bonded RNA such as strainer.The RNA that reclaims is increased with RT-PCR, used as with metacyclic template.By this operation is carried out about 10 times repeatedly, can obtain aptamers with target substance specificity bonded RNA.When the RNA aptamers that obtains promotes or suppress the function of target substance, this RNA aptamers can be applied to medicine etc.In fact, (the Japanese Patent spy opens the 2002-300885 communique with eIF4A as human translation initiation factor to use the preparation of SELEX method, Oguro etc., (2003) RNA 9,394-407) or eIF4E (the Japanese Patent spy opens the 2004-344008 communique, Mochizuki etc., (2005) RNA 11,77-89), acceptor RANK (ReceptorActivator of NF-κ B, Mori etc. relevant with bone metabolism, (2004) NucleicAcids Res.32, specificity bonded RNA aptamers such as 6120-6128).Also relevant for the report of the antigen recognition site bonded RNA aptamers by anti-DNA autoantibody (Kim etc., (2003) Biochemical and Biophysical Research Communication 300,516-523).

Summary of the invention

The objective of the invention is to, provide at the aptamers of IgG and utilize method etc.

The inventor etc. further investigate in order to solve above-mentioned problem, and the result has successfully prepared the good aptamers that highly designs at IgG, thereby finishes the present invention.

That is, the invention provides following invention etc.

[1] is incorporated into the aptamers of the Fc part of IgG.

[2] aptamers of above-mentioned [1] wherein, combines with Fc part specificity as the human IgG of the Fc of IgG part.

[3] aptamers of above-mentioned [1] or [2], wherein, the total nucleotide number that constitutes aptamers is below 40.

[4] any aptamers in above-mentioned [1] to [3], wherein, at least a Nucleotide that contains in the aptamers is selected from hydrogen atom, fluorine atom, hydroxyl and reaches for containing 2 ' of ribose-Nucleotide of at least 2 kinds of groups of O-Me base.

[5] aptamers of above-mentioned [3] wherein, contains the nucleotide sequence of GGUG (C/A) shown in (U/T).

[6] aptamers of above-mentioned [5], wherein, GGUG (C/A) (U/T) in the 3rd U be 2 ' the Nucleotide that hydroxyl is replaced by fluorine atom at ribose.

[7] aptamers of above-mentioned [6], wherein, GGUG (C/A) each Nucleotide (except the 3rd U) in (U/T) is identical or different respectively, in 2 ' Nucleotide that contains hydroxyl of ribose, or in 2 ' hydroxyl of ribose by hydrogen atom, fluorine atom or-Nucleotide of the basic replacement of O-Me.

[8] aptamers of above-mentioned [5], wherein, GGUG (C/A) is GGUGCU or GGUGAU (U/T).

[9] aptamers of above-mentioned [5] wherein, also contains the nucleotide sequence shown in the ANC (N is the Nucleotide that is selected from A, G, C, U and T).

[10] aptamers of above-mentioned [9], wherein, each Nucleotide among the ANC is identical or different respectively, in 2 ' Nucleotide that contains hydroxyl of ribose, or in 2 ' hydroxyl of ribose by hydrogen atom, fluorine atom or-Nucleotide of the basic replacement of O-Me.

[11] aptamers of above-mentioned [9] is following (i) any one in (iii):

(i) contain GGA in GGUG (C/A) 5 ' side (U/T), and contain UCC in the 3 ' side of ANC;

(ii) contain GGN in GGUG (C/A) 5 ' side (U/T) _X1A, and contain UN in the 3 ' side of ANC _X2CC (N _X1, N _X2Be respectively the Nucleotide that is selected from A, G, C, U and T); Or,

(iii) contain GGN in GGUG (C/A) 5 ' side (U/T) _X3N _X4A, and contain UN in the 3 ' side of ANC _X5N _X6CC (N _X3, N _X4, N _X5, N _X6Be respectively the Nucleotide that is selected from A, G, C, U and T).

[12] aptamers of above-mentioned [11], wherein, GGA, GGN _X1A or GGN _X3N _X4GG that A contains and UCC, UN _X2CC or UN _X5N _X6The CC that CC contains is respectively the Nucleotide that is replaced by hydrogen atom in 2 ' hydroxyl of ribose.

[13] aptamers of above-mentioned [6] wherein, has the potentiality secondary structure of following (I) to (III) expression:

[in the formula, N ¹, N ², N ³, N ⁴, N ⁵Respectively identical or different, for the Nucleotide that is selected from A, G, C, U and T,

N ²And N ³For mutual complementary Nucleotide,

N ⁴And N ⁵For mutual complementary Nucleotide,

(i) each Nucleotide (except the 3rd U) in (U/T) of GGUG (C/A), (ii) AN ¹Each Nucleotide among the C, (iii) N ²～N ⁵Each Nucleotide be respectively 2 ' Nucleotide that contains hydroxyl in ribose, or in 2 ' hydroxyl of ribose by hydrogen atom, fluorine atom or-Nucleotide that the O-Me base replaces].

[14] aptamers of above-mentioned [11], wherein, 2 ' the hydroxyl in ribose of all Nucleotide in ring (loop) structure is replaced by hydrogen atom.

[15] the potentiality secondary structure shown in each that the aptamers of above-mentioned [13], the aptamers that wherein, has a potentiality secondary structure shown in each in (I) to (III) have is following (I ') in (III '):

[in the formula, N ¹, N ², N ³, N ⁴, N ⁵Meaning identical with above-mentioned [13] respectively].

[16] aptamers of above-mentioned [3], wherein, be to contain (U/T) nucleotide sequence represented of C of useful AGGUG (C/A), AGGUG (C/A) is the Nucleotide that replaced by fluorine atom for 2 ' hydroxyl in ribose of the 4th U among the C (U/T), AGGUG (C/A) (U/T) each Nucleotide (except the 4th U) among the C is identical or different respectively, for in 2 ' Nucleotide that contains hydroxyl of ribose, or in 2 ' hydroxyl of ribose by hydrogen atom, fluorine atom or-Nucleotide that the O-Me base replaces.

[17] aptamers of above-mentioned [16], wherein, the nucleotide sequence that also contains GANCU (N is the Nucleotide that is selected from A, G, C, U and T) expression, each Nucleotide among the GANCU is identical or different respectively, for in 2 ' Nucleotide that contains hydroxyl of ribose, or in 2 ' hydroxyl of ribose by hydrogen atom, fluorine atom or-Nucleotide that the O-Me base replaces.

[18] aptamers of above-mentioned [6] wherein, has the potentiality secondary structure of following (Ia) to (IIIa) expression:

[in the formula, N ¹, N ², N ³, N ⁴, N ⁵, N ⁶, N ⁷Respectively identical or different, for the Nucleotide that is selected from A, G, C, U and T,

N ²And N ³For mutual complementary Nucleotide,

N ⁴And N ⁵For mutual complementary Nucleotide,

N ⁶And N ⁷For mutual complementary Nucleotide,

(i) each Nucleotide (except the 3rd U) in (U/T) of GGUG (C/A), (ii) AN ¹Each Nucleotide among the C, (iii) N ²To N ⁷Each Nucleotide be respectively 2 ' Nucleotide that contains hydroxyl in ribose, or in 2 ' hydroxyl of ribose by hydrogen atom, fluorine atom or-Nucleotide that the O-Me base replaces].

The potentiality secondary structure of each expression in (IIIa ') that [19] aptamers of above-mentioned [18], the aptamers that wherein, has a potentiality secondary structure of each expression in (Ia) to (IIIa) have is following (Ia '):

[in the formula, N ¹, N ², N ³, N ⁴, N ⁵Meaning identical with above-mentioned [18] respectively].

[20] aptamers of above-mentioned [19], wherein, N ⁴, N ⁶Be respectively the Nucleotide that is replaced by hydrogen atom in 2 ' hydroxyl, N ⁵, N ⁷Be respectively the Nucleotide that contains hydroxyl in 2 '.

The potentiality secondary structure of each expression in (IIIa " ') that [21] aptamers of above-mentioned [19], the aptamers that wherein, has a potentiality secondary structure of each expression in (Ia ') to (IIIa ') have is following (Ia " '):

[22] aptamers of above-mentioned [3] is in following (a) to (c) any one:

(a) aptamers of forming by the nucleotide sequence shown in each in the sequence numbering 1 to 23 (uridylic also can be thymus pyrimidine);

(b) by 1 in the nucleotide sequence of the nucleotide sequence shown in each in the sequence numbering 1 to 23 (uridylic also can be thymus pyrimidine) expression several Nucleotide are substituted, lack, insert or additional after the aptamers formed of nucleotide sequence;

(c) be selected from the concatenator of this (a), the concatenator of this (b) and the concatenator of this (a) and concatenator (b).

[23] complex body wherein, contains aptamers and and its bonded functional substance of each record in above-mentioned [1] to [22].

[24] complex body of above-mentioned [23], wherein, functional substance is affinity substance, mark material, enzyme, medicine, toxin or useful for drug delivery medium.

[25] solid phase carrier wherein, is fixed with the aptamers of each record in above-mentioned [1] to [22] or the complex body of above-mentioned [23] or [24].

[26] solid phase carrier of above-mentioned [25], wherein, solid phase carrier is substrate, resin, dish (plate), strainer, tube (cartridge), post or porous matter material.

[27] medical device wherein, contains the solid phase carrier of above-mentioned [25] or [26].

[28] equipment of above-mentioned [27], wherein, medical device is a blood purification equipment.

[29] diagnosis is used or is checked and use reagent, wherein, contains the complex body of the aptamers of each record in above-mentioned [1] to [22], above-mentioned [23] or [24] or the solid phase carrier of above-mentioned [25] or [26].

[30] medicine wherein, contains the aptamers of each record in above-mentioned [1] to [22], the complex body of perhaps above-mentioned [23] or [24].

[31] antibody purification or concentration method wherein, comprise the solid phase carrier that makes IgG antibody be adsorbed onto above-mentioned [25] or [26], make the step of adsorbed IgG antibody elution again by elutriant.

[32] method of above-mentioned [31], wherein, elutriant is a neutral solution.

[33] manufacture method of antibody purification wherein, comprises preparation IgG antibody, again with the IgG antibody of the preparation step by the solid phase carrier purifying of above-mentioned [25] or [26].

[34] IgG detects and/or quantivative approach, wherein, comprise the aptamers, the complex body of above-mentioned [23] or [24] or the solid phase carrier of above-mentioned [25] or [26] that use each record in above-mentioned [1] to [22], measure the step that has or not the amount of IgG and/or IgG in the sample.

Description of drawings

Fig. 1 represents the prediction secondary structure of the RNA shown in the sequence numbering 1.

Fig. 2 represents the prediction secondary structure of the RNA shown in the sequence numbering 2.

Fig. 3 represents the prediction secondary structure of the RNA shown in the sequence numbering 3.

Fig. 4 represents the prediction secondary structure of the RNA shown in the sequence numbering 4.

Fig. 5 represents the prediction secondary structure of the RNA shown in the sequence numbering 5.

Fig. 6 represents the prediction secondary structure of the RNA shown in the sequence numbering 6.

Fig. 7 represents the prediction secondary structure of the RNA shown in the sequence numbering 7.

Fig. 8 represents the prediction secondary structure of the RNA shown in the sequence numbering 8.

Fig. 9 represents the prediction secondary structure of the RNA shown in the sequence numbering 9.

Figure 10 represents the prediction secondary structure of the RNA shown in the sequence numbering 10.

Figure 11 represents the prediction secondary structure of the RNA shown in the sequence numbering 11.

Figure 12 represents the prediction secondary structure of the RNA shown in the sequence numbering 12.

Figure 13 represents the prediction secondary structure of the RNA shown in the sequence numbering 13.

Figure 14 represents the prediction secondary structure of the RNA shown in the sequence numbering 14.

Figure 15 represents the prediction secondary structure of the RNA shown in the sequence numbering 15.

Figure 16 represents the prediction secondary structure of the RNA shown in the sequence numbering 16.

Figure 17 represents the prediction secondary structure of the RNA shown in the sequence numbering 17.

Figure 18 represents the prediction secondary structure of the RNA shown in the sequence numbering 18.

Figure 19 represents the prediction secondary structure of the RNA shown in the sequence numbering 19.

Figure 20 represents the prediction secondary structure of the RNA shown in the sequence numbering 20.

Figure 21 represents the prediction secondary structure of the RNA shown in the sequence numbering 21.

Figure 22 represents the prediction secondary structure of the RNA shown in the sequence numbering 22.

Figure 23 represents the prediction secondary structure of the RNA shown in the sequence numbering 23.

Figure 24 shows the sensing figure that obtains by surperficial plasmon resonance analyzing, the figure shows the bonding state of the RNA shown in the sequence numbering 1 and human IgG-Fc.To be fixed in sensing chip with the A-dT key in 3 ' the terminal RNA that adds the PolyA that 16 residues are arranged, spray IgG-Fc, observe interaction with RNA.The RU of the longitudinal axis represents relative unit (Relative Unit), and Resp.Diff. represents response difference (Response Differences).Transverse axis is represented the time (second).These symbols in the longitudinal axis, the transverse axis are also identical in following Figure 25 to Figure 31,42.

Figure 25 shows the sensing figure that obtains by surperficial plasmon resonance analyzing, the figure shows RNA shown in the sequence numbering 3 and human IgG-Fc bonded state.To be fixed in sensing chip with the A-dT key in 3 ' the terminal RNA that adds the Poly A that 16 residues are arranged, spray IgG-Fc, observe interaction with RNA.

Figure 26 shows the sensing figure that obtains by surperficial plasmon resonance analyzing, the figure shows the RNA pond and the human IgG-Fc bonded state of stochastic sequence.To be fixed in sensing chip with the A-dT key in 3 ' the terminal RNA that adds the PolyA that 16 residues are arranged, spray IgG-Fc, observe interaction with RNA.

Figure 27 shows the sensing figure that obtains by surperficial plasmon resonance analyzing, the figure shows the state of the complex body formation of RNA shown in the sequence numbering 1 and human IgG1 and people Fc γ RI.To be fixed in sensing chip with the A-dT key in 3 ' the terminal RNA that adds the PolyA that 16 residues are arranged, and spray IgG1 it is incorporated into RNA, then spray Fc γ RI, observe interaction with IgG1.

Figure 28 shows the sensing figure that obtains by surperficial plasmon resonance analyzing, the figure shows the RNA pond and human IgG1 and the people Fc γ R bonded state that contain stochastic sequence.To be fixed in sensing chip with the A-dT key in 3 ' the terminal RNA that adds the Poly A that 16 residues are arranged, spray IgG1, spray Fc γ RI again.

Figure 29 shows the sensing figure that obtains by surperficial plasmon resonance analyzing, the figure shows the state of the complex body formation of RNA shown in the sequence numbering 1 and human IgG1 and ProteinA.To be fixed in sensing chip with the A-dT key in 3 ' the terminal RNA that adds the Poly A that 16 residues are arranged, and spray IgG1 it is incorporated into RNA, then spray Protein A, observe interaction with IgG1.

Figure 30 shows the sensing figure that obtains by surperficial plasmon resonance analyzing, the figure shows RNA aptamers shown in the sequence numbering 1 and Protein A bonded state.To be fixed in sensing chip with the A-dT key in 3 ' the terminal RNA that adds the PolyA that 16 residues are arranged, spray ProteinA, observe interaction with RNA.

Figure 31 shows the sensing figure that obtains by surperficial plasmon resonance analyzing, the figure shows the RNA aptamers shown in the sequence numbering 17-2 and human IgG1's bonded state.To be fixed in sensing chip with the A-dT key in 3 ' the terminal RNA that adds the Poly A that 16 residues are arranged, spray IgG1, observe interaction with RNA.

Figure 32 represents the RNA shown in sequence numbering 15 and 17 used with the part of separating agent as antibody purification and the result of SDS-PAGE during with IgG1 drop-down (pulldown).The RNA that will be combined with Poly (A) is fixed in the pearl that is combined with Poly (dT), carries out the drop-down of human IgG1.Swimming lane 1: be the situation that the RNA shown in the sequence numbering 15 is used as part.Swimming lane 2: be the situation that the RNA shown in the sequence numbering 17 is used as part.Swimming lane 3: be the situation that Protein A is used as part.Swimming lane 4: be the situation that rProteinA is used as part.

Figure 33 represents the RNA shown in the sequence numbering 15 as the part use of antibody purification with separating agent, the SDS-PAGE result during from human serum purifying human IgG.With the pearl that the RNA that is combined with vitamin H is fixed in streptavidin (Streptavidin), IgG is drop-down from human serum.Use 3 kinds of neutral elutriant elution of bound in the IgG of RNA.In order to confirm whether the neutral elutriant can add the sample damping fluid with the efficient wash-out of IgG in the pearl of finishing wash-out, heat, and analyzes with SDS-PAGE.Swimming lane 1: molecular weight marker (marker) protein.Swimming lane 2: use the elutriant of forming by 200mM Repone K and 10mM EDTA from using the IgG of RNA as the pearl wash-out of part.Swimming lane 3: use the elutriant of forming by 200mM Repone K, 10mM EDTA and 10% glycerine from using the IgG of RNA as the pearl wash-out of part.Swimming lane 4: use the elutriant of forming by 600mM Repone K, 10mM EDTA and 10% glycerine from using the IgG of RNA as the pearl wash-out of part.Swimming lane 5: use rProtein A sepharose 4B (Sepharose beads) with IgG when drop-down, with the IgG of pH3 glycine buffer wash-out.Swimming lane 6: be incorporated into IgG with the pearl after the elutriant processing of swimming lane 2.Swimming lane 7: be incorporated into IgG with the pearl after the elutriant processing of swimming lane 3.Swimming lane 8: be incorporated into IgG with the pearl after the elutriant processing of swimming lane 4.Swimming lane 9:, do not carry out wash-out and handle, and directly add the IgG that the sample damping fluid reclaims to pearl for the pearl of using RNA as part.Swimming lane 10: be incorporated into IgG with the pearl after the elutriant processing of swimming lane 5.

Figure 34 is illustrated in the SDS-PAGE result when carrying out test that the RNA shown in the sequence numbering 15 whether can be repeatedly uses with the part of separating agent as antibody purification.In antibody purification, used the separating agent of RNA once to clean with being combined with, carried out antibody purification once more with urea.Repeatable operation 2 times.Swimming lane 1: molecular weight marker protein.Swimming lane 2: the IgG of purifying acquisition for the first time.Swimming lane 3: the IgG of purifying acquisition for the second time.Swimming lane 4: the IgG of purifying acquisition for the third time.

Figure 35 represents the RNA shown in sequence numbering 16 and the sequence numbering 17-2 as the part use of antibody purification with separating agent, the SDS-PAGE result during from human serum purifying human IgG.Swimming lane 1: molecular weight marker protein.Swimming lane 2: when using the RNA shown in the sequence numbering 15 as part by drop-down IgG.Swimming lane 3: when using the RNA shown in the sequence numbering 16 as part by drop-down IgG.Swimming lane 4: when using the RNA shown in the sequence numbering 17-2 as part by drop-down IgG.Swimming lane 5: drop-down IgG when using rProtein A as part.Swimming lane 6: human serum.

Figure 36 represents to use the SDS-PAGE result when mercaptan coupler fixed RNA aptamers is carried out antibody purification.Swimming lane 1: molecular weight marker protein.Swimming lane 2: use the RNA shown in the sequence numbering 15 as part, when adding 5 μ L human serums by drop-down IgG.Swimming lane 3: use the RNA shown in the sequence numbering 15 as part, when adding 10 μ L human serums by drop-down IgG.Swimming lane 4: control group (blank) when 5 μ L human serums (in the pearl that does not have binding partner add by drop-down serum protein).Swimming lane 5: use rProtein A pearl, when adding 5 μ L human serums by drop-down IgG.Swimming lane 6: normal man IgG1.Swimming lane 7: human serum.

Figure 37 represents to use through amino coupler fixed RNA aptamers, the SDS-PAGE result when carrying out antibody purification.The recovery sample of application of sample half amount.Swimming lane 1: molecular weight marker protein.Swimming lane 2: use the RNA (immobilization amount 25 μ g) shown in the sequence numbering 15 as part, the IgG that reclaims from the human serum of 10 μ L.Swimming lane 3: use the RNA (immobilization amount 75 μ g) shown in the sequence numbering 15 as part, the IgG that reclaims from the human serum of 10 μ L.

Figure 38 represents to use through amino coupler fixed RNA aptamers, the SDS-PAGE result when carrying out antibody purification.Use the human serum of 10 μ L in drop-down.Swimming lane 1: molecular weight marker protein.Swimming lane 2: use the situation of the RNA shown in the sequence numbering 17-7 as part.Swimming lane 3: use the situation of the RNA shown in the sequence numbering 17-8 as part.Swimming lane 4: use the situation of the RNA shown in the sequence numbering 17-7-107 as part.Swimming lane 5: use the situation of the RNA shown in the sequence numbering 15 as part.Swimming lane 6: the situation of using the rProteinA resin.Swimming lane 7: normal man IgG1 (6 μ g).Swimming lane 8: human serum (0.2 μ L).

SDS-PAGE result when Figure 39 represents to use various elutriant wash-out.Swimming lane 1: molecular weight marker protein.The 10mMTris of swimming lane 2:200mM Repone K+10mM EDTA+pH 7.6.The 10mM Tris of swimming lane 3:200mM Repone K+pH 7.6.The 10mM Tris of swimming lane 4:300mM sodium-chlor+10mM EDTA+pH 7.6.The 10mM Tris of swimming lane 5:10mM EDTA+pH 7.6.

Figure 40 represents in order to assess through the characteristic of the aptamers resin of thermal regeneration and the result of the SDS-PAGE that carries out.With using 3 times aptamers resin 10 μ L to carry out heat treated with 2 kinds of methods, reuse 10 μ L human serums, carry out drop-down experiment.Neutral elutriated fraction is analyzed with SDS-PAGE.Swimming lane 1: the aptamers resin shown in the sequence numbering 17-18 is in ultrapure water, in 85 ℃ of heating 5 minutes.Swimming lane 2: the aptamers resin shown in the sequence numbering 17-17 is in 6M urea, in 65 ℃ of heating 15 minutes.

Figure 41 represents the IgG through resin mating-type oligo purifying is carried out the result of SDS-PAGE.To covalent attachment the human serum that adds 10 μ L among the resin mating-type oligo 10 μ L of the RNA shown in the sequence numbering 15 is arranged, to analyzing with SDS-PAGE by the fraction of neutral elutriant wash-out.

Figure 42 shows the sensing figure that obtains by surperficial plasmon resonance analyzing, the figure shows the RNA shown in the sequence numbering 17-7 and as the bonding state of the Rituximab (Rituxan) of antibody medicine.To be fixed in sensing chip with the A-dT key in 3 ' the terminal RNA that adds the Poly A that 16 residues are arranged, spray Rituximab, observe interaction with RNA.

Embodiment

The invention provides aptamers at immunoglobulin G (IgG).

So-called aptamers is that the target molecule of stipulating is had in conjunction with active nucleic acid molecule.Aptamers combines by the target molecule with regulation, also can have the active effect of target molecule that suppresses regulation.Aptamers of the present invention can be RNA, DNA, modification of nucleic acids or their mixture.Aptamers of the present invention can be straight chain shape or cyclic form.Aptamers of the present invention can be combined in the Fc part of IgG specifically.

The combinable IgG of aptamers of the present invention enumerates for example human IgG (for example IgG1, IgG2, IgG3, IgG4), hamster IgG, pig IgG.

Aptamers of the present invention can be incorporated into the arbitrary portion of the Fc part of IgG.The Fc part of IgG is known carries out combination to the receptor protein (Fc γ R) of expressing in immunocytes such as scavenger cell or neutrophilic leukocyte, aptamers of the present invention also can be incorporated into serve as in conjunction with the Fc of Fc γ R effect part different Fc part.In addition, known ProteinA is incorporated into the Fc part of IgG, aptamers of the present invention also can be incorporated into serve as in conjunction with the Fc of Protein A effect part different Fc part.

As long as aptamers of the present invention can combine with IgG, just there is no particular limitation, for example, is basis when assessing with dissociation constant (Kd value), can have about 1 * 10 ^-6Below the M, preferred about 1 * 10 ^-7Below the M, more preferably from about 1 * 10 ^-8The Kd that M is following.The Kd value for example can be calculated according to the method for utilizing surperficial plasmon resonance.

Length to aptamers of the present invention is not particularly limited, and can be about 16 usually to about 200 Nucleotide, below for example about 100 Nucleotide, below preferred about 50 Nucleotide, more preferably from about below 40 Nucleotide, more more preferably from about below 30 Nucleotide, most preferably from about below 25 Nucleotide.In addition, the length of aptamers of the present invention also can be for example more than about 18 Nucleotide, more than preferred about 20 Nucleotide.The total nucleotide number is few more, easy more chemosynthesis and mass production, and the advantage aspect cost is also big.In addition, think that chemically modified is also easy, the body internal stability is also high, and toxicity is also low.

Each Nucleotide that contains in the aptamers of the present invention is identical or different respectively, can be for the Nucleotide (that is unsubstituted Nucleotide) that contains hydroxyl in 2 ' of ribose or in the Nucleotide of the hydroxyl quilt of 2 ' in ribose atom or group replacement arbitrarily.As this atom or group arbitrarily, for example can enumerate by hydrogen atom, fluorine atom or-O-alkyl (for example-O-Me yl) ,-O-acyl group (for example-O-CHO yl), amino (for example-NH ₂Base) Nucleotide that replaces.

Aptamers of the present invention can contain the nucleotide sequence of (U/T) representing with GGUG (C/A).As GGUG (C/A) (U/T), can enumerate for example GGUGCU, GGUGAU, GGUGCT, GGUGAT, but from the viewpoint of RNA molecule, preferred GGUGCU, GGUGAU.Contain GGUG (C/A) (U/T) time in aptamers of the present invention, contained GGUG (C/A) number (U/T) can be 1 or a plurality of (for example 2 or 3) in the nucleic acid.Aptamers of the present invention can be in conjunction with 2 for 1 IgG.

Aptamers of the present invention can for GGUG (C/A) (U/T) in the 3rd U 2 in ribose by the aptamers after fluoridizing (promptly, 2 '-F modifies), perhaps with can keep aptamers of the present invention to IgG in conjunction with the aptamers of active mode after to 2 modifications that impose outside fluoridizing of ribose of the 3rd U.As such modification, for example enumerate-O-Meization, ammonification (NH ₂).

Aptamers of the present invention also can be chemosynthesis, is that 5 ' end can have the aspect of phosplate base, with different in the aptamers that 5 ' end has the triphosphoric acid ester group through transcribing (for example SELEX method) synthetic.At least a kind of aptamers of the present invention (for example 1,2,3 or 4 kind) Nucleotide also can be in 2 ' of ribose and contains the Nucleotide that hydroxyl or above-mentioned atom arbitrarily or group for example are selected from the group of hydrogen atom, fluorine atom, hydroxyl and-at least 2 kinds of the O-Me base (for example 2,3 or 4 kind).

When aptamers of the present invention contains the nucleotide sequence of (U/T) representing with GGUG (C/A), can have stem (stem) structure at its two ends.Stem structure can bring the sufficient stabilization of raised structures.For example, as stem structure, GGUG (C/A) (U/T) in 5 ' terminal G (the 1st Nucleotide) and the Nucleotide more than 1 that is adjacent in 5 ' side, 3 ' terminal U/T (the 6th Nucleotide) can form intramolecularly base pair (base pair) respectively with 1 the above Nucleotide that is adjacent in 3 ' side.At the Nucleotide of 5 ' side or 3 ' side adjacency so long as more than 1, just there is no particular limitation, can be for more than 2, preferred more than 3.

Aptamers of the present invention also can contain the nucleotide sequence shown in the ANC except the nucleotide sequence of above-mentioned GGUG (C/A) shown in (U/T).N among the ANC can be for being selected from any Nucleotide of A, G, C, U and T, and is preferable with A, G, C and U, better with A and G, with A the best.When aptamers of the present invention contains GGUG (C/A) (U/T) and during the nucleotide sequence shown in the ANC, can be that GGUG (C/A) (U/T) is present in 5 ' side, ANC is present in 3 ' side, perhaps can be that ANC is present in 5 ' side, GGUG (C/A) and (U/T) is present in 3 ' side.Aptamers of the present invention have GGUG (C/A) (U/T) in 5 ' terminal G and the C of ANC can form the structure of intramolecularly base pair, and/or GGUG (C/A) (U/T) in the A of the U/T of 3 ' end and ANC can form the structure of intramolecularly base pair.Aptamers of the present invention contain GGUG (C/A) (U/T) and ANC both the time, the GGUG that aptamers contains (C/A) (U/T) and the quantity of ANC be respectively 1 or a plurality of (for example 2 or 3).

Aptamers of the present invention can also be following (i) in (iii) any:

(i) GGUG (C/A) 5 ' side (U/T) contains GGA, and the 3 ' side of ANC contains UCC;

(ii) GGUG (C/A) 5 ' side (U/T) contains GGN _X1A, and the 3 ' side of ANC contains UN _X2CC (N _X1, N _X2Be respectively the Nucleotide that is selected from A, G, C, U and T); Or

(iii) GGUG (C/A) 5 ' side (U/T) contains GGN _X3N _X4A (for example GGACAG), and the 3 ' side of ANC contains UN _X5N _X6CC (N _X3, N _X4, N _X5, N _X6Be respectively the Nucleotide that is selected from A, G, C, U and T).GGA, GGN _X1A or GGN _X3N _X4A and UCC, UN _X2CC or UN _X5N _X6All Nucleotide of CC be 2 ' of ribose contain hydroxyl Nucleotide (promptly, be unsubstituted Nucleotide), perhaps be in 2 ' hydroxyl of ribose by hydrogen atom, fluorine atom or-Nucleotide that the O-Me base replaces, from in conjunction with active viewpoint, preferable by the Nucleotide that hydrogen atom replaces with hydroxyl in 2 '.

Aptamers of the present invention can also contain (U/T) nucleotide sequence of the nucleotide sequence shown in the C and/or GANCU (N is the Nucleotide that is selected from A, G, C, U and T) expression of AGGUG (C/A).AGGUG (C/A) (U/T) the 4th U among the C can be the Nucleotide that is replaced by fluorine atom in 2 ' hydroxyl, perhaps with can keep aptamers of the present invention to IgG in conjunction with the Nucleotide of active mode after to 2 ' modification that imposes except that fluoridizing of ribose of the 4th U.Nucleotide beyond the above-mentioned U is identical or different respectively, for the Nucleotide that contains hydroxyl in 2 ' of ribose or in 2 ' hydroxyl of ribose by hydrogen atom, fluorine atom or-Nucleotide of the basic replacement of O-Me.

Say that at length aptamers of the present invention has the potentiality secondary structure, 2 stem structures (S1, S2) and ring (loop) structure that this potentiality secondary structure contains raised structures and is present in these raised structures two ends.When using in this specification sheets, so-called " potentiality secondary structure " is can be in the secondary structure of stable existence under the physiological condition, and for example, whether having the potentiality secondary structure can determine by the structure prediction scheme of embodiment record.In the ring structure all Nucleotide can for the Nucleotide that contains hydroxyl in 2 ' of ribose (promptly, be unsubstituted Nucleotide), or the Nucleotide that is replaced by atom or group arbitrarily (for example hydrogen atom, fluorine atom or-O-Me yl) in 2 ' of ribose hydroxyl, from in conjunction with active viewpoint, preferable by the Nucleotide that hydrogen atom replaces with 2 ' hydroxyl in ribose.

Say that aptamers of the present invention can have the potentiality secondary structure of each expression in following (I) to (III) more detailed speech:

[in the formula, N ¹, N ², N ³, N ⁴, N ⁵Respectively identical or different, for being selected from the Nucleotide of A, G, C, U and T, and N ²And N ³Be mutual complementary Nucleotide, N ⁴And N ⁵Be mutual complementary Nucleotide].In (III), the Nucleotide that solid line (thick line) expression is selected from A, G, C, U and T links with random length above-mentioned (1), and solid line (fine rule) expression potentiality has complementary in conjunction with (base pairing) energy.S1, S2 represent stem structure respectively.In the stem structure of S1, S2, but the Nucleotide quantity of base pairing can be respectively more than 1, also can be for more than 2, more than 3 or more than 4.Curve part representative ring structure.Ring structure preferably is made of the Nucleotide more than 3, better is made of 4 Nucleotide.Be preferably, the structure of above-mentioned (I) to (III) expression can be the structure of above-mentioned (I ') to (III '), following (I ") to (III ") or (I " ') to (III " ') expression.

In addition, GGUG (C/A) (U/T) in the 3rd U can be 2 ' Nucleotide that is replaced by fluorine atom in ribose, other Nucleotide that contain in the aptamers of the present invention (except that above-mentioned U) are respectively identical or different, can be for the Nucleotide that contains hydroxyl in 2 ' of ribose or in the Nucleotide of the hydroxyl quilt of 2 ' in ribose atom or group (for example hydrogen atom, fluorine atom or-O-Me yl) replacement arbitrarily.

Aptamers of the present invention can have the potentiality secondary structure of each expression in following (Ia) to (IIIa):

[in the formula, N ¹, N ², N ³, N ⁴, N ⁵, N ⁶, N ⁷Respectively identical or different, for being selected from the Nucleotide of A, G, C, U and T, and N ²And N ³Be mutual complementary Nucleotide, N ⁴And N ⁵Be mutual complementary Nucleotide, N ⁶And N ⁷Be mutual complementary Nucleotide].In (IIIa), the Nucleotide that solid line (thick line) expression is selected from A, G, C, U and T links with random length above-mentioned (Ia), and solid line (fine rule) expression potentiality has complementary in conjunction with (base pairing) energy.S1, S2 represent stem structure respectively.In the stem structure of S1, S2, but the Nucleotide quantity of base pairing can be respectively more than 1, also can be for more than 2, more than 3 or more than 4.Curve part representative ring structure.Ring structure preferably is made of 3 above Nucleotide, to constitute preferable by 4 Nucleotide.Be preferably, the structure of above-mentioned (Ia) to (IIIa) expression can be the structure of above-mentioned (Ia ') to (IIIa '), following (Ia ") to (IIIa ") or (Ia " ') to (IIIa " ') expression.

In addition, GGUG (C/A) (U/T) in the 3rd U can be 2 ' Nucleotide that is replaced by fluorine atom in ribose, other Nucleotide that contain in the aptamers of the present invention (except that above-mentioned U) are respectively identical or different, can be for the Nucleotide that contains hydroxyl in 2 ' of ribose or in the Nucleotide of the hydroxyl quilt of 2 ' in ribose atom or group (for example hydrogen atom, fluorine atom or-O-Me yl) replacement arbitrarily.From in conjunction with active viewpoint, preferably, N ⁴, N ⁶Be respectively Nucleotide and the N that 2 ' hydroxyl in ribose is replaced by hydrogen atom ⁵, N ⁷Be respectively 2 ' Nucleotide that contains hydroxyl in ribose.

The aptamers that aptamers of the present invention can be made up of the nucleotide sequence shown in each in the sequence numbering 1 to 23 (uridylic also can be thymus pyrimidine) for (a); (b) in sequence numbering 1 to 23 1 in the nucleotide sequence of the nucleotide sequence shown in each (uridylic also can be thymus pyrimidine) expression several Nucleotide are substituted, lack, insert or additional after the aptamers formed of nucleotide sequence; (c) be selected from the concatenator of a plurality of concatenators of a plurality of concatenators of above-mentioned (a), above-mentioned (b), above-mentioned (a) and a plurality of concatenators (b).In above-mentioned (b), replacement, disappearance, insertion or additional Nucleotide quantity can be for example about below 10 as long as just there is no particular limitation for several, better about below 8, better about below 6, better more about below 5, be preferably 4,3,2 or 1.Binding can be by tandem formula (tandem) in conjunction with carrying out in above-mentioned (c).In addition, also can utilize during binding and connect base (linker).As connecting base, enumerate nucleotide chain (for example 1 to about 20 Nucleotide), non-nucleotide chain (for example-(CH ₂) n-connection base ,-(CH ₂CH ₂O) n-connect base, hexaethylene glycol connect base, TEG connect base, contain peptide the connection base, contain-the connection base of S-S-key, contain-the connection base of CONH-key, contain-OPO ₃The connection base of-key).A plurality of needing only in that just there is no particular limitation more than 2 in above-mentioned a plurality of concatenator for example can be 2 to 4.Each Nucleotide in above-mentioned (a) to (c) is identical or different respectively, can be for the Nucleotide that contains hydroxyl in 2 ' of ribose or in the Nucleotide of the hydroxyl quilt of 2 ' in ribose group (for example hydrogen atom, fluorine atom or-O-Me yl) replacement arbitrarily.

Aptamers of the present invention also can be regenerated, sterilize through heat treated.As such heat treated, for example can enumerate the processing of under 65 to 85 ℃, carrying out several minutes (for example 5 to 15 minutes).

In order to improve associativity to IgG, stability, useful for drug delivery etc., the saccharide residue of each Nucleotide of aptamers of the present invention (for example ribose) also can be modified.As adorned position in the saccharide residue, for example can enumerate 2 ', 3 ' of saccharide residue and/or 4 ' Sauerstoffatom are replaced to other atoms etc.As the kind of modifying, for example can enumerate fluoridize, O-alkylation (for example O-methylates, O-ethylizes), O-allylation, S-alkylation (for example S-methylates, S-ethylizes), S-allylation, amination (for example-NH ₂).The change of such saccharide residue can be carried out (reference example such as Sproat etc., (1991) Nucle.Acid.Res.19,733-738 according to known method itself; Cotton etc., (1991) Nucl.Acid.Res.19,2629-2635; Hobbs etc., (1973) Biochemistry 12,5138-5145).

In addition, in order to improve associativity for IgG etc., the purine of aptamers of the present invention, pyrimidine also can change (for example chemical replacement) person.Change as this, for example can enumerate that 5 pyrimidines change, 8 purine change, the replacement of the change of the outer amine of ring, 4-thiourdine, with the replacement of 5-bromine or 5-iodouracil.In addition, nuclease and hydrolysis are had patience, also can change the phosphate that contains in the aptamers of the present invention in order to make.For example P (O) O base also can be by P (O) S (thioester), P (S) S (dithio acid esters), P (O) NR ₂(amidate), P (O) R, R (O) OR ', CO or CH ₂(methyl acetal) or 3 '-amine (NH-CH ₂-CH ₂-) replace [, each R or R ' are independent separately, are hydrogen atom or the alkyl (for example methyl, ethyl) that is substituted or is unsubstituted] herein.Concatenating group can by-O-,-N-or-S-links, and is incorporated into the Nucleotide of adjacency.Change and also can comprise 3 ' and 5 ' the change that adds cap (capping) and so on.Change also and can carry out through polyoxyethylene glycol or other lipids are appended to end.This changes with reference to No. the 5 660 985, United States Patent (USP), No. the 5 756 703, United States Patent (USP).

Aptamers of the present invention can and be carried out chemosynthesis in the technology general knowledge of this technical field according to the disclosure in this specification sheets.As aptamers of the present invention, for example can also enumerate the aptamers that contains the nucleotide sequence of GGUG (C/A) shown in (U/T) (and the nucleotide sequence shown in the ANC in case of necessity), such aptamers is utilized SELEX method and other improved methods (Ellington etc. for example, (1990) Nature, 346,818-822; Tuerk etc., (1990) Science, 249,505-510) can highly design.For example use by following:

-(N) (U/T)-(N) b-of a-GGUG (C/A)

[in above-mentioned, (N) a represents the nucleotide chain be made up of a N, and (N) b represents the nucleotide chain be made up of b N, and N is identical or different respectively, for being selected from the Nucleotide of A, G, C, U and T (being preferably A, G, C and U).A, b are identical or different respectively, can be arbitrarily and count, it for example is 1 to about 100, be preferably 1 to about 50, more preferably 1 to about 30, more preferably 1 to about 20 or 1 to about 10 again] shown in the nucleic acid molecule of the single kind formed of nucleotide sequence or multiple nucleic acid molecule (for example library (library) of different nucleic acid molecule such as the number of a, b) and primer right with sequence (i), (ii) corresponding respectively primer, can highly design the aptamers of the present invention that contains the nucleotide sequence of GGUG (C/A) shown in (U/T) thus.The present invention also provides the manufacture method of this aptamers that can highly design.

Aptamers of the present invention for example can be used as antibody purification with the part of separating agent, with antibody is connected with the mark substance bonded base, antibody fixing agent, be connected basic with modification material bonded antibody.Specifically utilize method and the antibody purification method of using Protein A much at one as antibody purification with the part of separating agent, but because available neutral solution wash-out antibody, therefore with need be with the use of acidic solution wash-out antibody the method for Protein A compare, have the advantage that can prevent the antibody sex change.When antibody being connected base with the mark substance bonded and using, aptamers of the present invention need have not can be from the high combination activity of the degree of antibiody dissociation with aptamers of the present invention.On the other hand, when using with separating agent as antibody purification, because must be with the antibody elution of primary sorption, therefore may not be high more good more in conjunction with activity.By using different sequences, different lengths and different modifying method, provide to have IgG there are different bonding forces or stability and cheaply wait the sharp aptamers of putting among the present invention.Aptamers of the present invention also has various availability described later.

The present invention also provides the complex body of the functional substance that contains aptamers of the present invention and combine with it.Combining between the aptamers in the complex body of the present invention and the functional substance can be covalent attachment or non-covalent combination.Complex body of the present invention can form for aptamers of the present invention combines with the of the same race or xenogeneic functional substance of (for example 2 or 3) more than 1.As functional substance, for example can enumerate protein, peptide, amino acid, lipid, saccharic, monose, polynucleotide, Nucleotide.As functional substance, for example can also enumerate affinity substance, mark material, enzyme, medicine, toxin, useful for drug delivery medium.

As affinity substance, the affinity of polynucleotide, antibody, glutathione agarose (glutathionesepharose), Histidine enumerate biological example element, streptavidin, have to(for) the target complementary sequence.

Serve as a mark and use material, enumerate for example fluorescent substance, luminophore, radio isotope.As fluorescent substance, enumerate for example the green I of SYBR (SYBR Green I), the green II of SYBR, SYBR gold (SYBR Gold), SYPRO ruby red (SYPRO Ruby), SYPRO tangerine look (SYPROOrange), SYPRO orange (SYPRO Tangerine), FITC, FAM, EGFP, ECFP, AttoPhos, SYPRO red (SYPRO Red), Cy3, TAMRA, ROX, HEX, Alexa fluorescence 532 (Alexa Fluor 532), Alexa fluorescence 546, dark purple (Deep Purple), Pro-QDiamond, rhodamine reds, BODIPY 576/589, NED, the R-phycoerythrin, RFP, HNPP, Alexa fluorescence 633, Alexa fluorescence 635, Alexa fluorescence 647, Cy5, glimmering (BODIPY) 650/665 of fluorine boron, DiD, TOTO-3, phosphoric acid DDAO, ethidium bromide, SYPRO rose-red (SYPRORose), Cy7, luciferin.As luminophore, enumerate for example luminol,3-aminophthalic acid cyclic hydrazide (luminol), luciferin (luciferin), lucigenin (Lucigenin).As radio isotope, for example enumerate ³H, ¹⁴C, ³²P, ³⁵S, ⁹⁰Y, ¹²³I, ¹²⁵I, ¹³¹I.

As enzyme, enumerate for example Western horseradish peroxidase or alkaline phosphatase (alkaliphosphatase).

As medicine, for example can enumerate carcinostatic agent.As carcinostatic agent, enumerate the medicine that uses in calicheamicin for example or enlightening Europe Ka-7038 guided missile (missile) therapies such as (duocarmycin), endoxan, melphalan (merphalan), ifosfamide (ifosfamide) or Z-4828 mustargen (nitrogen mustard) analogues such as (trofosfamide), tespamin ethyleneimine classes such as (thio-TEPA), carmustine nitrosourea such as (carmustine), Temozolomide (temozolomide) or Dacarbazine reast agent such as (dacarbazine), folic acid class metabolic antagonists such as methotrexate (methotrexate) or Raltitrexed, Tioguanine (thioguanine), CldAdo (cladribin) or fludarabine purine analogues such as (fludarabine), Fluracil (fluorouracil), Tegafur (tegafur) or gemcitabine pyrimidine analogues such as (gemcitabine), vinealeucoblastine(VLB) (vinblastine), vincristine(VCR) (vincristine) or vinorelbine Changchun alkaloid and analogues thereof such as (Vinorelbin), Etoposide (etoposide), taxol (taxane), Docetaxel (docetaxel) or taxol podophyllotoxin (podophyllotoxin) derivatives such as (paclitaxel), Dx (doxorubicin), epirubicin (epirubicin), idarubicin (edarubicin) and mitoxantrone anthracycline antibiotics (anthracycline) class and analogues such as (mitoxantrone), bleomycin (bleomycin) and mitomycin (mitomycin) wait other cytotoxic antibiotics, cis-platinum (cisplatin), carboplatin (carboplatin) and oxaliplatin platinic compound such as (oxaliplatin), pentostatin (pentostatin), miltefosine (miltefocin), estramustine (estramstin), Hycamtin (topotecan), irinotecan (irinotecan) and bicalutamide (bicaltamide) wait other antineoplastic agents.

As toxin, enumerate for example Ricin (Ricin), Leah toxin (リ ァ toxin).

As the useful for drug delivery medium, enumerate for example rrna, microsphere (microsphere), polyoxyethylene glycol, cholesterol, peptide.

Aptamers of the present invention and/or complex body of the present invention can be used as for example medicine or reagent (for example diagnose medicine, check medicine (comprising experiment reagent)) use.Medicine for example of the present invention or diagnosis medicine, for example can be used for the overexpression of unusual IgG and/or IgG is that (for example rheumatosis, ephritis, Ka Siluman disease (Castleman ' s disease), Wei Genashi (Wegener ' s) granuloma, glomerulosclerosis, renal glomerular disease, multiple arteritis, anaphylactoid purpura, lupus erythematosus (erythematosus), organ transplant's rejection), the disease (for example B cell lymphoma) relevant with the IgG generation etc. comprise the IgG relative disease of autoimmune disease for the disease of reason; The perhaps treatment of cancer or diagnosis (monitoring of for example state of an illness assurance, result of treatment).In addition, in the treatment of cancer, use complex body of the present invention (for example make the aptamers of the present invention that is incorporated into carcinostatic agent or toxin be incorporated into antibody drug and complex body), cancer cells can be killed.

Reagent of the present invention replaces the antibody except using aptamers of the present invention, can use by the method identical with immunological method.Therefore, the aptamers of the application of the invention replaces antibody, through with enzyme immunoassay (EIA) (direct competitive ELISA for example, indirect competitive ELISA, Sanming City therapy ELISA), radioimmunoassay (RIA), fluorescent immunoassay (FIA), immunochromatographic method, luminescence immunoassay, the rotation immunoassay, Western blotting (secondary antibodies that for example substitutes Western blotting is used), immunohistochemistry staining method, the cell sorting method identical methods of method such as (cell sorting), the diagnosis or the IgG described later that can carry out above-mentioned disease detect quantitatively.The present invention also provides use the present invention to diagnose and uses compositions and methods, at this moment, also can use solid phase carrier of the present invention.

Medicine of the present invention can be mixed with pharmaceutically acceptable carrier.As pharmaceutically acceptable carrier, enumerate for example sucrose, starch, mannitol, Sorbitol Powder, lactose, glucose, Mierocrystalline cellulose, talcum, calcium phosphate, vehicle such as lime carbonate, Mierocrystalline cellulose, methylcellulose gum, hydroxypropylcellulose, polypropylpyrrolidone, gelatin, gum arabic, polyoxyethylene glycol, sucrose, tackiness agents such as starch, starch, carboxymethyl cellulose, hydroxypropylated starch, sodium-ethylene glycol-starch, sodium bicarbonate, calcium phosphate, disintegrating agents such as citrate of lime, Magnesium Stearate, aerosol (aerosol), talcum, lubricants such as sodium lauryl sulphate, citric acid, menthol, Potenlini (glycyrrhizin) ammonium salt, glycine, perfume compound such as orange powder, Sodium Benzoate, sodium bisulfite, methyl hydroxybenzoate, preservativess such as nipasol, citric acid, Trisodium Citrate, stablizers such as acetate, methylcellulose gum, polyvinylpyrrolidone, clouding agents such as aluminum stearate, dispersion agents such as tensio-active agent, water, physiological saline, thinners such as orange juice, theobroma oil, polyoxyethylene glycol, matrix waxes such as kerosene etc., but be not limited thereto.

Peroral administration desirable preparation is the liquid preparation that makes after the part of significant quantity is dissolved in the diluent of water, physiological saline, orange juice and so on, the part that contains significant quantity makes solid or particulate capsule, granule or tablet, the effective constituent of significant quantity is suspended in suspension preparation in the suitable dispersion medium, makes the solution of the effective constituent that is dissolved with significant quantity be scattered in emulsive emulsion etc. in the suitable dispersion medium.

The desirable preparation of non-oral administration (for example intravenous injection, subcutaneous injection, intramuscular injection, local injection, intraperitoneal administration etc.) is water-based and nonaqueous etc. oozes aseptic injecting fluid preparation, wherein also can contain antioxidant, damping fluid, fungistat, isotonic agent etc.In addition, water-based and nonaqueous sterile suspensions agent can be enumerated, wherein also suspension agent, solubilizing agent, tackifier, stablizer, sanitas etc. can be contained.Said preparation can be enclosed in the container of ampoule or bottle and so on unit dosage or multiple dosing amount.In addition, can be with effective constituent and pharmaceutically acceptable carrier lyophilize, with face use before as long as dissolving or be suspended in get final product in the suitable aseptic vehicle the state preservation.

The dosage of medicine of the present invention is according to the difference at the drug tolerance of the species of the severity of the kind activity of effective constituent, disease, administration object, administration object, body weight, age etc. and difference, usually the effective constituent amount of being grown up every day can be about 0.0001 to about 2.0g/kg, for example about 0.0001 to about 0.1g/kg, and better about 0.005 to about 0.05g/kg.

The present invention also provides immobilization that the solid phase carrier of aptamers of the present invention and/or complex body of the present invention is arranged.As solid phase carrier, enumerate for example substrate, resin, plate (for example porous plate), strainer, tube, post, porous matter material.Substrate can be the substrate of use in DNA chip or the protein chip etc. etc., for example nickel-PTFE (tetrafluoroethylene (polytetrafluoroethylene)) substrate or glass substrate, phosphatic rock substrate, silicon substrate, aluminum oxide substrate etc., and the plate after the coating of implementing polymkeric substance etc. on these substrates.As resin, enumerate for example be filled in the antibody purification chromatography or with antibody as the resin in the post that uses in the affinity chromatography of part etc. and be used for batch process with antibody purification or immobilized resin, in addition, comprise the agarose particle of various concentration or through highly cross-linked agarose particle, silicon dioxide granule, acrylamide and N, the multipolymer of N '-methylene-bisacrylamide, the crosslinked divinylbenzene particle of polystyrene, with the particle of dextran after with epichlorohydrin cross-linked, cellulosic fibre, allyl group dextran and N, the cross-linked polymer of N '-methylene-bisacrylamide, the monodisperse system synthetic polymer, the monodisperse system hydrophilic polymer, Sepharose (trade(brand)name) agarose, Toyopearl (trade(brand)name) chromatographic resins etc. also comprise the resin that makes after various functional groups are attached to these resins.

Aptamers of the present invention and/or complex body of the present invention can be fixed in solid phase carrier by known method itself.The functional group of for example enumerating affinity substance (for example above-mentioned affinity substance) or regulation imports aptamers of the present invention and/or complex body of the present invention, utilizes the functional group of this affinity substance or regulation again, is fixed in the method for solid phase carrier.The invention provides such method.The functional group of regulation can be enumerated for example amino, thiol group, hydroxyl, carboxyl for supplying with the functional group of coupled reaction.The present invention also provides the aptamers that imports this functional group.

Solid phase carrier of the present invention can be used for the detection of the purifying of IgG for example and IgG, quantitatively.Solid phase carrier of the present invention also can be used in the disease that the above-mentioned unusual IgG of treatment or IgG surplus are expressed as reason.Use liquid-feeding pump, from the patient vessel blood is flowed into solid phase carrier of the present invention (for example tube), absorption is back to the patient with the blood that purifies after removing the IgG of specified amount.At this moment, useful is to add the resisting blood coagulation agent blood is not solidified.The amount of removing of IgG can be regulated according to the loading capacity that sees through blood flow volume and solid phase carrier of the present invention.Solid phase carrier of the present invention can be sterilized through heating or uviolizing etc. by cleaning with neutral elutriant, regenerates.When in the purification of blood, utilizing solid phase carrier of the present invention, can or use with reference to dialysis therapy about detailed using method or result of treatment as Prosorba that has used the IgG remover of Protein A (trade(brand)name, Fresenius company makes) and Immunosorba (trade(brand)name; The manufacturing of Fresenius company) blood purification method is carried out.Therefore, also provide can be with the medical device that contains solid phase carrier of the present invention of this blood purification in the present invention.

The invention provides purifying antibody and/or concentration method.Purifying of the present invention and/or concentration method can comprise IgG antibody is adsorbed in solid phase carrier of the present invention, make the step of the IgG antibody elution of absorption again by elutriant.Purifying of the present invention and/or concentration method also can be and solid phase carrier of the present invention is filled in container (for example flask, developmental tube, test tube) carries out purifying or spissated static method, and the solution that will contain IgG is delivered to solid phase carrier of the present invention (for example post) and carried out purifying or spissated dynamic approach.

IgG antibody can carry out according to known method itself in the absorption of solid phase carrier of the present invention.The sample (for example blood, blood plasma, serum, ascites, cell culture supernatant, tissue extract) that for example will contain IgG is directed into solid phase carrier of the present invention or is filled with the container or the carrier of solid phase carrier.During static method, for IgG being incorporated into solid phase carrier of the present invention in about about 1 to 60 minute by placing in room temperature while stirring.During dynamic approach,, IgG is incorporated into solid phase carrier of the present invention for by importing sample with about about 0.1 to 20mL/ minute flow velocity.Also can before being fed to solid phase carrier of the present invention, will contain the sample dilution of IgG.The preferred solution that contains sodium-chlor and magnesium chloride that uses of dilution.After IgG is incorporated into solid phase carrier of the present invention,, clean solid phase carrier of the present invention with scavenging solution in order to remove impurity.Scavenging solution preferably contains the solution of sodium-chlor and magnesium chloride.

The wash-out of IgG antibody can use neutral solution to carry out.In the IgG purifying antibody, use in the method in the past of Protein A, owing to must carry out wash-out, therefore the shortcoming of the easy sex change of antibody is arranged with acidic solution.On the other hand, aptamers of the present invention can be carried out wash-out with neutral solution, and is therefore different with in the past method, has the advantage that can prevent the antibody sex change.

There is no particular restriction for neutral elutriant, for example can be pH about 6 to about 9, better about 6.5 to 8.5, better about 7 to about 8.In addition; neutral solution comprises sylvite (for example Repone K (KCl)); potassium acetate; potassium formiate; potassium primary phosphate; dipotassium hydrogen phosphate; Tripotassium phosphate; saltpetre; vitriolate of tartar; potassium sulfite; potassium perchlorate; Tripotassium Citrate; potassium malate; potassium oxalate; potassium cyanide); magnesium salts (magnesium chloride for example; magnesium acetate; magnesium formiate; sal epsom; magnesium oxalate); calcium salt (calcium chloride for example; lime acetate; calcium formiate; calcium sulfate; caoxalate); ammonium salt (ammonium chloride for example; ammonium acetate; ammonium formiate; ammonium phosphate; ammonium nitrate; ammonium sulfate; ammonium sulphite; ammoniumper chlorate; ammonium citrate; ammonium cyanide; ammonium oxalate); sequestrant (ethylenediamine tetraacetic acid (EDTA) (EDTA) for example; Citrate trianions such as Trisodium Citrate; malates such as sodium malate; oxalate such as sodium oxalate; quadrol; ethanoyl acetyl sodium; EGTA); properties-correcting agent or tensio-active agent (guanidine; SDS; Tween 20; NP-40; Triton X-100); from the cost aspect, preferable to contain Repone K.The concentration of Klorvess Liquid is 100 to 1000mM, and better 200 to 800mM, and better 300 to 600mM.The concentration of EDTA solution is 1 to 100mM, and better 5 to 50mM, and better 10 to 20mM.

Purification process of the present invention also can be included in the step of solid phase carrier being cleaned after the IgG antibody absorption.Scavenging solution is enumerated the solution that for example contains urea, highly basic (for example sodium hydroxide, potassium hydroxide), weak base (for example ammonia), strong acid (for example hydrochloric acid, nitric acid, sulfuric acid, trifluoroacetic acid), weak acid (for example acetate, formic acid).Urea for example can be 1 to 10M.Highly basic and weak base are preferably 0.01 to 10N, and more preferably 0.01 to 1N, more more preferably 0.01 to 0.1N.Strong acid and weak base are preferably 0.01 to 10N, and more preferably 0.01 to 1N, more more preferably 0.01 to 0.1N.

Purification process of the present invention can also comprise the step of solid phase carrier being carried out heat treated.By described operation, renewable, the sterilization of solid phase carrier.This heat treated is for example enumerated in about 50 to about 100 ℃, and better about 60 to about 90 ℃, better about 65 to 85 ℃ were carried out several minutes for example 1 to 30 minute, and better 1 to 20 minute, better 5 to 15 minutes processing.Heat treated can be carried out in urea (for example 1 to 10M).

The present invention also provides the manufacture method of antibody purification.Manufacture method of the present invention can comprise preparation IgG antibody, utilizes aptamers of the present invention and complex body (for example solid phase carrier of the application of the invention) step with the IgG antibody purification that makes.

Antibody through manufacture method preparation of the present invention can be IgG.Antibody can be polyclonal antibody or monoclonal antibody.Polyclonal antibody or monoclonal antibody can prepare by known method itself.Antibody also can be humanized antibodies or people's antibody, and is preferable with humanized antibodies or people's antibody.But the flat 4-506458 communique of the special table of humanized antibodies's reference example such as Japanese Patent, the Japanese Patent spy opens preparations such as clear 62-296890 communique, but people's antibody reference example is as " Nature Genetics; Vol.15; p.146-156; 1997 ", " Nature Genetics; Vol.7; p.13-21; 1994 ", the flat 4-504365 communique of the special table of Japanese Patent, the open WO94/25585 communique of international application, " Nikkei science; June number, the 40th to the 50th page; nineteen ninety-five ", " Nature, Vol.368; p.856-859,1994 ", the flat 6-500233 communique of the special table of Japanese Patent waits and prepares.

Now, the antibody that makes can use the aptamers purifying.The detailed method of purifying is identical with purification process of the present invention.

The present invention also provides detection and/or the quantivative approach of IgG.Detection of the present invention and/or quantivative approach comprise the step of utilizing aptamers of the present invention (for example complex body of the application of the invention and/or solid phase carrier) to measure IgG.This method as in diagnosis of the present invention with as described in the content of reagent, substitute the antibody except using aptamers of the present invention, detect and/or quantitatively by the method identical with immunologic method.

The present invention also provides the modifying method of antibody.Modifying method of the present invention can comprise by aptamers of the present invention, makes the step of functional substance and antibodies.The present invention also provides the modified antibodies that makes by this modifying method.

The content of all publications records that comprises the patent enumerated in this specification sheets and patent application specification is by the quoting of this specification sheets, all to introduce in this specification sheets with expressing with degree ground.

Below enumerate embodiment and illustrate in greater detail the present invention, the present invention is not subjected to any qualification of following embodiment etc.

[embodiment]

The preparation of [embodiment 1] and IgG specificity bonded nucleic acid

Use the SELEX method to make and IgG specificity bonded nucleic acid.SELEX carries out people's such as people's such as Ellington method (Ellington and Szostak, Nature 346,818-822,1990) and Tuker method (Tuerk and Gold, Science 249,505-510,1990) improvement.Target substance uses with the mosaic of the human IgG1's of histidine mark (tag) Fc part (Pro 100 is to Lys330) and RANK (receptor activation of NF-κ B (Receptor activator)) (IgG1-Fc, An Di biotech firm makes).This mosaic is expressed for using murine myeloma cell.The RNA that initial bout uses is to use DuraScribe ^TMThe DNA that T7 transcript reagent box (a Chinese mugwort cent (Epicentre) company makes) obtains chemosynthesis transcribes and gets.Through the RNA that this method obtains, its ribosomal 2 ' of Nucleotide of containing pyrimidine bases is fluoridized.Dna profiling uses the DNA that has length 90 residues of primer sequence in the stochastic sequence both sides of 40 residues.Dna profiling and primer make (manufacturing of Bo Ao company) by chemosynthesis.The sequence and the primer sequence of dna profiling are as follows.

Dna profiling:

5 '-ctctcatgtcggccgtta-40N-cgtccattgtgtccctatagtgagtcgtatta-3 ' (sequence numbering 24)

Primer-A:5 '-taatacgactcactatagggacacaatggacg-3 ' (sequence numbering 25)

Primer-B:5 '-ctctcatgtcggccgtta-3 ' (sequence numbering 26)

Primer A contains the promoter sequence of T7 RNA polymerase.The variation (variation) in the RNA pond that initial bout uses is 10 in theory ¹⁴

Make IgG1-Fc as target substance be adsorbed in Ni-NTA affinity resin (be in harmony root (Qiagen) company make) or BD Talon ^TMAffinity resin (manufacturing of BD Biological Science Co., Ltd) and fixing, to wherein adding the RNA pond, the RNA that will not be incorporated into IgG1-Fc after keeping 30 minutes under the room temperature washes with solution A.Solution A at this is the mixing solutions of 145mM sodium-chlor, 5.4mM Repone K, 1.8mM calcium chloride, 0.8mM magnesium chloride, 20mM pH7.6 Tris.The RNA that is incorporated into IgG1-Fc is added elutriant, reclaim, after the RT-PCR amplification, use DuraScribe ^TMT7 transcript reagent box is transcribed, and is used for second leg.Elutriant uses the solution that adds the 250mM imidazoles in solution A.After the 7th bout and the 10th bout are finished, the PCR product is cloned (Cloning) in pGEM-T Easy carrier (vector) (manufacturing of Pu Luomaige company), transform at e. coli strains DH5 α (Japan spins company and makes).From mono-clonal,, use DNA sequencer (ABIPRISM3100, ABI company makes) definite kernel nucleotide sequence with after the plasmid extraction.Clone with the sequence shown in the sequence numbering 1 is 10 clones among 48 clones.In addition, have being cloned in of the sequence shown in the sequence numbering 2,3,4,5,6,7,8 and have 2,7,14,2,5,4,4 clones among 48 clones respectively.

Use the secondary structure (M.Zuker of the RNA shown in the MFOLD program forecasting sequence numbering 1 to 8, Mfold web server of nucleic acid folding and hybridization prediction.Nucleic Acids Res.31 (13), 3406-15, (2003)).Its structure is shown in Fig. 1 to Fig. 8.As shown in the figure, this RNA contains the common sequences of GGUGCU, and this common sequences forms projection.

Change combination of primers, carry out SELEX once more by method same as described above.Primer sequence is as described below.

Primer C:5 '-taatacgactcactatagggccacagcgag-3 ' (sequence numbering 27)

Primer D:5 '-ccgaccacacgcg-3 ' (sequence numbering 28)

After the 8th bout was finished, there was 1 clone in the RNA shown in the sequence numbering 9 in 48 clones that investigate sequence.This RNA combines with human IgG1's specificity, has the sequence of GGUGCU.Use the MFOLD program to predict the secondary structure of this RNA, there is not the raised structures of GGUGCU in the result.Use vsfold4 program ( Http:// www.rna.it-chiba.ac.jp/vsfold4/) secondary structure of the RNA shown in the forecasting sequence numbering 9, found that the raised structures (Fig. 9) of GGUGCU.On the other hand, other 47 clones do not combine with IgG1.

Then, be fixed with the SELEX of the Fc fragment (IgG-Fc) of IgG by amino coupling.Human IgG-Fc (A Sensi research science and technology (Athens Research with 100 μ g; Technology) company makes) be fixed in the NHS-activatory sepharose 4B (activated Sepharosebeads) (peace agate West Asia Biological Science Co., Ltd (Amersham Bioscience) makes) of 30 μ L.The IgG-Fc solution of buying is owing to contain the Tris damping fluid, and therefore will be replaced into 20mM HEPES damping fluid (manufacturing of sigma company) carries out coupling afterwards again.Coupling is carried out according to specification sheets.The immobilization amount is by confirming with IgG-Fc solution before the SDS-PAGE investigation immobilization and the supernatant liquor after the immobilization just.If do not detect the bands of a spectrum of IgG-Fc, think that then the IgG-Fc that uses is almost entirely by coupling from supernatant liquor.RNA uses 2 ' RNA after fluoridizing of the ribose of the Nucleotide that contains pyrimidine bases same as described above.As the dna profiling that is used to prepare RNA initial stage pond, use the sequence after the stochastic sequence utilize following primer sequence clamping 40 residues.

Primer E:5 '-taatacgactcactatagggtacgagtctggacttgcaa-3 ' (sequence numbering 29)

Primers F: 5 '-gcctgttgtgagcctca-3 ' (sequence numbering 30)

After the 7th bout was finished, the RNA shown in the sequence numbering 19,20,21 had 13,9,6 clones respectively in 48 clones that investigate sequence.These RNA contain the common sequences of GGUGCU.Use the MFOLD program to predict this secondary structure, sequence numbering 20 and 21 RNA contain the raised structures identical with the RNA of sequence numbering 1 as a result, and the RNA of sequence numbering 19 does not then contain (Figure 19 to Figure 21).Then, for only existing 1 clone's RNA sequence to carry out detailed investigation among 48 clones, the RNA shown in the sequence numbering 22 and 23 contains the common sequences of GGUGCU as a result.Use MFOLD program prediction secondary structure, the RNA of sequence numbering 22 contains same raised structures as a result, and the RNA of sequence numbering 23 does not then contain.Other 1 clone has 15 sequences.They all do not contain GGUGCU.Investigate the combination activity of 8 sequences in these sequences, the result does not all have in conjunction with active.

Use by 2 ' of ribose by fluorizated contain the Nucleotide of pyrimidine bases and natural type the purine-containing nucleotide base and RNA, carry out different SELEX 3 times, from wherein any, select to contain the RNA of GGUGCU common sequences.Do not have special feature in the sequence of these common sequences both sides, prediction with the combining of IgG in GGUGCU play an important role.Use MFOLD program prediction secondary structure, the selected RNA of result nearly all contains the raised structures of GGUGCU, predicts that all RNA with common sequences all have the raised structures of GGUGCU.

[embodiment 2] are in conjunction with active assessment

Active with the RNA shown in the surperficial plasmon resonant method research sequence numbering 1 to 9 to the combination of human IgG-Fc.Measure and use the BIAcore 2000 that makes than Acker (BIAcore) company.Sensing chip uses the immobilized SA chip of streptavidin.Thereon in conjunction with being combined with the Poly dT of 16 residues of vitamin H in 5 ' end about 1000 RU.Become the Poly A of the RNA of part, be fixed on the SA chip with combining of A by dT at 3 ' terminal additional 16 residues.Its immobilization amount makes it to become about 1000 RU for spraying 60 μ L with 0.01 μ g/ μ L concentration.The IgG-Fc (A Sensi research scientific ﹠ technical corporation makes) that analyzes usefulness is for being adjusted into concentration the surge 70 μ L of 0.6 μ M.The composition of the running buffer (Running Buffer) that uses is identical with SELEX solutions employed A.

Sensing figure when the RNA shown in the fixed sequence program numbering 1 or 3 sprays the IgG-Fc injection is shown in Figure 24 or Figure 25 respectively.Show RNA and IgG-Fc bonded state.Control group carries out and will contain the immobilized mensuration in RNA pond of stochastic sequence, and the result is not in conjunction with IgG-Fc (Figure 26).Carry out identical mensuration for the RNA shown in the sequence numbering 2 to 9, the result all combines with IgG-Fc.

Then, use the same method and investigate the activity that combines with the human IgG1 of total length.RNA shown in the sequence numbering 1 to 9 all combines with IgG1.In addition, the RNA pond of containing stochastic sequence does not show in conjunction with active.

Then, the IgG (0.6 μ M to 0.05 μ M) that working concentration is different carries out the analysis of rate theory, obtains the dissociation constant (Kd) of each RNA aptamers.Dissociation constant is tried to achieve by the following method: utilize the A-dT key to spray the different IgG (0.6 μ M to 0.05 μ m) of concentration in 3 ' the terminal RNA solidification that adds the PolyA that 16 residues are arranged in sensing chip, obtain through surperficial plasmon resonant method.The results are shown in table 1.

Table 1

It is active with combining of human IgG1 to investigate the RNA shown in the sequence numbering 19 to 23 with surperficial plasmon resonant method.By the result as can be known, all RNA have in conjunction with active IgG1.The secondary structure of RNA shown in the sequence numbering 19 and 23 of use MFOLD program prediction does not contain the common raised structures, but all the human IgG1 is had in conjunction with active.

Use Biacore2000 (trade(brand)name) (making) to measure the combination activity of aptamers than Acker company.Be assembled with the parsing software of rate theory among the Biacore2000,, can obtain dissociation constant by shape and the theoretical formula match (fitting) of the sensing figure that will obtain.Long aptamers shown in the sequence numbering 1 to 9 and 19 to 23 extremely meets the theoretical formula of 1: 1 combination model, for short aptamers such as sequence numbering 17 grades, is more suitable for the theoretical formula of the Bivalent model of 1: 2 combination model.Therefore antibody it has been generally acknowledged that two aptamers of an antibodies owing to be symmetric structure.

Confirmed to have the combination of human IgG active by the RNA shown in the sequence numbering 1 to 9 and 19 to 23 of SELEX method preparation by above.This GGUGCU pair of common sequences of expression is very important with combining of IgG.

The miniaturization of [embodiment 3] RNA aptamers

RNA length shown in the sequence numbering 1 to 9 and 19 to 23 is about 70 residues, if can foreshorten to the following length of about 40 residues, then the RNA aptamers can be prepared by chemosynthesis.Attempted the RNA miniaturization shown in sequence numbering 1 to 9 and 19 to 23.At this, 2 ' in the ribose of the Nucleotide that contains pyrimidine bases of the RNA shown in the sequence numbering 1 to 9 and 19 to 23 (U, C) is fluoridized, and the Nucleotide (A, G) that contains purine bases is natural RNA type.In addition, 2 ' in the ribose of all pyrimidine bases of freshly prepd short rna is fluoridized in the present embodiment.

At first the RNA shown in the sequence numbering 1 is attempted miniaturization as the basis.RNA shown in the sequence numbering 10 cuts off 5 ' of the RNA shown in the sequence numbering 1 terminal GGGACAC and 3 ' terminal GAGAG, again in order to transcribe at 5 ' terminal additional GG.RNA shown in the sequence numbering 11 gets 5 ' of the RNA shown in the sequence numbering 10 terminal GGAAU and 3 ' terminal ACAU cut-out.RNA shown in the sequence numbering 12 cuts off 5 ' of the RNA shown in the sequence numbering 11 terminal GGACGAGUU and 3 ' terminal AACGGCCG, gets in 5 ' terminal additional GG and at 3 ' terminal additional CC.RNA shown in the sequence numbering 13 gets for the loop-stem structure that the loop-stem structure in the back of the GGUGCU projection of the RNA shown in the sequence numbering 12 is changed to the RNA shown in the sequence numbering 2.RNA shown in the sequence numbering 14 gets for the loop section with the RNA shown in the sequence numbering 13 is changed to GAAA tetranucleotide ring.RNA shown in the sequence numbering 15 gets for remove 2 base pairs shortening stems from the initial stem of RNA shown in the sequence numbering 13.RNA shown in the sequence numbering 16 gets for remove 3 base pairs shortening stems from the 2nd stem of the RNA shown in the sequence numbering 13.RNA shown in the sequence numbering 17 gets for remove 2 base pairs shortening stems from the initial stem of RNA shown in the sequence numbering 16.RNA shown in the sequence numbering 18 gets for remove 1 base pair shortening stem from the 2nd stem of the RNA shown in the sequence numbering 17.

Use surperficial plasmon resonant method confirm through the RNA of miniaturization in conjunction with active.Measure identically with embodiment 1, the RNA that utilizes the A-pT key will add the Poly A of 16 residues fixes, and sprays IgG thereon.By the result as can be known, the RNA that contains the GGUGCU common sequences shown in the sequence numbering 10 to 18 has in conjunction with active the human IgG1.Wherein, the RNA shown in the sequence numbering 18 is formed by 21 residues.Their dissociation constant is shown in table 1 respectively.

Preparation is replaced into the C of the 8th Nucleotide of RNA of sequence numbering 17 expressions the varient of U.This C is the C of common sequences GGUGCU.By the result of surperficial plasmon resonance analyzing as can be known, this varient does not have in conjunction with active the human IgG1.This fact represents that GGUGCU pair as common sequences is very important with combining of IgG.

By with the RNA miniaturization shown in the sequence numbering 1, can make the RNA aptamers of the length of energy chemosynthesis according to above-mentioned.In addition, active as long as existence as the raised structures of the GGUGCU of common sequences, just can keep with combining of IgG.

[embodiment 4] species specific assessment

Whether the RNA aptamers of using surperficial plasmon resonant method institute to make also has associativity for hypotype IgG beyond the human IgG1 or the animal IgG beyond the people.Measure identically with embodiment 1, the nucleic acid that utilizes the A-pT key will add the Poly A of 16 residues is fixed, and sprays IgG thereon.The RNA aptamers is used the nucleic acid shown in the sequence numbering 1 and 17.Antibody end user IgG1 (manufacturing of Ka Er biological chemistry (Calbiochem) company), human IgG2's (manufacturing of Ka Er Biochemics Inc.), human IgG 3 (manufacturing of Ka Er Biochemics Inc.), human IgG 4 (manufacturing of Ka Er Biochemics Inc.), mouse IgG1 (manufacturing of the Ka Mikang world (Chemicon International) company), mouse IgG2a (manufacturing of Ka Mikang international corporation), mouse IgG2b (diligent plum moral is tested (Zymed Laboratories) company and made), mouse IgG3 (times Shi Er test (Bethyl Laboratories) company makes), rat IgG1 (manufacturing of An Di biotech firm), rat IgG2a (diligent the test of plum moral company make), rat IgG2b (diligent the test of plum moral company make), rat IgG2c (manufacturing of Britain serum science and technology (UK-Serotec) company), rabbit IgG (diligent the test of plum moral company make), ox IgG1 (times Shi Er test company makes), ox IgG2 (times Shi Er test company makes), chicken IgG (manufacturing of Roc orchid (Rockland) company), dog IgG (the blue company of Roc makes), cat IgG (times Shi Er test company makes), cavy IgG (manufacturing of biological gene (Biogenesis) company), hamster IgG (the blue company of Roc makes), pig IgG (the blue company of Roc makes).The result is shown in table 2.

Table 2

* the data referencing of Protein A is pacified the specification sheets of agate West Asia Biological Science Co., Ltd.+ expression bonded intensity ,+many persons represent in conjunction with strong.-represent not combination.Nd represents undetermined.

As shown in Table 2, though the RNA shown in sequence numbering 1 and 17 has in conjunction with active human IgG1, human IgG2, human IgG 3, human IgG 4, hamster IgG, pig IgG, other kinds animal is not then had in conjunction with active.In addition, the nucleic acid shown in the sequence numbering 1 and 17 does not have in conjunction with active people IgD (Biogen, Inc.'s manufacturing) and people IgE (manufacturing of Ka Er Biochemics Inc.).Though the nucleic acid shown in the sequence numbering 17 does not have in conjunction with active people IgA (times Shi Er test company makes), people IgM (manufacturing of Ka Mikang international corporation) is shown very weak in conjunction with active.

As from the foregoing, RNA aptamers provided by the invention is the IgG specificity bonded RNA with people, hamster, pig.In addition, irrelevant about human IgG and hypotype as can be known, combine with IgG1 to IgG4 is equal.This be with the Protein A that uses with the part of resin as present antibody purification different characteristic.

The investigation 1 of [embodiment 5] RNA aptamers combining site (Fc γ R)

The Fc of IgG partly is incorporated into the receptor protein (Fc γ) of expressing in immunocytes such as scavenger cell or neutrophilic leukocyte, promote the activation or the inhibition of cell.Use surperficial plasmon resonant method to investigate the Fc γ R combining site whether RNA provided by the invention is combined in IgG.At first, use the method identical to fix the additional RNA aptamers that the Poly A of 16 residues arranged, spray human IgG thereon and make it with after the RNA aptamers combine injection Fc γ R with embodiment 1.If the combining site of the RNA aptamers combining site of IgG and Fc γ R is whole or major portion repeats, can think that then Fc γ R can not be incorporated into the IgG that combines with the RNA aptamers.In addition,, when causing the replacement(metathesis)reaction of RNA aptamers and Fc γ R, infer that IgG dissociates from the RNA aptamers that is fixed, form complex body with Fc γ R and flow out when IgG is stronger than the bonding force of IgG and RNA aptamers with the bonding force of Fc γ R.

Use the RNA shown in the sequence numbering 1 as RNA aptamers, human IgG-Fc (A Sensi research scientific ﹠ technical corporation makes) as IgG, people Fc γ RI (R﹠amp; D Systerns company makes) measure as Fc γ R.The result observes IgG-Fc and rises (Figure 27) in conjunction with the signal in conjunction with generation of back by Fc γ RI, therefore as can be known, forms RNA aptamers, IgG-Fc, Fc γ RI three's complex body.In addition, control group uses when containing the RNA pond of stochastic sequence, and IgG-Fc does not combine (Figure 28) with Fc γ RI.

As from the foregoing, the RNA aptamers is combined in the Fc γ RI combining site institute distinct portions with IgG.

The investigation 2 (Protein A) of [embodiment 6] RNA aptamers combining site

As other materials that are incorporated into IgG-Fc, knowing has Protein A.Therefore Protein A is used as the part of antibody purification with separating agent owing to combine with the Fc part specificity of IgG.Investigate the ProteinA combining site whether RNA provided by the invention is incorporated into IgG by the method identical with embodiment 5.At first, to fix by the method identical in the RNA aptamers shown in the sequence numbering 1 of 3 ' the terminal additional PolyA that 16 residues are arranged with embodiment 1, after its inflow human IgG1 (manufacturing of Ka Er Biochemics Inc.) makes it to be incorporated into the RNA aptamers, spray Protein A (manufacturing of MPBiomedicals company).The result observes IgG1 and rises (Figure 29) in conjunction with the signal in conjunction with generation of back by Protein A, therefore forms RNA aptamers, IgG1, Protein A three's complex body as can be known.In addition, control group carries out the mensuration with the fixing back injection of the RNA aptamers shown in the sequence numbering 1 ProteinA, and the result does not see the combination (Figure 30) of ProteinA.

As from the foregoing, the RNA aptamers is incorporated into the Protein A combining site institute distinct portions with IgG-Fc.

2 ' the modifying method of [embodiment 7] RNA aptamers and active to the combination of IgG

2 ' in the ribose of the Nucleotide that contains pyrimidine bases of the RNA aptamers of embodiment 1 preparation is fluoridized.In the present embodiment, the preparation and the different RNA of modifying method of 2 ' in ribose use surperficial plasmon resonant method to investigate combination activity to IgG.

At first, make the natural type RNA shown in the sequence numbering 11 and 13, investigate the combination of IgG active.Natural type RNA transcribes and makes by chemosynthesis template DNA (manufacturing of Ou Beilong company), use T7 RNA polymerase (precious biotech firm makes).In conjunction with active identical, measure by surperficial plasmon resonant method with embodiment 1.End user IgG1 (manufacturing of Ka Er Biochemics Inc.) is as IgG.By the result as can be known, the binding capacity of IgG1 reduces, and the natural type RNA of sequence numbering 11 and 13 expressions has in conjunction with active IgG.

Then, based on sequence numbering 17, be produced as follows the RNA shown in the different sequence numbering 17-1 to 17-14 of described modification.

Sequence numbering 17

G(OH)G(OH)A(OH)G(OH)G(OH)U(F)G(OH)C(F)U(F)C(F)C(F)G(OH)A(OH)A(OH)A(OH)G(OH)G(OH)A(OH)A(OH)C(F)U(F)C(F)C(F)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)

Sequence numbering 17-1 (23F1)

G(OH)G(OH)A(OH)G(OH)G(OH)U(F)G(OH)C(F)U(F)C(H)C(H)G(OH)A(OH)A(OH)A(OH)G(OH)G(OH)A(OH)A(OH)C(F)T(H)C(H)C(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)

Sequence numbering 17-2 (23F2)

G(H)G(H)A(OH)G(OH)G(OH)U(F)G(OH)C(F)U(F)C(F)C(F)G(H)A(H)A(H)A(H)G(OH)G(OH)A(OH)A(OH)C(F)U(F)C(H)C(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)

Sequence numbering 17-3 (23F3)

G(H)G(H)A(H)G(OH)G(OH)U(F)G(OH)C(F)U(F)C(H)C(H)G(H)A(H)A(H)A(H)G(H)G(H)A(OH)A(OH)C(F)T(H)C(H)C(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)

G(H)G(H)A(OH)G(OH)G(OH)U(F)G(OH)C(F)U(F)C(F)C(H)G(H)A(H)A(H)A(H)G(OH)G(OH)A(OH)A(OH)C(F)U(F)C(H)C(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)

Sequence numbering 17-5 (23F11)

G(H)G(H)A(OH)G(OH)G(OH)U(F)G(OH)C(F)U(F)C(F)C(F)G(H)A(H)A(H)A(H)G(H)G(H)A(OH)A(OH)C(F)U(F)C(H)C(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)

Sequence numbering 17-6 (23F12)

G(H)G(H)A(OH)G(OH)G(OH)U(F)G(H)C(F)U(F)C(F)C(F)G(H)A(H)A(H)A(H)G(OH)G(OH)A(OH)A(OH)C(F)U(F)C(H)C(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)

Sequence numbering 17-7 (23F23)

G(H)G(H)A(OH)G(OH)G(OH)U(F)G(OH)C(H)U(F)C(H)C(H)G(H)A(H)A(H)A(H)G(OH)G(OH)A(H)A(H)C(F)U(F)C(H)C(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)

Sequence numbering 17-8 (23F25)

G(OMe)G(OMe)A(OH)G(OH)G(OH)U(F)G(OH)C(F)U(F)C(F)C(F)G(OMe)A(OMe)A(OMe)A(OMe)G(OH)G(OH)A(OH)A(OH)C(F)U(F)C(OMe)C(OMe)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)

Sequence numbering 17-9 (23F32)

G(H)G(H)A(OH)G(OH)G(OH)U(F)G(OH)C(H)U(F)C(H)C(H)G(H)A(H)A(H)A(H)G(OMe)G(OH)A(H)A(H)C(F)U(F)C(H)C(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)

Sequence numbering 17-10 (23F33)

G(H)G(H)A(OH)G(OH)G(OH)U(F)G(OH)C(H)U(F)C(H)C(H)G(H)A(H)A(H)A(H)G(OH)G(OMe)A(H)A(H)C(F)U(F)C(H)C(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)

Sequence numbering 17-11 (23F41)

G(OMe)G(OMe)A(OH)G(OH)G(OH)U(F)G(OH)C(F)U(OMe)C(F)C(F)G(OMe)A(OMe)A(OMe)A(OMe)G(OH)G(OH)A(OH)A(OH)C(F)U(F)C(OMe)C(OMe)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)

Sequence numbering 17-12 (23F42)

G(OMe)G(OMe)A(OH)G(OH)G(OH)U(F)G(OH)C(F)U(F)C(OMe)C(F)G(OMe)A(OMe)A(OMe)A(OMe)G(OH)G(OH)A(OH)A(OH)C(F)U(F)C(OMe)C(OMe)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)

Sequence numbering 17-13 (23F43)

G(OMe)G(OMe)A(OH)G(OH)G(OH)U(F)G(OH)C(F)U(F)C(F)C(OMe)G(OMe)A(OMe)A(OMe)A(OMe)G(OH)G(OH)A(OH)A(OH)C(F)U(F)C(OMe)C(OMe)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)

Sequence numbering 17-14 (23F31)

G(H)G(H)A(OH)G(OH)G(OH)U(F)G(OH)C(H)U(OMe)C(H)C(H)G(H)A(H)A(H)A(H)G(OH)G(OH)A(H)A(H)C(F)U(F)C(H)C(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)A(H)

RNA makes (manufacturing of Gene Design company) by chemosynthesis.In conjunction with active identical, measure by surperficial plasmon resonant method with embodiment 1.IgG end user IgG1 (manufacturing of Ka Er Biochemics Inc.).Measurement result is that the RNA shown in the sequence numbering 17-1 shows that with the RNA shown in the sequence numbering 17 combining of same degree is active.In addition, the RNA in conjunction with shown in the specific activity sequence numbering 17 of the RNA shown in sequence numbering 17-2, the 17-4 to 17-14 is higher.On the other hand, the RNA in conjunction with shown in the specific activity sequence numbering 17 of the RNA shown in the sequence numbering 17-3 is low.

With the nucleic acid shown in the sequence numbering 15 serves as that the change body is modified in the basic preparation of operation equally, and it is active to investigate its combination to human IgG.

Sequence numbering 15

G(OH)G(OH)A(OH)G(OH)G(OH)U(F)G(OH)C(F)U(F)C(F)U(F)G(OH)C(F)G(OH)A(OH)G(OH)C(F)C(F)A(OH)C(F)G(OH)C(F)G(OH)G(OH)A(OH)A(OH)C(F)U(F)C(F)C(F)

Sequence numbering 15-1 (30F-1)

G(H)G(H)A(OH)G(OH)G(OH)U(F)G(OH)C(F)U(F)C(F)U(F)G(OH)C(F)G(OH)A(H)G(H)C(H)C(H)A(H)C(F)G(OH)C(F)G(ON)G(OH)A(OH)A(OH)C(F)U(F)C(H)C(H)

Sequence numbering 15-2 (30F-2)

G(H)G(H)A(OH)G(OH)G(OH)U(F)G(OH)C(F)U(F)C(F)U(H)G(OH)C(F)G(OH)A(H)G(H)C(H)C(H)A(H)C(H)G(OH)C(H)G(OH)G(OH)A(OH)A(OH)C(F)U(F)C(H)C(H)

Sequence numbering 15-3 (30F-3)

G(H)G(H)A(OH)G(OH)G(OH)U(F)G(OH)C(F)U(F)C(F)C(H)G(OH)C(H)G(OH)A(H)G(H)C(H)C(H)A(H)C(H)G(OH)C(H)G(OH)G(OH)A(OH)A(OH)C(F)U(F)C(H)C(H)

Sequence numbering 15-4 (30F-4)

G(H)G(H)A(OH)G(OH)G(OH)U(F)G(OH)C(F)U(F)C(F)C(F)G(OH)C(H)G(OH)G(H)A(H)A(H)A(H)C(H)G(OH)C(H)G(OH)G(OH)A(OH)A(OH)C(F)U(F)C(H)C(H)

By the result who measures with surperficial plasmon resonant method as can be known, the nucleic acid shown in the sequence numbering 15-1 to 15-3 have with the nucleic acid shown in the sequence numbering 15 equal combine activity.

Sum up above result and be shown in table 3-1 and table 3-2.In the table 3 with+represent in conjunction with active height ,+number more multilists show that activity is high more.In addition, RNA that sequence numbering 17-2 is represented and IgG bonded state are shown in Figure 31.

Table 3-1

Table 3-2

The raised structures of [embodiment 8] GGUGCU

In order to investigate whether GGUGCU sequence in addition is the sequence that has affinity with IgG, carry out optimization SELEX.The RNA with the part random alignment of GGUGCU is used in initial pond.This RNA pond is a template by the DNA that makes with the chemosynthesis shown in following, uses DuraScribe ^TMT7 transcript reagent box (a Chinese mugwort cent company makes) is transcribed and is made.

Dna profiling:

5 '-tgtcggccgttacagttccggtttcccgg-6N-tgtaactcgtccattgtccc-3 ' (sequence numbering 31)

Primer G:5 '-taatacgactcactatagggacaatggacgagttac-3 ' (sequence numbering 32)

Primer H:5 '-tgtcggccgttacagttc-3 ' (sequence numbering 33)

The theoretic variation in RNA pond is 4096.Based on specification sheets, the human IgG (diligent plum moral test) of 40 μ g is fixed in the NHS-activatory sepharose 4B (peace agate West Asia Biological Science Co., Ltd makes) of 40 μ L.SELEX and embodiment 1 carry out in the same manner.

Investigate the sequence after 3 bouts are finished, 36 sequences contain GGUGCU in 48 sequences as a result.Use the MFOLD program to investigate secondary structure, do not exist with different sequences to form the raised structures identical with GGUGCU with GGUGCU.In addition, use surperficial plasmon resonant method to investigate in conjunction with active, the result does not exist for the sequence different with GGUGCU and human IgG is had in conjunction with active sequence.

Then, the sequence after finishing for the 2nd bout is investigated 48 sequences.There is 1 sequence in the sequence that contains GGUGCU.Use the secondary structure of MFOLD program prediction all sequences, the sequence that the result contains GGUGAU forms the raised structures identical with GGUGCU.Use surperficial plasmon resonant method to investigate the affinity of this clone and IgG, result this clone as can be known has in conjunction with active IgG.In addition, though the ACCGAC sequence is found in 2 clones, this sequence is not incorporated into IgG.If use the MFOLD program, then find except GGUGCU and GGUGAU, also to form the sequence of the raised structures identical with GGUGCU.Chemosynthesis contains the nucleic acid that such sequence replaces the GGUGCU of the nucleic acid shown in the sequence numbering 17-7, and it is active with combining of human IgG1 to use surperficial plasmon resonant method to measure.The sequence that replaces GGUGCU to use is as described below.

The sequence of sequence numbering 17-7 jut:

G(OH)G(OH)U(F)G(OH)C(H)U(OH)

The sequence of sequence numbering 17-7-1 jut:

G(OH)A(OH)U(F)G(OH)C(H)U(OH)

The sequence of sequence numbering 17-7-2 jut:

G(OH)C(F)U(F)G(OH)C(H)U(OH)

The sequence of sequence numbering 17-7-3 jut:

G(OH)G(OH)C(F)G(OH)C(H)U(OH)

The sequence of sequence numbering 17-7-4 jut:

G(OH)G(OH)U(F)A(OH)C(H)U(OH)

The sequence of sequence numbering 17-7-5 jut:

G(OH)G(OH)U(F)U(F)C(H)U(OH)

Do not exist among the sequence numbering 17-7-1 to 17-7-5 human IgG is had in conjunction with active sequence.

The GGUGCU of the nucleic acid shown in the sequence numbering 17-7 is shown in G (OH) G (OH) U (F) G (OH) C (F) U (F), and 2 ' in the ribose that contains the Nucleotide of pyrimidine bases is fluoridized.Whether investigation utilizes other modifying method to exist human IgG is had in conjunction with active sequence.The nucleic acid that experiment is used is as described below, makes through chemosynthesis.It is active to use surperficial plasmon resonant method to investigate human IgG1's combination.

The sequence of sequence numbering 17-7 jut:

G(OH)G(OH)U(F)G(OH)C(H)U(F)

The sequence of sequence numbering 17-7-101 jut:

G(OH)G(F)U(F)G(OH)C(H)U(F)

The sequence of sequence numbering 17-7-102 jut:

G(OH)G(OH)U(OH)G(OH)C(H)U(F)

The sequence of sequence numbering 17-7-103 jut:

G(OH)G(OH)U(H)G(OH)C(H)U(F)

The sequence of sequence numbering 17-7-104 jut:

G(OH)G(OH)U(F)G(F)C(H)U(F)

The sequence of sequence numbering 17-7-105 jut:

G(OH)G(OH)U(F)G(OH)C(OH)U(F)

The sequence of sequence numbering 17-7-106 jut:

G(OH)G(OH)U(F)G(OH)C(H)U(OH)

The sequence of sequence numbering 17-7-107 jut:

G(OH)G(OH)U(F)G(OH)C(H)U(OMe)

By as can be known in conjunction with active measurement result, 17-7-101,17-7-104 to 107 have with 17-7 equal combine active.In addition, 17-7-102 and 17-7-103 do not have in conjunction with active.

Be summarized in table 4 more than inciting somebody to action.In the table 4 with+represent in conjunction with active height ,+number more multilists show that activity is high more.

Table 4

As from the foregoing, the raised structures of GGUGCU and GGUGAU is for very important with combining of IgG.In addition, also as can be known, when natural ribonucleotide (2 ' of ribose is OH) or deoxyribose Nucleotide (2 ' of ribose is H), the 3rd the U of GGUGCU disappears in conjunction with active.[embodiment 9] have used the related experiment of the IgG method of purification of RNA aptamers

RNA shown in sequence numbering 15 and 17 is fixed on the pearl, carries out drop-down (pull-down) experiment of human IgG1.In the pipe (the love company that pursues progress makes) of each 200 μ L, put into oligomeric (dT)-cellulose bead (Oligo (dT)-Cellulose beads) (peace agate West Asia Biological Science Co., Ltd makes) 10 μ l, with bovine serum albumin (rich Linear mannheim (Boehringet Mannheim) company makes) coating.Fix to wherein being added in 3 ' the terminal about 10 μ g of RNA with 16 A.RNA re-uses DuraScribe by chemical synthesising DNA template and primer (manufacturing of Bo Ao company) ^TMT7 transcript reagent box (a Chinese mugwort cent company makes) is transcribed and is made it.After unconjugated RNA being removed with the solution A cleaning, add human IgG1's (Ka Er biological chemistry) of 20 μ g, under room temperature, kept 30 minutes.The human IgG1's cleaning that will not be incorporated into RNA with solution A is removed.Then, in pearl, add the sample damping fluid, heated 15 minutes down, utilize SDS-PAGE to analyze in 65 ℃.6 * sample damping fluid be mix 1.3g sodium lauryl sulphate (SDS), 3mL 2 mercapto ethanol, 4.2mL glycerol, the 1.5mg bromophenol is blue and prepare.The result of SDS-PAGE is shown in Figure 32.Swimming lane 1 is the situation of the aptamers of part use sequence numbering 15, and swimming lane 2 uses the situation of the aptamers of sequence numberings 17.Top band is the band of the heavy chain (H chain) of IgG, and following band is the band of light chain (L chain).As can be known, use as the part of antibody purification with separating agent by RNA with sequence numbering 15 or 17 expressions, can IgG is drop-down.

Get the pearl (peace agate West Asia Biological Science Co., Ltd makes) that is combined with Protein A and be combined with pearl (peace agate West Asia Biological Science Co., Ltd makes) the 10 μ L that removed the Protein A (rProtein A) in albumin bound zone by gene recombination, the human IgG1 who adds 20 μ g similarly carries out the purifying of IgG.Use the pH3 glycine buffer as elutriant.Utilize SDS-PAGE to analyze the Figure 32 that the results are shown in of elutriant.Swimming lane 3 is with the situation of ProteinA as part, and swimming lane 2 is with the situation of rProteinA as part.Aptamers can be with drop-down with IgG with the equal performance of Protein A.

Whether investigation can use the RNA of sequence numbering 15 expressions from human serum purifying human IgG.Whether in addition, also investigate neutral elutriant can be with the IgG wash-out.In the pipe (the love company that pursues progress makes) of each 200 μ L, put into sepharose 4B (peace agate West Asia Biological Science Co., Ltd makes) the 10 μ L that are combined with streptavidin, apply with bovine serum albumin.Fix to wherein being added in the about 10 μ g of RNA (manufacturing of gene design company) that 5 ' end is combined with vitamin H.Remove the human serum (manufacturings of Ka Mikang international corporation) that adds 20 μ L behind the unconjugated RNA, in room temperature maintenance 30 minutes.Remove the human serum composition that is not incorporated into RNA with sodium-chlor-magnesium chloride buffer solution for cleaning.Sodium-chlor-magnesium chloride damping fluid is the 20mM Tris damping fluid of 150mM sodium-chlor, 2.5mM magnesium chloride, pH 7.6.With neutral elutriant elution of bound in the IgG of RNA.Neutral elutriant uses the mixing solutions of (1) 200mM Repone K+mixing solutions of 10mM EDTA, the mixing solutions of (2) 200mM Repone K+10mM EDTA+10% glycerine, (3) 600mM Repone K+10mM EDTA+10% glycerine.In order to study the IgG amount of recovery, utilize SDS-PAGE to analyze elutriant.In order to investigate the IgG amount that is not incorporated into pearl, add the sample damping fluid in the pearl after removing elutriant in addition,, utilize SDS-PAGE to analyze in 65 ℃ of heating 15 minutes by wash-out.The Figure 33 that the results are shown in SDS-PAGE.Use the aptamers resin as can be known by swimming lane 2 to 4, can be human IgG is drop-down with high purity from serum.In addition, almost do not detect IgG as can be known by swimming lane 6 to 8, can the wash-out human IgG by using neutral elutriant.

Get the pearl 10 μ L that are combined with rProtein A, add human serum 20 μ L, carry out the purifying of IgG equally.Elutriant uses the glycine buffer of pH3.To utilize SDS-PAGE to analyze the Figure 33 that the results are shown in of elutriant and pearl.As can be known, though the loading capacity of IgG is variant, the aptamers resin can with the rProteinA resin with the purity of degree with the IgG purifying.Can be during from use aptamers resin aspect the neutral wash-out IgG, the aptamers resin is more superior than rProtein A resin.

Use mice serum (manufacturing of Ka Mikang international corporation) to carry out same experiment.IgG down-drawable when using rProtein A then can not be drop-down when using the RNA shown in the sequence numbering 15.As can be known, antibody purification of the present invention is merely able to the antibody with high purity purifying people with the RNA part.

Whether can test with the part use of separating agent as antibody purification repeatedly for the RNA shown in the sequence numbering 15.As mentioned above, the RNA that is combined with about 10 μ g of vitamin H is fixed on the pearl of streptavidin of 10 μ L, adds human serum, with neutral solution wash-out IgG.Afterwards, pearl is cleaned 3 times, in order to remove urea, again with after the pearl cleaning 3 times, added human serum 20 μ L once more, with neutral solution wash-out IgG with sodium-chlor-magnesium chloride damping fluid with 50 μ L 6M urea.This step is carried out once again, utilize SDS-PAGE to confirm the yield (Figure 34) of IgG in 3 times the antibody purification.By its result as can be known, the amount through drop-down IgG there is no big-difference in 3 times antibody purification.Represent that thus the RNA part that antibody purification is used can clean, regenerate with urea.Similarly clean with 0.1M sodium hydroxide.Repetitive operation is carried out purifying 3 times, and the yield of IgG there is no significantly and reduces.

Then, 5 ' of the RNA that represents at RNA shown in the sequence numbering 16 and sequence numbering 17-2 terminally combines vitamin H, as mentioned above, from human serum with the IgG purifying.Use this RNA part as can be known by its result, but high purity IgG purification (Figure 35).

As from the foregoing, by the RNA aptamers is used as part, can be under neutrallty condition from human serum efficient and high purity ground IgG purification.

[embodiment 10] have used the related experiment by the IgG method of purification of mercaptan coupler fixed RNA aptamers

RNA shown in the sequence numbering 15 is fixed in pearl with the mercaptan coupler, carries out the drop-down experiment identical with embodiment 9.Connection base at 5 ' the terminal C18 of the RNA shown in the sequence numbering 15 makes it to combine with thiol group (manufacturing of gene design company).The about 20 μ g of this RNA are fixed in activatory sepharose 4B (peace agate West Asia Biological Science Co., Ltd makes) 10 μ L.Anchor root carries out according to specification sheets.Fixed amount by the RNA amount before utilizing the absorbancy instrumentation fixing surely with just fixing after supernatant liquor in RNA measure and estimate.By its result as can be known, the RNA more than 90% is fixed among the RNA of coupling use.Use this RNA aptamers pearl, carry out the drop-down experiment identical with embodiment 9.Add human serum 5 μ L and 10 μ L to pearl 10 μ g, clean,, utilize SDS-PAGE to analyze (Figure 36) based on the wash-out of neutral elutriant.By its result as can be known IgG by high purity drop-down (Figure 36 swimming lane 2,3).

The drop-down experiment of rProtein A pearl is used in operation similarly to Example 9.Add human serum 5 μ L to rProteinA pearl 10 μ L, clean the back and add SDS-PAGE sample damping fluid,, utilize SDS-PAGE to analyze (Figure 36 swimming lane 5) in 65 ℃ of heating 15 minutes.

By this drop-down result of experiment as can be known, by using through mercaptan coupler fixed RNA aptamers pearl, can be under neutrallty condition, efficient and high purity ground IgG purification from human serum.

Has used through amino coupling and the related experiment of the IgG method of purification of fixed RNA aptamers [embodiment 11]

5 ' the terminal connection base that inserts C12 at RNA makes it to combine with amino, by amino coupling RNA is fixed on the resin.Be combined with amino RNA and make (manufacturing of gene design company) via chemosynthesis.The fixing use Tresyl-TOYOPEARL resin of RNA (eastern Cao company make).Every 1mL resin uses the RNA of 10mg, and the RNA of about 8mg is fixed.The amount that fixed amount is decided RNA in the supernatant liquor before and after the coupling by the absorbancy instrumentation is tried to achieve.Use this aptamers resin, operation is carried out from the experiment of the drop-down IgG of human serum similarly to Example 9.RNA (Figure 38) shown in part use sequence numbering 15 (Figure 37) and sequence numbering 17-7,17-8, the 17-7-107,15.Its result shows any aptamers resin from these, all with the equal purity of rProtein A resin with IgG drop-down (Figure 37,38).

When using the aptamers resin among the embodiment 9, demonstrating can be with the IgG wash-out with the neutral elutriant of 200mM Repone K+10mMEDTA.At this, use through amino coupling and the aptamers resin that fixed sequence numbering 17-7 represents carries out the IgG wash-out experiment based on the neutral elutriant of heterogeneity.Elutriant uses (1) 200mM Repone K+10mM Tris of 10mM EDTA+pH7.6, the 10mM Tris of the 10mM Tris of (2) 200mM Repone K+pH7.6, (3) 300mM sodium-chlor+10mMEDTA+pH7.6, the 10mM Tris of (4) 10mM EDTA+pH7.6.Operation is drop-down from human serum with IgG similarly to Example 9, uses the eluate of SDS-PAGE analysis with above-mentioned elutriant wash-out.By its result as can be known, only can be with IgG wash-out (Figure 39) with Repone K or EDTA.

Whether investigate can be with the IgG wash-out with the 1M sodium chloride solution.Because nucleic acid has negative charge, therefore it is generally acknowledged with combination of proteins in mainly be ionic linkage.Thereby, will separate with combination of proteins as long as use the solution of high salt concentration.Use the 1M sodium chloride solution as elutriant, carry out experiment same as described above, but do not detect IgG in the elutriant, almost be adsorbed in the aptamers resin entirely.In order to confirm the following aptamers of sodium-chlor existence of high density and combining of IgG, use the experiment of surperficial plasmon resonant method.Running buffer uses the Tris mixed solution of 500mM sodium-chlor+2mM magnesium chloride+10mM pH7.6.By its result as can be known, even there is the sodium-chlor of 500mM, all do not reduce in conjunction with activity.

As from the foregoing, the IgG that is incorporated into the aptamers resin can use 200mM Klorvess Liquid wash-out, but can not use 1M sodium chloride solution wash-out.

Then, carry out the aptamers resin through the regeneration of heating and the related experiment of sterilization.To use 3 times sequence numbering 17-17 or the aptamers resin (1) shown in the 17-18 to add ultrapure water, add 6M urea,, carry out drop-down experiment once more in 65 ℃ of heating 15 minutes in 85 ℃ of heating 5 minutes or (2).Use the human serum of 10 μ L, elutriant uses the 10mM Tris solution of 200mM Repone K+10mMEDTA+pH7.6.Utilize SDS-PAGE to analyze elutriant, the result as can be known, the heat treated through (1) and (2), the aptamers resin almost worsens (Figure 40).

But investigation IgG purification whether under moving state.Aptamers resin 100 μ L are filled in small-sized post (rubbing than the mobicols of scientific ﹠ technical corporation (MoBiTec)), add human serum 100 μ L.Add solution A with injection tube afterwards, clean resin (solution A: 4mL, the about 1mL/ of flow velocity minute) at once.Then, use neutral elutriant, will be incorporated into the IgG wash-out (neutral elutriant: 2mL, the about 1mL/ of flow velocity minute) of aptamers part.With the fraction of SDS-PAGE investigation wash-out, results verification IgG is by wash-out.In addition, measure the absorbancy of each fraction, obtain the dynamic purification amount of IgG, the result as can be known, by 1 purifying, but the IgG of every 1mL resin purifying 3.5mg.

[embodiment 12] use the drop-down experiment of IgG of resin mating-type oligo

Hereto, the aptamers that chemosynthesis is made is fixed on the resin and uses via polyA-polydT combination, vitamin H-streptavidin combination, mercaptan coupling, amino coupling.Nucleic acid is synthetic to be fixed in state of resin, and synthetic back re-uses from resin isolation.With the process that shortens from resin isolation and be attached to resin again is purpose, synthesize finish after not with nucleic acid from resin isolation, and be directly used in drop-down experiment (resin mating-type oligo).Resin mating-type oligo uses the RNA shown in the sequence numbering 15 is gone up synthetic (manufacturing of gene design company) at low affinity upholder (Oligo affinity support) (manufacturing of high grace research (Glen Research) company).It is drop-down with IgG to add human serum 10 μ L in resin 10 μ L, and with neutral elutriant wash-out, IgG is purified (Figure 41) with high purity as a result.

[embodiment 13] assessment is active to the combination of chimera antibody

Whether the antibody that uses surperficial plasmon resonant method to confirm that this aptamers is used for the pharmaceuticals that use gene recombination technology to make has in conjunction with active.Antibody uses the Rituximab (Roche Holding Ag's manufacturing) that uses as pharmaceuticals, and part uses the nucleic acid shown in the sequence numbering 17-7.By the result who measures as can be known, the nucleic acid shown in the sequence numbering 17-7 has in conjunction with active (Figure 42) Rituximab.

[industrial utilizability]

The invention provides the nucleic acid ligands that IgG is had the combination energy. Nucleic acid ligands provided by the invention IgG is kept high combination activity and specificity. In addition, because can chemical synthesis, therefore can hold Changing places changes the nucleotides sequence, perhaps increases and modifies. Thereby, antibody is used in pharmaceutical reagent examines During disconnected medicine, can change in conjunction with active or stable easily according to various needs, perhaps make it knot Close fluorescent substance or anticancer dose etc. and increase new function. In recent years with the monoclonal antibody of peopleization as Molecular targeted agents and practical carries out the exploitation of worldwide antibody preparation. Therefore, expectation exploitation Substitute the high functionality release agent of the Protein A resin isolation agent of present antibody purification use, this branch Reach about about 50,000,000,000 yen from the agent market prediction. Nucleic acid ligands provided by the invention can be used as anti-The body purifying uses with the part of release agent, can resist by easy high-purity ground purification of target under neutral condition Body. This purifying under acid condition with in the past use Protein A is greatly different, in the purifying The possibility of antibody inactivation is little. In addition, nucleic acid ligands provided by the invention can be widely used as for knot That closes antibody and fluorescent substance or enzyme novelly is connected base, is used for antibody is fixed in substrate or resin Novel fixing agent, be used for novel connection base that antibody is combined with anticancer dose or toxin. The present invention Can expect to be widely used as the novel release agent relevant with antibody, reagent, medicinal industry and research uses Instrument, its economic effect is also big.

Present patent application proposes the 2005-195717 of patent application and the U.S. Provisional Application US60/749026 that filed an application in the U.S. on December 12nd, 2005 based on July 5th, 2005 in Japan, and its content is applied in this specification sheets.

Sequence table

＜110〉Ribomic Inc.

＜120〉with immunoglobulin G bonded nucleic acid and utilize method

<130>09944

<150>JP?2005-195717

<151>2005-07-05

<150>US?60/749,026

<151>2005-12-12

<160>33

<170>PatentIn?version?3.3

<210>1

<211>73

<212>RNA

＜213〉artificial

<220>

＜223〉synthetic RNA

<400>1

gggacacaau?ggacgaguua?caggugcucc?aucaacaaaa?uguuacaugg?aacuguaacg 60

gccgacauga?gag 73

<210>2

<211>71

<212>RNA

＜213〉artificial

<220>

＜223〉synthetic RNA

<400>2

gggacacaau?ggacgucaaa?gaagaggugc?ucugcgagcc?acgcggaacu?cuauaacggc 60

cgacaugaga?g 71

<210>3

<211>74

<212>RNA

＜213〉artificial

<220>

＜223〉synthetic RNA

<400>3

gggacacaau?ggacggcgua?aaauggaacc?uggguuagaa?uaucgggggu?gcuccguaac 60

ggccgacaug?agag 74

<210>4

<211>71

<212>RNA

＜213〉artificial

<220>

＜223〉synthetic RNA

<400>4

gggacacaau?ggacggccaa?cguuaacugg?aacuguaaau?caggugcucg?agauaacggc 60

cgacaugaga?g 71

<210>5

<211>72

<212>RNA

＜213〉artificial

<220>

＜223〉synthetic RNA

<400>5

gggacacaau?ggacgacuac?aaggugcucc?uugaaauguu?aaaugaggaa?cuuguaacgg 60

ccgacaugag?ag 72

<210>6

<211>73

<212>RNA

＜213〉artificial

<220>

＜223〉synthetic RNA

<400>6

gggacacaau?ggacggcugg?uaaggugcuc?ggaauggaac?ucgucauucg?gaacuuaacg 60

gccgacauga?gag 73

<210>7

<211>72

<212>RNA

＜213〉artificial

<220>

＜223〉synthetic RNA

<400>7

gggacacaau?ggacgggugu?uacaggugcu?ugauaaaggu?agaaaaucaa?acuguaacgg 60

ccgacaugag?ag 72

<210>8

<211>72

<212>RNA

＜213〉artificial

<220>

＜223〉synthetic RNA

<400>8

gggacacaau?ggacgaaagg?ugcuccaacu?aaauuuggaa?cuuuccaccc?auaauaacgg 60

ccgacaugag?ag 72

<210>9

<211>67

<212>RNA

＜213〉artificial

<220>

＜223〉synthetic RNA

<400>9

gggccacagc?gaggugcucc?accauucacg?uggaacucgu?gggcagcccg?uccccgcgug 60

uggucgg 67

<210>10

<211>63

<212>RNA

＜213〉artificial

<220>

＜223〉synthetic RNA

<400>10

ggaauggacg?aguuacaggu?gcuccaucaa?caaaauguua?cauggaacug?uaacggccga 60

cau 63

<210>11

<211>54

<212>RNA

＜213〉artificial

<220>

＜223〉synthetic RNA

<400>11

ggacgaguua?caggugcucc?aucaacaaaa?uguuacaugg?aacuguaacg?gccg 54

<210>12

<211>41

<212>RNA

＜213〉artificial

<220>

＜223〉synthetic RNA

<400>12

ggacaggugc?uccaucaaca?aaauguuaca?uggaacuguc?c 41

<210>13

<211>34

<212>RNA

＜213〉artificial

<220>

＜223〉synthetic RNA

<400>13

ggacaggugc?ucugcgagcc?acgcggaacu?gucc 34

<210>14

<211>33

<212>RNA

＜213〉artificial

<220>

＜223〉synthetic RNA

<400>14

ggacaggugc?ucugcggaaa?cgcggaacug?ucc 33

<210>15

<211>30

<212>RNA

＜213〉artificial

<220>

＜223〉synthetic RNA

<400>15

ggaggugcuc?ugcgagccac?gcggaacucc 30

<210>16

<211>27

<212>RNA

＜213〉artificial

<220>

＜223〉synthetic RNA

<400>16

ggacaggugc?uccgaaagga?acugucc 27

<210>17

<211>23

<212>RNA

＜213〉artificial

<220>

＜223〉synthetic RNA

<400>17

ggaggugcuc?cgaaaggaac?ucc 23

<210>18

<211>21

<212>RNA

＜213〉artificial

<220>

＜223〉synthetic RNA

<400>18

ggaggugcuc?gaaagaacuc?c 21

<210>19

<211>73

<212>RNA

＜213〉artificial

<220>

＜223〉synthetic RNA

<400>19

ggguacgagu?cuggacuugc?aacaaggugc?uccaccuacc?uaguggaacu?ugcugaugag 60

gcucacaaca?ggc 73

<210>20

<211>79

<212>RNA

＜213〉artificial

<220>

＜223〉synthetic RNA

<400>20

ggguacgagu?cuggacuugc?aaggugcucc?guuagcauug?cggaacuugc?aauauauccu 60

auugaggcuc?acaacaggc 79

<210>21

<211>79

<212>RNA

＜213〉artificial

<220>

＜223〉synthetic RNA

<400>21

ggguacgagu?cuggacuugc?aacggauaca?ggugcuccga?ucuucggaac?uggcguuggc 60

ggugaggcuc?acaacaggc 79

<210>22

<211>79

<212>RNA

＜213〉artificial

<220>

＜223〉synthetic RNA

<400>22

ggguacgagu?cuggacuugc?aauacgaggu?gcuccaagga?aucgcccugg?aacucgacga 60

cgugaggcuc?acaacaggc 79

<210>23

<211>79

<212>RNA

＜213〉artificial

<220>

＜223〉synthetic RNA

<400>23

ggguacgagu?cuggacuugc?aaccuaaugc?ggugcuccuc?uggaaccauu?agcuguugca 60

aaugaggcuc?acaacaggc 79

<210>24

<211>90

<212>DNA

＜213〉artificial

<220>

＜223〉synthetic DNA

<220>

<221>misc_feature

<222>(19)..(58)

<223>n?is?a，c，g，or?t

<400>24

ctctcatgtc?ggccgttann?nnnnnnnnnn?nnnnnnnnnn?nnnnnnnnnnn?nnnnnnnncg 60

tccattgtgt?ccctatagtg?agtcgtatta 90

<210>25

<211>32

<212>DNA

＜213〉artificial

<220>

＜223〉synthetic DNA

<400>25

taatacgact?cactataggg?acacaatgga?cg 32

<210>26

<211>18

<212>DNA

＜213〉artificial

<220>

＜223〉synthetic DNA

<400>26

ctctcatgtc?ggccgtta 18

<210>27

<211>30

<212>DNA

＜213〉artificial

<220>

＜223〉synthetic DNA

<400>27

taatacgact?cactataggg?ccacagcgag 30

<210>28

<211>13

<212>DNA

＜213〉artificial

<220>

＜223〉synthetic DNA

<400>28

ccgaccacac?gcg 13

<210>29

<211>39

<212>DNA

＜213〉artificial

<220>

＜223〉synthetic DNA

<400>29

taatacgact?cactataggg?tacgagtctg?gacttgcaa 39

<210>30

<211>17

<212>DNA

＜213〉artificial

<220>

＜223〉primer

<400>30

gcctgttgtg?agcctca 17

<210>31

<211>50

<212>DNA

＜213〉artificial

<220>

＜223〉synthetic DNA

<220>

<221>misc_feature

<222>(30)..(30)

<223>n?is?a，c，g，or?t

<400>31

tgtcggccgt?tacagttccg?gtttcccggn?tgtaactcgt?ccattgtccc 50

<210>32

<211>36

<212>DNA

＜213〉artificial

<220>

＜223〉primer

<400>32

taatacgact?cactataggg?acaatggacg?agttac 36

<210>33

<211>18

<212>DNA

＜213〉artificial

<220>

＜223〉primer

<400>33

tgtcggccgt?tacagttc 18

Claims (34)

1. an aptamers is characterized in that, is incorporated into the Fc part of IgG.

2. aptamers as claimed in claim 1 is characterized in that, combines with Fc part specificity as the human IgG of the Fc of IgG part.

3. aptamers as claimed in claim 1 or 2 is characterized in that, the total nucleotide number that constitutes aptamers is below 40.

4. as each described aptamers in the claim 1 to 3, it is characterized in that, at least a Nucleotide that described aptamers contains is selected from hydrogen atom, fluorine atom, hydroxyl and reaches for containing in 2 ' of ribose-Nucleotide of at least 2 kinds of groups of O-Me base.

5. aptamers as claimed in claim 3 is characterized in that, described aptamers contains the nucleotide sequence of GGUG (C/A) shown in (U/T).

6. aptamers as claimed in claim 5 is characterized in that, described GGUG (C/A) (U/T) in the 3rd the Nucleotide of U for being replaced by fluorine atom in 2 ' hydroxyl of ribose.

7. aptamers as claimed in claim 6, it is characterized in that, described GGUG (C/A) each Nucleotide except that the 3rd U in (U/T) is identical or different respectively, for in 2 ' Nucleotide that contains hydroxyl of ribose, or in 2 ' hydroxyl of ribose by hydrogen atom, fluorine atom or-Nucleotide that the O-Me base replaces.

8. aptamers as claimed in claim 5 is characterized in that, described GGUG (C/A) is GGUGCU or GGUGAU (U/T).

9. aptamers as claimed in claim 5 is characterized in that, also contains the nucleotide sequence shown in the ANC, and wherein N is the Nucleotide that is selected from A, G, C, U and T.

10. aptamers as claimed in claim 9, it is characterized in that, each Nucleotide among the ANC is identical or different respectively, in 2 ' Nucleotide that contains hydroxyl of ribose, or in 2 ' hydroxyl of ribose by hydrogen atom, fluorine atom or-Nucleotide of the basic replacement of O-Me.

11. aptamers as claimed in claim 9 is characterized in that, any one that described aptamers is following (i) in (iii):

(i) contain GGA in GGUG (C/A) 5 ' side (U/T), and contain UCC in the 3 ' side of ANC;

(ii) contain GGN in GGUG (C/A) 5 ' side (U/T) _X1A, and contain UN in the 3 ' side of ANC _X2CC, wherein, N _X1, N _xBe respectively the Nucleotide that is selected from A, G, C, U and T; Or,

(iii) contain GGN in GGUG (C/A) 5 ' side (U/T) _X3N _X4A, and contain UN in the 3 ' side of ANC _X5N _X6CC, wherein, N _X3, N _X4, N _X5, N _X6Be respectively the Nucleotide that is selected from A, G, C, U and T.

12. aptamers as claimed in claim 11 is characterized in that, described GGA, GGN _X1A or GGN _X3N _X4The GG that contains among the A, and described UCC, UN _X2CC or UN _X5N _X6The CC that contains among the CC is respectively the Nucleotide that is replaced by hydrogen atom in 2 ' hydroxyl of ribose.

13. aptamers as claimed in claim 6 is characterized in that, has the potentiality secondary structure of following (I) to (III) expression:

In the formula, N ¹, N ², N ³, N ⁴, N ⁵Respectively identical or different, for being selected from the Nucleotide of A, G, C, U and T,

N ²And N ³Be mutual complementary Nucleotide,

N ⁴And N ⁵Be mutual complementary Nucleotide,

(i) each Nucleotide except that the 3rd U in (U/T) of GGUG (C/A), (ii) AN ¹Each Nucleotide among the C, (iii) N ²To N ⁵Each Nucleotide be respectively 2 ' Nucleotide that contains hydroxyl in ribose, or in 2 ' hydroxyl of ribose by hydrogen atom, fluorine atom or-Nucleotide that the O-Me base replaces.

14. aptamers as claimed in claim 11 is characterized in that, all Nucleotide in the ring structure are replaced by hydrogen atom in 2 ' hydroxyl of ribose.

15. aptamers as claimed in claim 13 is characterized in that, the potentiality secondary structure shown in each that the aptamers with potentiality secondary structure shown in each in (I) to (III) has is following (I ') in (III '):

In the formula, N ¹, N ², N ³, N ⁴, N ⁵Meaning identical with claim 13 respectively.

16. aptamers as claimed in claim 3, it is characterized in that, contain (U/T) nucleotide sequence shown in the C of AGGUG (C/A), AGGUG (C/A) Nucleotide that (U/T) the 4th U is replaced by fluorine atom for 2 ' hydroxyl in ribose among the C, AGGUG (C/A) (U/T) each Nucleotide except that the 4th U among the C is identical or different respectively, for in 2 ' Nucleotide that contains hydroxyl of ribose, or in 2 ' hydroxyl of ribose by hydrogen atom, fluorine atom or-Nucleotide that the O-Me base replaces.

17. aptamers as claimed in claim 16, it is characterized in that, also contain the nucleotide sequence shown in the GANCU, each Nucleotide among the GANCU is identical or different respectively, for in 2 ' Nucleotide that contains hydroxyl of ribose, or in 2 ' hydroxyl of ribose by hydrogen atom, fluorine atom or-Nucleotide that the O-Me base replaces, wherein, N is the Nucleotide that is selected from A, G, C, U and T.

18. aptamers as claimed in claim 6 is characterized in that, has the potentiality secondary structure of following (Ia) to (IIIa) expression:

In the formula, N ¹, N ², N ³, N ⁴, N ⁵, N ⁶, N ⁷Respectively identical or different, for being selected from the Nucleotide of A, G, C, U and T,

N ²And N ³Be mutual complementary Nucleotide,

N ⁴And N ⁵Be mutual complementary Nucleotide,

N ⁶And N ⁷Be mutual complementary Nucleotide,

(i) the Nucleotide except that 3rd U of GGUG (C/A) in (U/T), (ii) AN ¹Each Nucleotide of C, (iii) N ²To N ⁷Each Nucleotide be respectively 2 ' Nucleotide that contains hydroxyl in ribose, or in 2 ' hydroxyl of ribose by hydrogen atom, fluorine atom or-Nucleotide that the O-Me base replaces.

19. aptamers as claimed in claim 18 is characterized in that, the potentiality secondary structure shown in each that the aptamers with potentiality secondary structure shown in each in (Ia) to (IIIa) has is following (Ia ') in (IIIa '):

In the formula, N ¹, N ², N ³, N ⁴, N ⁵Meaning and claim 18 identical.

20. aptamers as claimed in claim 19 is characterized in that, described N ⁴, N ⁶Be respectively the Nucleotide that is replaced by hydrogen atom in 2 ' hydroxyl, described N ⁵, N ⁷Be respectively the Nucleotide that contains hydroxyl in 2 '.

21. the aptamers as claim 19 is characterized in that, the potentiality secondary structure shown in each that the aptamers with potentiality secondary structure shown in each in (Ia ') to (IIIa ') has is following (Ia " ') in (IIIa " '):

22. aptamers as claimed in claim 3 is characterized in that, is in following (a) to (c) any one:

(a) aptamers of forming by the nucleotide sequence shown in each in the sequence numbering 1 to 23, wherein, uridylic also can be thymus pyrimidine;

(b) by 1 in the nucleotide sequence shown in the nucleotide sequence shown in each in the sequence numbering 1 to 23 several Nucleotide are substituted, lack or additional after the aptamers formed of nucleotide sequence, wherein, uridylic also can be thymus pyrimidine;

(c) be selected from the concatenator of the concatenator of described (a), described (b) and the concatenator of described (a) and concatenator (b).

23. a species complex is characterized in that, contains each described aptamers and and described aptamers bonded functional substance in the claim 1 to 22.

24. complex body as claimed in claim 23 is characterized in that, described functional substance is affinity substance, mark material, enzyme, medicine, toxin or useful for drug delivery medium.

25. a solid phase carrier is characterized in that, is fixed with each described aptamers or claim 23 or 24 described complex bodys in the claim 1 to 22.

26. solid phase carrier as claimed in claim 25 is characterized in that, described solid phase carrier is substrate, resin, plate, strainer, tube, post or porous matter material.

27. a medical device is characterized in that, contains claim 25 or 26 described solid phase carriers.

28. equipment as claimed in claim 27 is characterized in that, described medical device is a blood purification equipment.

29. a diagnosis with or check and use reagent, it is characterized in that, contain each described aptamers in the claim 1 to 22, claim 23 or 24 described complex bodys or claim 25 or 26 described solid phase carriers.

30. a medicine is characterized in that, contains each described aptamers or claim 23 or 24 described complex bodys in the claim 1 to 22.

31. antibody purification or concentration method is characterized in that, comprise making IgG antibody be adsorbed to claim 25 or 26 described solid phase carriers, make the step of adsorbed IgG antibody elution again with elutriant.

32. method as claimed in claim 31 is characterized in that, elutriant is a neutral solution.

33. the manufacture method of an antibody purification is characterized in that, comprises preparation IgG antibody, the IgG antibody of preparation is passed through the step of claim 25 or 26 described solid phase carrier purifying again.

34. the detection of an IgG and/or quantivative approach, it is characterized in that, comprise and use each described aptamers, claim 23 or 24 described complex bodys or claim 25 or 26 described solid phase carriers in the claim 1 to 22, measure the step that has or not the amount of IgG and/or IgG in the sample.

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