JPH0822875B2 - Method for producing purified RNA-binding protein - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention is directed to the prediction, diagnosis, and diagnosis of autoimmune diseases and autoimmune phenomena.
The present invention relates to a method for producing an RNA-binding protein effectively used as an antigen reagent in an autoantibody test performed as a part of follow-up observation.

[Prior art] As a protein that specifically binds to low molecular weight RNA, it binds to low molecular weight RNA in the cell nucleus, a group of proteins that contribute to RNA splicing, and low molecular weight RNA in the cytoplasm, A group of proteins that control regulation are known.

In a systemic autoimmune disease, the RNA is
Antibodies against the binding protein are produced, which causes lesions associated with various autoimmune phenomena such as inflammation, ulcer, skin rash, and dryness. Furthermore, RNA-binding proteins corresponding to antibodies appearing in the sera of patients with autoimmune diseases are diverse among the disease groups, for example, in systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD), nuclear Within R
In spite of NA-binding protein, in dryness syndrome (SjS), antibodies are frequently produced against RNA-binding protein in the cytoplasm.

Therefore, the essential clinical significance of detecting various autoantibodies appearing in the sera of patients with these autoimmune diseases lies in the diagnosis, follow-up observation and prediction of autoimmune phenomena of the disease.

Conventionally, as an antigen used to detect the antibody group, an extract of human cultured cells or mammalian cells or the cells themselves have been used as a crude antigen. This is because the antigenicity of the protein is common across species, and antibodies derived from any mammal can be used as an antigen for detecting an autoantibody against the self component found in the serum of an autoimmune disease patient, that is, an autoantibody. It is. Also,
Antibodies against the RNA-binding protein in the cell nucleus include the anti-ribonucleoprotein antibody group and the anti-Smith antibody group, and as antibodies against the RNA binding protein in the cytoplasm, the anti-SSA / Ro antibody group and the
The SSB / La antibody group is widely known, and the detection of these antibody groups can be detected by, for example, the two-way immunodiffusion method in comparison with the reaction mode between the conventionally controlled reference serum and the above-mentioned crude antigen solution. It was detected only by the fusion phenomenon of sedimentation lines or by reading the stained image of the cultured cells by the fluorescent antibody method, and it was not necessary that the antigen was purified.

In recent years, the properties and functions of antigens against the antibody group have been elucidated by molecular biology or protein chemistry, and it has become necessary to establish a sensitive and quantitative clinical test method using purified antigen as a detection reagent. Is coming.

Many scientific literatures describe methods for separating and purifying RNA-binding proteins (Clin Exp. Immunol., 54, 731-738,
(1983), J. Biol. Chem., 258, 2604-2613 (1983), etc.). In most methods, purification is carried out by biochemical separation means such as ion exchange chromatography and molecular sieve chromatography based on the physicochemical properties of the protein to be purified. Further, depending on the method, purification is carried out by adsorption chromatography using an adsorbent such as a dye as a carrier (Scand. J. Immunol., 15, 1-7, (1982)).

[Problems to be Solved by the Invention]

As described above, the RNA-binding protein to be isolated is separated based on the difference in the molecular weight of the protein, the difference in the charge state, or the difference in the adsorptivity to a substance. Is inevitable, and a dramatic increase in purification rate cannot be expected. This is because proteins exhibiting the same physicochemical behavior exist in the biomaterial. Therefore, in general, when a specific protein is separated by the above-described method, it is necessary to increase the purification magnification by combining a plurality of separation means, and the number of days of purification is long.

On the other hand, as a method for greatly increasing the purification magnification in a single-step operation, there is a method utilizing biological affinity, for example, a substance having biological affinity for an isolation target is immobilized as a ligand. And affinity chromatography using a modified carrier.

The inventors of the present invention have conducted intensive studies on such a method of greatly increasing the purification ratio in one step, and have completed the present invention.

[Means for solving the problem]

That is, the present invention extracts RNA-binding proteins from animal tissues, cultured cells, or processed products thereof, and then uses the uridylic acid polymer as a ligand in a solution environment containing a salt or chaotropic ion in the resulting extract. A purified RNA comprising a step of contacting with an affinity gel containing a carrier, and then eluting the RNA-binding protein adsorbed on the affinity gel by increasing the salt concentration or chaotropic ion concentration in the solution. The present invention relates to a method for producing a binding protein.

In the process of extracting the dilute salt-soluble fraction containing RNA-binding protein from animal tissues, cultured cells or their processed products, to protect against proteolytic enzyme invasion by all possible proteolytic enzymes that are simultaneously solubilized The extraction is preferably performed with an extraction buffer containing an inhibitor. Furthermore, regarding this extraction operation, in order to obtain as much RNA-binding protein as possible, it is preferable to perform extraction from the object to be extracted twice and combine the two extracts. The salt concentration of the extraction buffer used in the present extraction operation is not particularly limited as long as the protein in the globulin fraction is easily dissolved and the histone in the nucleus is not dissolved, but it is preferably performed at a physiological salt concentration. . Further, since the detection of the RNA-binding protein in the extract is easy, it is preferable to carry out the detection by the dual immunodiffusion method using an antiserum which specifically recognizes the RNA-binding protein of interest.

Then, the extract (unpurified fraction) extracted by the above-mentioned method is sufficiently dialyzed against a weak alkaline buffer solution containing a predetermined concentration of salt or chaotropic ion. The chaotropic ion is an anion having a large ionic radius, and the ion increases the aqueous solution of the hydrophobic molecule and acts to weaken the hydrophobic bond between the carrier and the separation target. Chaotropic The ion _{^{SCN -, I -, ClO 4}} -, NO 3 -, B
r ⁻ , Cl ⁻ , CH ₃ CO ₂ ⁻ , F ⁻ , SO ₄ ^{2− and the} like are mentioned, and the order of magnitude of their action (chaotropy) is the same as the above order.

Specific examples of salts or compounds containing chaotropic ions include highly soluble monovalent or divalent metal chlorides such as sodium chloride, potassium chloride and magnesium chloride, and metal thiocyanates such as sodium thiocyanate and potassium thiocyanate. Compounds, etc.,
Of these, magnesium chloride is particularly preferable. The concentration varies depending on the type of salt, for example magnesium chloride is about
About 0.1M is preferable. Then, the RNA-binding protein in the unpurified fraction is bound to the carrier by bringing the uridylic acid polymer equilibrated with the buffer solution in advance into the carrier as a ligand. As the carrier, polyuridylic acid agarose (commercially available), affinity gel obtained by coupling polyuridylic acid as a ligand to a sepharose gel, and the like are used. The amount of carrier to contact is such that the total protein concentration is 10 mg / ml.
It is preferable that the amount of the extract is 5 ml or more per 1 ml. After that, the carrier is washed with the weak alkaline buffer solution used for equilibrating the carrier having the uridylic acid polymer as a ligand. Subsequently, the RNA-binding protein is eluted by increasing the concentration of salt or chaotropic ion in the solution in which the carrier is suspended more than when the extract is in contact with the carrier. The method of eluting and collecting is not particularly limited, and for example, a chromatography method, a batch method and the like can be used. Further, the method of increasing the salt or chaotropic ion concentration of the solution in which the carrier having the uridylic acid polymer bound with the RNA-binding protein as the ligand is suspended is not particularly limited, and the gradient method or the stepwise method can be employed.

By the steps described above, the ratio of the RNA-binding protein to be separated and purified to the total amount of protein can be dramatically increased. That is, most of other contaminant proteins can be removed.

Here, specific RNA-binding proteins that can dramatically increase the purification rate by this method include SSB / La protein, SSA / Ro protein, which constitutes the YRNA complex protein, and respective molecular weights which constitute the U _sn RNA-binding protein. Protein.

The RNA-binding protein obtained by the above steps can be used as it is as an antigen for use in some immunoassays such as the Octaloni method. Furthermore, a highly purified RNA-binding protein preparation can be obtained by combining at least one step of operations such as molecular sieve chromatography, ion exchange chromatography, adsorption chromatography, and certain group-specific chromatography. It can be used as an antigen for use in any diagnostic agent for measuring autoantibodies whose immunochemical reaction is the measurement principle.

Next, a method for measuring an anti-RNA binding protein antibody for detecting or quantifying an antibody present in a test subject using the obtained RNA binding protein as an antigen will be described.

The measuring method of the present invention may be any measuring method as long as the above-mentioned antigen is used. For example, enzyme immunoassay, radioimmunoassay, immunoturbidimetric method, immunonephelometric method, latex agglutination method, hemagglutination method, fluorescent immunoassay method, immunochemiluminescence method, dye immunoassay method and the like can be performed. .
As a preferred example, an immunoassay using a labeled secondary antibody will be described.

A solid surface, eg polystyrene pores, is covered with the polypeptide chain. Usually, this coating operation has a buffering effect in the alkaline range. For example, the polypeptide chain is dissolved in sodium carbonate buffer and used as a 0.01 to 100 μg / ml solution, which is performed overnight at low temperature. Thereafter, the polypeptide chains not physically adsorbed on the solid surface are removed by suction together with a buffer solution, and then the hydrophilic globular protein having no immunochemical cross-reactivity with the polypeptide chains, for example, 0.01 to 1% of milk casein. Blocking with (weight / volume) solution at room temperature for about 1 hour. This is achieved by covering the solid surface not coated with the polypeptide chain or the easily adsorbable site on the molecular surface of the polypeptide chain physically adsorbed on the solid surface, so that the test object solution or the labeled secondary antibody to be added subsequently This is for preventing the protein component in the solution from being non-specifically adsorbed.

Thereafter, in order to remove the polypeptide chains or protein components that have not been used for coating or blocking from the solid surface, the substrate is sufficiently washed with a neutral washing solution containing a nonionic surfactant. As described above, the polypeptide chain serving as the antigen is immobilized on the carrier, and then the antibody is detected or quantified.

A subject to be appropriately diluted with a physiological buffer containing a nonionic surfactant and a hydrophilic globular protein having no immunochemical crossability, for example, a patient surface and a solid surface coated with the polypeptide chain and an antigen Contact for a sufficient time to complete the antibody binding reaction.

Thereafter, the solid surface is sufficiently washed with a neutral washing solution containing a nonionic surfactant to complete the antigen-antibody binding reaction of the solid surface with a physiological solution containing an excessive amount of a labeled secondary antibody. Contact for a sufficient period of time. Here, the labeling substance is not particularly limited and may be an enzyme, a radioisotope, a fluorescent substance or the like, but an enzyme label is particularly preferable.

Then, the solid surface is sufficiently washed with a neutral washing solution containing a nonionic surfactant to detect the presence or amount of the labeled secondary antibody. In the case of enzyme labeling, the solid surface is contacted with a substrate solution specific for the enzyme for a time sufficient to detect the product of the enzymatic reaction. In this case, the amount of product produced by the enzymatic reaction is proportionally dependent on the amount of antibody to the antigenic determinant on the polypeptide chain contained in the test subject, and thus indirectly is the amount of the product in the test subject. Antibodies can be quantified.

〔Example〕

Hereinafter, the present invention will be described in detail with reference to Examples regarding a method for separating and purifying SSB / La protein among RNA binding proteins.

Example Buffer A: 11 in sodium chloride 8g, potassium chloride 0.2g,
Phosphate buffer solution containing 2.7 g of disodium phosphate dodecahydrate and 0.2 g of 1 potassium phosphate.

Buffer B: as proteolytic enzyme inhibitors, ethylene glycol -OO'- bis (2-aminomethyl) -NNN'N'-4 acetate (EDTA) 10 ^-3 M, fluoride phenylmethylsulfonyl (PMSF) 10 ^- Buffer A further containing ³ M, leupeptin 0.05% (weight / volume), antipain 0.05% (weight / volume), chymostatin 0.05% (weight / volume), peptantine A 0.05% (weight / volume).

Buffer C: Tris buffer 10 mM x HCl pH 8.0.

Buffer D: Buffer C containing 0.1 M magnesium chloride.

Eluent E1: Buffer C containing 0.2M magnesium chloride.

Eluent E2: Buffer C containing 0.3M magnesium chloride.

Eluent E3: Buffer C containing 0.4M magnesium chloride.

Eluent E4: Buffer C containing 0.5 M magnesium chloride.

 The following operations were all performed at 4 ° C.

1) Acquisition of tissue extract containing RNA-binding protein 300 ml of buffer B was added to 30 g of rabbit thymus acetone powder (Pel Freeze) and the mixture was dissolved overnight. Then, the suspension was centrifuged at 10,000 × g for 30 minutes, and the supernatant was used as the extract A. In order to perform the second extraction from this precipitate, 50 ml of buffer solution B was added to the precipitate and stirred for 4 hours. Then, the suspension is added to 10,000 × g at 30
After centrifuging, the supernatant was used as the extract B and the extract A was combined with it to obtain the extract C.

2) Fractionation by carrier having uridylic acid polymer as a ligand 1) The extract C obtained in 1) is dialyzed against the buffer D for 2 days and night, and then 0.1 volume of the extract and 0.9 volume of the buffer D are added. Then, 1 volume of a carrier (polyuridylic acid agarose, manufactured by Sigma) in which the uridylic acid polymer was previously equilibrated with the buffer solution D as a ligand was added, and the mixture was gently mixed by inversion for 15 minutes or more.
Subsequently, the supernatant was removed by centrifugation, the buffer D1 volume was added, and the carrier was washed by centrifugation. Furthermore, subsequently, an E11 volume of eluent was added, and the RNA-binding protein which was spun down and eluted in the supernatant was recovered. Similarly, eluents E2, E3, and E4 were used in this order, and the same operation was performed to recover the RNA-binding protein in each eluent.

3) Measurement of SSB / La protein in each eluent by enzyme immunoassay: 200 μl of diluted SSB / La protein solution (each eluent) was added to the well of 96-well ELISA microplate. Four
Adsorption was carried out at 0 ° C overnight. A 0.1 M sodium carbonate buffer, pH 8.6 was used for dilution. The next day, 0.2% milk solution 40
The plate was blocked with 0 μl at room temperature for 1 hour to coat unreacted sites on the plate and easily adsorbed sites on the surface of the adsorbed protein.
Subsequently, 200 μl of the primary antibody solution (anti-SSB / La serum diluted 1 / 1,000 with Dulbecco's phosphate buffered saline containing 0.1% milk and 0.1% Tween 20) was added,
The reaction was carried out at room temperature for 2 hours to bind the anti-SSB / La antibody to the coated antigen. After the primary antibody reaction, the plate was washed (using Dulbecco's phosphate buffered saline containing 0.1% Tween 20 as a wash buffer for 5 minutes for 3 minutes), secondary antibody solution (phosphatase-labeled anti-human IgG + A + M antibody) Serum (KPL) 0.1% milk and 0.1% Tween 20
20 μl of Dulbecco's Phosphate Buffered Saline Solution containing 1) was added and further reacted for 2 hours at room temperature to bind the secondary antibody to the primary antibody (anti-SSB / La antibody) on the plate. It was Following the secondary antibody reaction, the plate was washed in the same manner as above, 200 μl of the substrate solution (1 mg / ml p-nitrophenyl phosphate, 1 M diethanolamine buffer) was added, and the labeled secondary antibody captured by the primary antibody was added. Was determined by measuring the absorbance at a wavelength of 405 nm with a spectrophotometer. Since this enzyme activity is in a proportional relationship with the amount of SSB / La antigen on the plate, the amount of antigen in the sample can be measured by the magnitude of the enzyme activity.

The obtained results are shown in FIG. The amount of protein in each eluent was determined by the Lowry method, but the activity of SSB / La antigen in each eluent (MgCl ₂ 0.2M, 0.3M, and 0.4M) per amount of protein increased markedly. Was there.

4) Analysis by SDS polyacrylamide gel electrophoresis method Each eluent was developed as a migration sample in 12.5% acrylamide gel (degree of crosslinking 0.8) according to the method of Laemmli et al. The electrophoresis conditions were 40 mA at the start of electrophoresis, 4 V / cm during concentrated electrophoresis, and 8 V / cm during separation electrophoresis. In addition, the sample to be electrophoresed was a buffer for samples (312.4 mM Tris-HCl, pH 6.8,0.
25% by volume of 1% bromphenol blue, 10% sodium dodecyl sulfate, 20% glycerin) was added, and the mixture was treated at room temperature for 30 minutes. After the electrophoresis, the gel was stained with 0.05% Coomassie Brilliant Blue overnight, and the next day, it was destained with 0.7% acetic acid. As molecular weight markers, 92.5K Dalton, 66.2K Dalton, 4
5.0K Dalton, 31.0K Dalton, 21.5K Dalton and 14.4K
A molecular weight marker manufactured by BIO-RAD having a Dalton marker was used.

As a result, the magnesium chloride concentration was 0.2M, 0.3M and 0.4M.
In the case of M, the ratio of the density of the band having a molecular weight of about 50 K daltons to the total stained protein band was increased.

〔The invention's effect〕

Conventionally, a cell extract has been used as it is as an antigen for measuring an antibody against an RNA-binding protein.

However, as a cell constituent that can become an autoantigen in recent years,
The molecular properties of RNA-binding proteins are being clarified,
Relationship between these functions and the function and etiology of antibodies to them that appear in the serum of patients with autoimmune diseases, or the relationship between autoimmune phenomena observed during the course of disease and changes in serum antibody titers or disease days Various theories have been discussed regarding the relationship with the above, and it has become important to understand the contribution of anti-RNA binding protein antibody as a causative agent and the relationship with the clinical picture.

Therefore, as in the present invention, RNA can be specifically and efficiently
The establishment of a method for separating a binding protein from other proteins or components is effective as a method for preparing a reagent component used in a clinical test performed for diagnosis or follow-up of an autoimmune disease or prediction of an autoimmune phenomenon. It is a thing. Then, the method for measuring the anti-RNA binding protein antibody of the present invention using the obtained RNA binding protein is clinically very effective.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the RNA binding protein (SSB in the example of the present invention.
/ La protein) is a diagram showing the elution from polyuridylate agarose, the vertical axis, the absorbance at a wavelength of 405 nm when the amount of SSB / La antigen in each supernatant was quantified by enzyme immunoassay, The axis shows the concentration of magnesium chloride used.

Claims (4)

[Claims] 1. An affinity gel containing a carrier having a polyuridylic acid polymer as a ligand, in which an RNA-binding protein is extracted from an animal tissue, a cultured cell or a processed product thereof, and the resulting extract is then placed in a solution environment containing a salt. A method for producing a purified RNA-binding protein, which comprises a step of elution of the RNA-binding protein adsorbed on the affinity gel by increasing the salt concentration in the solution. 2. An RNA binding protein is extracted from animal tissues, cultured cells or processed products thereof, and the resulting extract is then subjected to a solution environment containing chaotropic ions, and an affinity containing a carrier having a polyuridylic acid polymer as a ligand. A method for producing a purified RNA-binding protein, which comprises a step of contacting with a gel and then eluting the RNA-binding protein adsorbed on the affinity gel by increasing a chaotropic ion concentration in a solution. 3. The method for producing a purified RNA-binding protein according to claim 1 or 2, wherein the RNA-binding protein to be purified is SSB / La protein. 4. The method for producing a purified RNA-binding protein according to claim 1 or 2, wherein the RNA-binding protein to be purified is an SSA / Ro protein.