JPH085915B2 - 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.

[Conventional technology]

As a protein that specifically binds to low-molecular-weight RNA, a group of proteins that bind to low-molecular-weight RNA in the cell nucleus and contribute to RNA splicing and to low-molecular-weight RNA in the cytoplasm that regulates RNA metabolism The protein is 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, since the antigenicity of the protein is common across species, any mammal-derived antigen can be used as an antigen for detecting an antibody against an autologous component found in the serum of an autoimmune disease patient, that is, an autoantibody. 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 sensitive and quantitative clinical test methods using purified antigens as detection reagents. Is coming.

Methods for separating and purifying RNA-binding proteins are described in many scientific literatures (Clin. Exp. Immunol., 54, 731-738,
(1983), J. Biol. Chem., 258, 2604-2613, (1983), etc.), the protein solution extracted from animal tissue or cultured cells is salted out and then subjected to each chromatography. Separation methods are common.

[Problems to be Solved by the Invention]

The salting-out method is a fractionation method that utilizes the phenomenon that the solubility of protein decreases with increasing salt concentration.However, when salting-out method is used for the crude fractionation of RNA-binding protein, It is not possible to effectively precipitate the protein. this is
It is considered that this is due to the large variety of RNA-binding proteins, and for the purpose of precipitating as many RNA-binding proteins as possible, the range of salt concentrations to be selected becomes extremely wide and the separation effect becomes markedly poor. Furthermore, in the salting-out method, the target RNA
It is unavoidable that many proteins other than the binding proteins are precipitated in a predetermined salt concentration range and these proteins are contaminated to a considerable extent. Therefore, the present inventors have focused their attention on zinc-binding finger sequences as an amino acid sequence characteristic of a protein capable of binding to RNA, and as a result of extensive studies on an efficient method for fractionating an RNA-binding protein, the present invention has been completed. .

[Means for Solving the Problems] The present invention is to extract an RNA-binding protein from animal tissues, cultured cells or processed products thereof, and then extract the resulting product with Z
Separation from other soluble proteins by adding n ₊₊ , Ni ₊₊ , Cu ₊₊ or Co ₊₊ and aggregating and precipitating RNA binding protein, then adding a chelating agent to redissolve RNA binding protein A purified RNA characterized by including the step of
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 prevent proteolytic enzymes from being attacked 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 this extraction operation is not particularly limited as long as the protein of the globulin fraction is easily dissolved and the histones in the nucleus are not dissolved, but it is preferable to perform it 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.

Next, the divalent metal ions are added to cause aggregation and precipitation. In order to facilitate this, it is preferable that the obtained extract is sufficiently dialyzed against a weak alkaline buffer having a low ionic strength.

As a method of adding the divalent metal ion, it is preferable to add a suspension of the divalent metal ion in the same volume as that of the above extract and leave it on ice for 30 minutes or longer. The solution of divalent metal ions added at this time is RN
Aggregation and precipitation of the A-binding protein occurs, but in order to facilitate aggregation and precipitation, it is preferable to use a suspension having a concentration exceeding the saturation concentration and use suspended particles as a carrier for aggregation and precipitation. The RNA-binding protein that has aggregated and precipitated is collected by a separation means such as centrifugation or filtration. The recovered precipitate is preferably washed with a saturated divalent metal ion solution.

Here, as the divalent metal ion, the ionic radius is 0.
Zn ₊₊ , Ni ₊₊ , Cu ₊₊ and Co _{++ of} 71 to 0.75Å are highly effective and preferred, and Zn ₊₊ is most preferred.

Specifically, as the salt containing a divalent metal ion to be added, chlorides such as zinc chloride, nickel chloride, cupric chloride, and cobalt chloride are preferable, but the salt is not limited thereto.

Then, subsequently, a chelating agent is added to the separated precipitate to redissolve the precipitate. At this time, the chelating agent is preferably dissolved in a buffer solution and used as a chelating agent solution. It is preferable that the solution is added again in the same volume as before precipitation collection and redissolved. In this case, by increasing the concentration of the chelating agent solution to be added stepwise, the redissolved RNA
The type of binding protein can be limited. For example, an SSB / La protein can be efficiently redissolved with an ethylenediamine tetraacetate (EDTA) solution having a final concentration of 30 mM or less, and an SSA / Ro protein can be efficiently redissolved with an ethylenediamine tetraacetate (EDTA) solution having a final concentration of 30 mM or more.

Here, as the chelating agent, ethylenediamine tetraacetate (EDTA), ethylene glycol-OO'-bis (2aminomethyl) -NNN'N'-4 acetate (EGTA), o-phenanthroline, 8-oxyquinoline Can be used, but ED
Most preferred is the use of TA.

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 proteins and SSA / Ro proteins that form YRNA complex proteins.

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. Can be used as an antigen used in any diagnostic agent for measuring autoantibodies whose immunochemical reaction is a 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.

After that, in order to remove the polypeptide chain or protein component not used for coating or blocking from the solid surface, it is thoroughly 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 fixed surface is thoroughly washed with a neutral washing solution containing a nonionic surfactant to complete the antigen-antibody binding reaction on the solid surface with a physiological solution containing an excess amount of the labeled secondary antibody. Allow sufficient time for contact. 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 of a method for separating and purifying SSB / La protein.

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.

Suspension D: Buffer C in which 0.2 M zinc chloride was suspended.

Chelating solution E1: Ethylenediamine tetraacetate (EDTA) 10 mM
Buffer C containing.

Chelating solution E2: Ethylenediamine tetraacetate (EDTA) 20 mM
Buffer C containing.

Chelating solution E3: Ethylenediamine tetraacetate (EDTA) 30 mM
Buffer C containing.

Chelating solution E4: Ethylenediamine tetraacetate (EDTA) 40 mM
Buffer C containing.

Chelating solution E5: Ethylenediamine tetraacetate (EDTA) 50 mM
Buffer C containing.

Chelating solution E6: Ethylenediamine tetraacetate (EDTA) 100 mM
Buffer C containing.

 The following operations were all performed at 4 ° C.

1) Acquisition of Tissue Extract Containing RNA Binding Protein Used 300 ml of buffer B was added to 30 g of rabbit thymus acetone powder (manufactured by 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 carry out 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) Recovery of Precipitate and Precipitation of RNA-Binding Protein with Zinc Chloride Suspension Extract C was dialyzed against buffer C for a sufficient time, and then Suspension D in the same volume was added and left for 30 minutes or longer. afterwards,
The aggregated and precipitated protein was centrifuged at 10,000 × g for 20 minutes.

3) Re-dissolution of RNA-binding protein with a chelating agent solution 2) From the precipitate obtained in 2), chelating solution E1, E2, E3, E4, E5, E
150 ml of 6 was sequentially added, and the amount of redissolved protein was measured at each stage. The obtained protein was used as an RNA-binding protein fraction preparation.

4) Measurement of SSB / La in each eluate by protein enzyme immunoassay 200 μl of 1/1000 diluted SSB / La protein solution (each fraction preparation) was put into the well of 96-well ELISA microplate. Adsorption was carried out at 4 ° C. overnight. A 0.1 M sodium carbonate buffer, pH 8.6 was used for dilution. The next day, it was blocked with 400 μl of 0.2% milk solution at room temperature for 1 hour to coat unreacted sites on the plate and easily adsorbed sites on the surface of the adsorbed protein. Next, the primary antibody solution (antibody SSB / La serum, 0.
20 μl of Dulbecco's phosphate buffered saline containing 1% milk and 0.1% Tween 20) was added and reacted 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 a Dulbecco's phosphate buffered saline containing 0.1% Tween 20 as a washing buffer for 5 minutes for 3 minutes),
Secondary antibody solution (phosphatase-labeled anti-human IgG + A +
M antibody serum (manufactured by KPL) in Dulbecco's phosphate buffered saline containing 0.1% milk and 0.1% Tween 20 at 1.00
Add 20 μl (diluted to 1/0) and further incubate for 2 hours at room temperature to use the secondary antibody as the primary antibody (anti-SSB / La) on the plate.
Antibody). Following the secondary antibody reaction, the plate was washed in the same manner as above and the substrate solution (1 mg / ml p-nitrophenyl phosphate, 1M diethanolamine buffer) 200
μl was added, and the enzyme activity of the labeled secondary antibody captured by the primary antibody was determined by measuring the absorbance at a wavelength of 405 nm with a spectrophotometer. This enzyme activity is
Since there is a proportional relationship with the amount of SSB / La antigen, 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 fraction preparation was determined by the Lowry method. When the amount of EDTA was 20 mM or 30 mM, the activity of SSB / La antigen per amount of protein was remarkably increased.

5) Analysis by SDS polyacrylamide gel electrophoresis method According to the method of Laemmli et al., Each fraction preparation was developed as a migration sample in 12.5% acrylamide gel (crosslinking degree 0.8). 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 is a sample buffer solution (312.5 mM Tris-HCl, pH 6.8, which does not contain a reducing agent in advance,
25% by volume of 0.1% bromphenol blue, 10% sodium dodecyl sulfate, 20% glycerin) was added, and the mixture was treated at room temperature for 30 minutes. The gel after electrophoresis was stained with 0.05% CBB overnight, and then decolorized with 0.7% acetic acid the next day.

As a molecular weight marker, 92.5K Dalton, 66.2K
BIO-RAD molecular weight markers with markers of Dalton, 45.0K Dalton, 31.0K Dalton, 21K Dalton and 14.4K Dalton were used.

As a result, in the cases of EDTA concentrations of 20 mM and 30 mM, 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, which appear in the sera of patients suffering from autoimmune diseases, or the relationship or disease between the autoimmune phenomenon observed during the course of the diseases and the fluctuation of serum antibody titers. Various theories have been discussed regarding the relationship with Japan, 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 fractionating a binding protein from other proteins and components was established as a reagent component used in clinical tests performed for the diagnosis and follow-up of autoimmune diseases, or for the prediction of autoimmune phenomena. It will provide purification to prepare highly active antigens. And the resulting RNA
The method for measuring an anti-RNA binding protein antibody of the present invention using a binding protein is clinically very effective.

[Brief description of drawings]

FIG. 1 shows the RNA binding protein (SSB in the example of the present invention.
/ La protein) is a diagram showing the amount of redissolved by a chelating agent solution from zinc precipitation, the vertical axis at the wavelength of 405 nm when the amount of SSB / La antigen in the lysis supernatant was quantified by enzyme immunoassay. The absorbance is shown, and the horizontal axis shows the EDTA concentration of the chelating agent solution used.

Claims (3)

[Claims] 1. An RNA-binding protein is extracted from animal tissues, cultured cells or processed products thereof, and then Z is added to the resulting extract.
Separation from other soluble proteins by adding n ₊₊ , Ni ₊₊ , Cu ₊₊ or Co ₊₊ and aggregating and precipitating RNA binding protein, then adding a chelating agent to redissolve RNA binding protein A purified RNA characterized by including the step of
Method for producing binding protein. 2. The method for producing a purified RNA-binding protein according to claim 1, wherein the RNA-binding protein to be purified is SSB / La protein. 3. The method for producing a purified RNA-binding protein according to claim 1, wherein the RNA-binding protein to be purified is SSA / Ro protein.