JP2012139106A - Method for producing soluble sr-a protein, and evaluation method using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an evaluation system for a ligand such as denatured LDL, highly useful in screening a soluble SR-A protein forming a polymeric conformation and an agonist or antagonist to the protein.SOLUTION: This invention provides an SR-A protein having a mutation or lacking in a base sequence from the N-terminus to the transmembrane domain, inserted into the downstream of a secretion signal, and retaining the activity as a soluble protein. This invention also provides the evaluation system for direct interaction among proteins such as binding of ligand such as a denatured LDL to an SR-A or screening of an agonist or antagonist of the SR-A by using the prepared protein having the polymeric conformation.

Description

  The present invention relates to a method for producing and evaluating a class A scavenger receptor (solubilized SR-A protein) which is a solubilized recombinant membrane protein having a multimeric conformation. The present invention also relates to serum or antibodies against the solubilized SR-A protein.

  Membrane proteins, in particular membrane receptors, play an important role in activating the uptake of various substances into cells and the cascade of information transmission by binding to ligands on the cell surface. Since agonists (agonists) and antagonists (antagonists) of membrane receptors are drug candidates, it is very important in drug discovery research to develop efficient production techniques for membrane receptors and evaluation methods using them. is important.

  In addition, the market for antibody drugs for membrane receptors is growing year by year, but the types of membrane receptors that are targets of antibody drugs that have been put to practical use are limited. The development of anti-membrane receptor antibodies requires recombinant membrane receptors for both the immunogen membrane receptor and the evaluation of antibodies produced by the immune response. For example, efficient production techniques for 7-transmembrane membrane receptors such as G protein-coupled receptors (GPCRs) have not been developed, and development of pharmaceuticals including antibodies targeting them has been delayed. ing.

  Conventionally, mammalian membrane receptors, which are targets for drug discovery, have been attempted to be expressed on cell membranes using animal cells such as CHO cells and insect cells such as sf9 (Patent Literature). 1, 2, 3). However, the expression efficiency in the cell membrane fraction is low, and the presence of many other types of membrane proteins present on the cell membrane even when expressed on the cell membrane has been a problem in purification. Furthermore, in the purification of membrane proteins from membrane fractions, it is very difficult to solubilize while maintaining the activity while taking into account the effects of complex factors such as ionic strength, pH, surfactant type and concentration. It is. In addition, as a simple method, there is a method using bacteria as a host (Patent Document 4). However, in bacteria, membrane receptors derived from mammals are difficult to express, and even if expressed, they are recovered in an insoluble fraction or modified after translation. There were many problems such as lack of activity due to the absence of

  The scavenger receptor (SR) is a group of factors that are deeply associated with macrophage foaming and vascular endothelial cell damage by binding to modified low-density lipoprotein (LDL) modified by oxidation and acetylation. Among them, the SR-A protein is a scavenger receptor present in macrophages, vascular endothelial cells, and smooth muscle cells, and has a main function among the scavenger receptors (Non-patent Document 1).

Special table 2003-516109 gazette JP 2005-507245 Gazette JP 2002-525095 gazette Special table 2007-531502 gazette

Journal of Lipid Research vol. 46, 2005, pp.11-20

  However, SR-A protein, which is a membrane receptor, is a trimeric transmembrane protein, and it has been difficult to express and purify it by conventional methods and has not existed so far. Therefore, it is still difficult to construct an evaluation system using the standard.

  In view of the above situation, the present invention has developed means for obtaining a solubilized SR-A protein that forms a multimeric conformation, and is very useful for screening for agonists and antagonists of the protein. It is to provide the construction of an evaluation system for.

  In the present invention, as a result of intensive studies to solve the above problems, a base sequence encoding SR-A protein in which a transmembrane domain has been mutated or deleted from the N-terminus is inserted downstream of the secretion signal, and further, Solubilized SR secreted into the medium by using a DNA structure in which a tag sequence is inserted downstream of the base sequence encoding the DNA side and culturing animal cells having this DNA structure in a serum-free medium -A method has been developed that can be purified with high purity while retaining activity with an affinity carrier using the tag sequence of the A protein. By using the solubilized SR-A protein thus obtained, direct protein such as binding of a ligand such as denatured LDL and SR-A protein, or screening for an agonist or antagonist of SR-A protein This is the first successful construction of an evaluation system for interaction between the two.

That is, the present invention relates to the following (1) to (4).
(1) A method for producing a highly pure solubilized class A scavenger receptor (SR-A) protein, comprising the following steps a and b.
a: Solubilized SR by culturing animal cultured cells into which a recombinant SR-A gene having a mutation or deletion from the N-terminal to the transmembrane domain of the SR-A protein which is a scavenger receptor is introduced in a serum-free medium -Producing the A protein b: Purifying the culture solution containing the solubilized SR-A protein or the culture treatment solution thereof (2) As the recombinant SR-A gene, the SR-A protein is downstream of the secretion signal. A DNA structure in which an SR-A gene having a mutation or deletion from the N-terminal to the transmembrane domain is inserted and a tag sequence is inserted downstream of the base sequence encoding the C-terminal side of the SR-A gene is used. The manufacturing method according to (1) above,
(3) a solubilized SR-A protein produced by the production method according to (1) or (2),
(4) A method for evaluating affinity for SR-A, comprising a step of allowing a test substance to act on the solubilized SR-A protein according to (3).
(5) Inhibiting the test substance against the binding of SR-A and denatured LDL, comprising the step of allowing the test substance and denatured LDL to act on the solubilized SR-A protein according to (3) Evaluation method for promoting action.
(6) Serum or antibody produced by immunizing an animal species using the solubilized SR-A protein described in (3) above.

  The present invention is the first to enable high-purity purification of a solubilized SR-A protein that forms a multimeric conformation that retains the same LDL binding activity as the SR-A protein. Therefore, according to the present invention, it is possible to use a high-purity solubilized SR-A protein that retains its activity, and it is possible to evaluate the affinity of test substances such as agonists and antagonists for the SR-A protein. In addition, inhibition or promotion of a test substance for binding of SR-A protein and denatured LDL can be directly evaluated by protein-protein interaction. Therefore, these techniques are used in the medical field where SR-A protein is said to be related, for example, in the research of diseases such as arteriosclerosis, inflammation, infectious diseases, Alzheimer's disease, and in the field of drug discovery for these diseases. Can contribute widely.

FIG. 1 shows the results of detecting the solubilized SR-A protein secreted into the medium obtained by the method of Example 1 using an anti-histidine tag antibody. FIG. 2 shows the results of evaluating the affinity for acetylated LDL and oxidized LDL by enzyme immunoassay (ELISA) using the solubilized SR-A protein obtained by the method of Example 1 and the method of Example 2. It is a graph to show. The vertical axis indicates the actual 450 nm value, and the higher the value, the more denatured LDL is bound. FIG. 3 is a graph showing the results of evaluating the binding inhibition reaction with acetylated LDL by a plurality of extracts by ELISA using the solubilized SR-A protein obtained by the method of Example 1 and the method of Example 2. It is. In addition, the negative and positive of each extract are tests using different cells in advance, and the extract having no affinity for the solubilized SR-A protein is shown as negative, and the extract with affinity is shown as positive. FIG. 4 is a graph showing the results of verifying the molecular weight of the solubilized SR-A protein obtained by the method of Example 1 in a non-denaturing state using a gel filtration column. Calibration curves were prepared using 4 types of marker proteins.

The production method of the present invention includes the following steps a and b:
a: Solubilized SR by culturing animal cultured cells into which a recombinant SR-A gene having a mutation or deletion from the N-terminal to the transmembrane domain of the SR-A protein which is a scavenger receptor is introduced in a serum-free medium -Producing the protein A b: It comprises a step of purifying a culture solution containing the solubilized SR-A protein or a culture treatment solution thereof.

(Step a)
The SR-A protein, which is a scavenger receptor targeted in step a, is mainly expressed in macrophages and is a glycosylated type II transmembrane protein that binds to modified LDL and oxidized LDL. It has been known.

Types of SR-A proteins include type 1 (SR-A1), type 2 (SR-A2), type 3 (SR-A3) and MARCO. Hereinafter, these are collectively referred to as the SR-A family.
In any case, from the N-terminal side, a monomer having an amino acid sequence composed of an intracellular domain, a transmembrane domain, a spacer domain, and a collagen-like domain forms a trimer. Type 1 (SR-A1), Type 2 (SR-A2) and Type 3 (SR-A3) have an α-helical coiled coil domain between the spacer domain and the collagen-like domain. SR-A1 further has a cysteine-rich region on the C-terminal side of the collagen-like domain.

  Examples of the origin of these SR-A proteins include mammals such as humans, cows, rabbits, horses, rats, and pigs.

  The “solubilized SR-A protein” as used in the present invention is a protein in which the amino acid sequence from the N-terminal to the transmembrane domain is mutated or deleted, and is a normal SR-A protein. A protein that forms a multimeric conformation, such as a protein.

The degree of the mutation or deletion is as long as one or several amino acids are deleted, substituted or added in the amino acid sequence from the N-terminal to the transmembrane domain. The whole amino acid sequence may be deleted.
Examples of the mutation or deletion method include, for example, deletion of the base sequence of the SR-A gene encoding a transmembrane domain from the N-terminus of the SR-A protein by a general genetic engineering method. Any known technique may be used that introduces one or more mutations such as substitution or insertion. Moreover, you may perform the variation | mutation which deletes all the base sequences which code the amino acid sequence from the said N terminal to a transmembrane domain so that it may mention later.

  In the solubilized SR-A protein of the present invention, one or several amino acids may be deleted, substituted or added to the extracellular domain to such an extent that a multimeric conformation is formed. It is preferable that the amino acid sequence of the domain part is not modified.

  The base sequence of the SR-A gene encoding the SR-A protein is, for example, the base sequence database GenBank provided by the National Center for Biotechnology Information or the Japan DNA Data Bank (DNA). Data Bank of Japan) and published. In addition, the amino acid sequence of the SR-A protein can be easily determined from the base sequences registered in these institutions.

  In the present invention, the recombinant SR-A gene encoding the SR-A protein in which the N-terminal to transmembrane domain is mutated or deleted as described above is used as a predetermined primer set using various cDNA libraries as templates. It is obtained by performing polymerase chain reaction (PCR) using

  Any cDNA library may be used as long as it is related to cells expressing or inducing expression of the SR-A family. Commercially available products may be used, and they are prepared from various animal cells based on known methods. You can also.

A primer set refers to a combination of primers used simultaneously during PCR. In the present invention, one primer is capable of binding before and after the base sequence encoding the transmembrane domain on the N-terminal side of the SR-A protein, and the other primer is an amino acid located on the C-terminal side of the collagen-like domain. What can be selected is one that can bind to the base sequence encoding the sequence.
In addition, primary PCR is performed with a primer set that can amplify a base sequence encoding SR-A protein including from the transmembrane domain to the C-terminal side, and then secondary PCR is performed with a primer set that deletes only the transmembrane domain. Also good.

  Further, as a primer, a primer set to which an appropriate restriction enzyme site is added may be used, or by inserting into a pCR 8 / GW / TOPO vector (manufactured by Invitrogen) by TA cloning as in the present invention, There is an advantage that a plasmid vector can be constructed easily.

  The PCR conditions may be adjusted as appropriate according to the manual of the PCR device to be used.

  In the present invention, the PCR product obtained as described above is inserted into a predetermined vector, and the obtained recombinant vector is introduced into various animal cultured cells.

  The vector may be an expression vector containing a secretion signal. In the present invention, the secretory signal refers to a signal sequence that allows the desired protein translated from the vector to be secreted outside the host animal cultured cell. The type of secretory signal is the same as the type of animal cultured cell. What is necessary is just to determine suitably according to. The secretion signal may be introduced into a desired plasmid vector by known means, but from the viewpoint of simplicity, it is preferable to use a plasmid vector containing a secretion signal. For example, a plasmid vector includes, but is not limited to, pSecTag2 / Hygro vector (Invitrogen). If necessary, a cosmid vector, virus vector, artificial chromosome vector or the like may be used as a vector containing a secretion signal.

  The PCR product can be inserted into the vector by, for example, mixing a PCR product previously cleaved with a restriction enzyme and a vector cleaved with the same restriction enzyme. Here, any restriction enzyme may be used as long as it exists at the cloning site of the vector without cleaving the internal sequence of the PCR product so that the expressed protein can form a multimeric conformation. The ligation conditions may be appropriately adjusted according to the manual when a commercially available ligation kit is used.

  The vector obtained as described above has, as a recombinant SR-A gene, a DNA structure comprising a secretion signal and an SR-A gene in which the N-terminal to transmembrane domain of SR-A protein is mutated or deleted. It is a thing. In addition, the DNA construct can further efficiently purify the expressed solubilized SR-A protein by including a tag sequence as necessary.

  The animal cultured cells may be any cells capable of introducing the vector and corresponding to the secretory signal sequence. Among them, as the animal cultured cells that can secrete the solubilized SR-A protein outside the cells, cells capable of high-density culture are preferable, and examples thereof include freestyle 293F cells (manufactured by Invitrogen). Further, a stable expression strain may be obtained by selection even in transient expression.

  For introduction of the recombinant vector into the animal cultured cells, a known transfection technique such as a commercially available transfection reagent, electroporation method, calcium phosphate method, DEAE-dextran method, lipofection method or the like can be used. Good.

  In the present invention, cultured animal cells into which the recombinant vector has been introduced are cultured in a serum-free medium, so that the solubilized SR-A protein secreted from the animal cultured cells into the serum-free medium is preliminarily obtained as a serum-derived protein. Can be separated.

  As described above, since the transmembrane domain is mutated or deleted from the intracellular domain, the solubilized SR-A protein of the present invention does not remain at the cell membrane even if expressed in the animal cultured cells. Since it is excreted out of animal cultured cells by a signal, there is no physical restriction such as the surface area of the cell membrane as compared with the conventional production method in which the expressed SR-A protein is present in the cell membrane. The solubilized SR-A protein can be recovered very easily simply by separating the culture supernatant from the cultured animal cells. In addition, centrifugation etc. are mentioned as a supernatant-separation means.

  The serum-free medium includes a protein-free medium and a chemically synthesized medium. In addition, regarding the composition of the serum-free medium and the culture conditions for the animal cultured cells, conditions appropriate for the cell type may be selected as appropriate.

In the present invention, in the next step b, the culture solution containing the solubilized SR-A protein or the culture treatment solution thereof is purified.
Specifically, with respect to the culture solution collected from the serum-free medium or the culture treatment solution thereof, column chromatography, organic solvent fractionation, salt fractionation, isoelectric point fractionation, density gradient centrifugation, crystallization method The solubilized SR-A protein can be separated and concentrated by using such a method.
The culture treatment solution means a solution obtained by centrifuging the culture solution to remove insoluble components.
When a vector containing a tag sequence is used, the solubilized SR-A protein can be easily separated and concentrated by using a carrier having affinity for the tag sequence. Examples of the carrier include a Ni-NTA agarose column.

  The solubilized SR-A protein obtained as described above has a molecular weight of about 40 to 70 kDa under SDS-PAGE conditions and a molecular weight of about 100 kDa or more under non-denaturing conditions. It is a protein forming a conformation and can be said to have a three-dimensional structure similar to that of a normal SR-A protein. Further, the solubilized SR-A protein is capable of binding to denatured LDL in the same manner as normal SR-A protein, as confirmed in Examples described later.

  Therefore, by using the solubilized SR-A protein, it is possible to construct an evaluation system for the SR-A protein, which has been difficult in the past.

Examples of the evaluation system include an evaluation system for affinity for SR-A protein.
As a method for evaluating the affinity for SR-A protein, a test substance is allowed to act on the solubilized SR-A protein. By using this method, the test substance can not only evaluate the affinity of a substance expected to bind to a normal SR-A protein such as denatured LDL, oxidized LDL, etc., but the affinity is unknown. Substances can also be screened.

  Further, since the solubilized SR-A protein has a high binding rate to denatured LDL, binding of the SR-A protein and LDL is inhibited by allowing the test substance and denatured LDL to act on the solubilized SR-A protein. It is possible to construct an evaluation system for the action to be performed or promoted. By using such an evaluation method, an unknown substance that inhibits or promotes the binding between denatured LDL and SR-A protein can be screened.

Further, according to the present invention, since the solubilized SR-A protein can be produced in a large amount, serum against the SR-A protein can be obtained by immunizing animal species using the solubilized SR-A protein. Alternatively, antibodies can be obtained.
For example, sera of rabbits, mice, sheep, goats, monkeys, etc., and mammals generally used for obtaining antibodies are immunized by injecting the solubilized SR-A protein, Serum and antibodies may be collected by a known method.

Next, the present invention will be specifically described with reference to examples thereof.
Example 1
Polymerase chain reaction (PCR) was performed using a human spleen cDNA library as a template and the following primer set.
Primer 1: 5'-tccttcaaagctgcactg-3 '(SEQ ID NO: 1 in the sequence listing)
Primer 2: 5'-agagggccctgccctaatatg-3 '(SEQ ID NO: 2 in the sequence listing)
The extension time was 1 minute, the annealing temperature was 54 ° C., and the number of cycles was 30 cycles.
By this PCR, the base sequence encoding the SR-A2 protein from the transmembrane domain to the C-terminus can be amplified.

When the base sequence of the obtained PCR product was examined based on a conventional method, it was found to contain the 142th to 1074th base sequences out of 1074 base sequences encoding the SR-A protein (SR-A2). It was.
Next, the PCR product was inserted into the pCR8 / GW / TOPO vector (Invitrogen) by TA cloning. The plasmid in which the insert was correctly inserted was recovered, and this plasmid was used as a template DNA for the following.

Next, PCR was performed using this template DNA and the following primer set.
Primer 3: 5'-aaccaagcttatgctgaagtgggaaacg-3 '(SEQ ID NO: 3 in the sequence listing)
Primer 4: 5′-ataagaatgcggccgcagagggccctgccctaatatg-3 ′ (SEQ ID NO: 4 in the sequence listing)
The extension time was 1 minute, the annealing temperature was 63 ° C., and the number of cycles was 30 cycles.
By this PCR, a base sequence (226th to 1074th) encoding the SR-A2 protein from the spacer domain to the C-terminal excluding the transmembrane domain can be amplified.

The obtained PCR product was treated with HindIII and NotI restriction enzymes and inserted into the multicloning site of a pSecTag2 / Hygro vector (manufactured by Invitrogen) pretreated with the same restriction enzymes so that the frames matched. The plasmid in which the insert was correctly inserted was recovered after confirmation by sequencing. This plasmid is a DNA structure in which the PCR product is inserted downstream of the secretion signal and a tag sequence is inserted downstream thereof. The amino acid sequence of the solubilized SR-A protein obtained from this DNA structure is normal. Compared to the SR-A2 protein, the N-terminal to transmembrane domain is deleted, and all of the extracellular domain, spacer domain, α-helix coiled-coil domain, and collagen-like domain are included. On the side, it was expected that the amino acid derived from the tag sequence was also added.
This plasmid was transfected into FreeStyle 293-F cells (Invitrogen) to perform transient expression. The conditions for inserting the PCR product into the plasmid vector, transfection, and transient expression were in accordance with the manual of Invitrogen.

  After 4 days of culture, the medium was collected by centrifugation, and the precipitate fraction was removed by passing through a 0.45 μm filter. After sampling a portion, it was loaded onto a 2 ml Ni-NTA agarose column (Qiagen) equilibrated in advance with phosphate buffered saline (PBS). The column after loading was washed with PBS and then eluted with a solution containing 250 mM imidazole. The eluted fraction was further dialyzed with PBS and used as a solubilized SR-A protein in the following experiments.

  The sampled medium and the solubilized SR-A protein as the final sample were subjected to SDS polyacrylamide gel electrophoresis (SDS-PAGE) according to a conventional method. Furthermore, after electrophoresis, it was transferred to a PVDF (polyvinylidene fluoride) membrane according to a conventional method, and an anti-mouse conjugated with horseradish peroxidase as a secondary antibody using an anti-histidine tag antibody (manufactured by GE Healthcare) as a primary antibody. Detection was performed using an IgG antibody (Bio-Rad). These results are shown in FIG.

  As shown in FIG. 1, the expression of the solubilized SR-A protein secreted into the culture supernatant is confirmed on the transfer membrane, and the solubilized SR-A protein is purified and concentrated from the culture medium using a Ni-NTA agarose column. I was able to.

(Example 2)
The evaluation system for the solubilized SR-A protein obtained in Example 1 was constructed as follows. However, this embodiment is an example of the evaluation system and is not particularly limited.
This was performed using a maxi soap immunoplate (96 well type, manufactured by NUNC). The solubilized SR-A protein purified as described above was prepared in PBS buffer to 5 μg / ml, and 100 μl was applied to each well. After allowing to stand at 4 ° C. overnight, each well was washed with 400 μl × 2 times with PBS buffer, and 300 μl of PBS buffer containing 25% Block Ace (Dainippon Sumitomo Pharma Co., Ltd.) was applied to each well. After standing overnight at 4 ° C., each well was washed with 400 μl × 2 times with PBS buffer, and each antagonist prepared at a suitable dilution ratio with PBS buffer containing 1% bovine serum albumin (BSA) 100 μl of sample such as agonist was applied to each well. After allowing to stand at 4 ° C. overnight, each well was washed with PBS buffer at 400 μl × 3 times, and 100 μl of denatured LDL prepared with PBS buffer was applied to each well at 5 μg / ml. After allowing to stand at 4 ° C. overnight, each well was washed with PBS buffer 400 μl × 3 times, and anti-apolipoprotein B antibody conjugated with horseradish peroxidase (manufactured by The Binding Site) was 1000 times with PBS buffer. Dilute and apply 100 μl to each well. After standing at room temperature for 2 hours, each well was washed with PBS buffer 400 μl × 5 times, and 3,3 ′, 5,5′-tetramethylbenzidine (TMB) -peroxidase-enzyme immunoassay (EIA) -Substrate-kit reagent (Biorad) 100 μl was applied to each well. After an appropriate reaction time, 50 μl of 2MH ₂ SO ₄ was applied to each well to stop the reaction. Finally, detection was performed at 450 nm, and the affinity for SR-A was quantified as the amount of denatured LDL bound. The results are shown in FIG.

  As shown in FIG. 2, it was confirmed that the obtained solubilized SR-A protein bound to these modified LDLs in a concentration-dependent manner. The binding of these modified LDLs was not confirmed to BSA immobilized as a negative control instead of the solubilized SR-A protein.

Further, by using the solubilized SR-A protein obtained by the method of Example 1, the binding inhibition reaction with acetylated LDL by a plurality of extracts was evaluated by ELISA according to the method of Example 2. The results are shown in FIG. The negative and positive of each extract are shown in FIG. 3 in the test using different cells in advance, with the extract having no affinity for the solubilized SR-A protein being negative and the extract having affinity being positive.
As shown in FIG. 3, by using the obtained solubilized SR-A protein, it is possible to screen a substance that inhibits or promotes the binding between the SR-A protein and the denatured LDL in multiple samples. Is shown.

In addition, the molecular weight of the solubilized SR-A protein obtained by the method of Example 1 in a non-denaturing state was verified using a gel filtration column. A calibration curve was prepared using 4 types of marker proteins. The results are shown in FIG.
As shown in FIG. 4, the molecular weight predicted from the amino acid sequence of the solubilized SR-A protein was about 35 kDa, and was confirmed as a molecular weight of about 40 to 70 kDa due to glycosylation on SDS-PAGE. The molecular weight in the non-denaturing state calculated from the gel filtration column was about 100 kDa or more, suggesting that this was not a monomer but a multimer.

  In the above examples, the SR-A2 protein was used as the SR-A protein, but referring to this example, the SR-A1, SR-A3, and MARCO proteins, which are other SR-A families, were used. Also, a recombinant SR-A gene having a mutation or deletion from the N-terminal side to the transmembrane domain is prepared, and animal cultured cells into which this recombinant SR-A gene has been introduced are cultured in a serum-free medium, A person skilled in the art can sufficiently produce a solubilized SR-A protein.

Claims (6)

A method for producing a high-purity solubilized class A scavenger receptor (SR-A) protein, comprising the following steps a and b.
a: Solubilized SR by culturing animal cultured cells into which a recombinant SR-A gene having a mutation or deletion from the N-terminal to the transmembrane domain of the SR-A protein which is a scavenger receptor is introduced in a serum-free medium -Producing the A protein b: Purifying the culture solution containing the solubilized SR-A protein or the culture treatment solution thereof   As the recombinant SR-A gene, an SR-A gene in which the N-terminal to transmembrane domain of SR-A protein is mutated or deleted downstream of the secretion signal is inserted, and the C-terminal side of the SR-A gene The production method according to claim 1, wherein a DNA structure in which a tag sequence is inserted downstream of a base sequence that encodes a DNA.   A solubilized SR-A protein produced by the production method according to claim 1 or 2.   A method for evaluating affinity for an SR-A protein, comprising a step of allowing a test substance to act on the solubilized SR-A protein according to claim 3.   An action of inhibiting or promoting the test substance on the binding of SR-A protein and denatured LDL, comprising the step of allowing the test substance and denatured LDL to act on the solubilized SR-A protein according to claim 3. Evaluation methods.   A serum or an antibody produced by immunizing an animal species using the solubilized SR-A protein according to claim 3.