JP2009050227A - Recombinant apolipoprotein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a new recombinant apolipoprotein, to obtain recombinant apolipoprotein C-I and an antibody against the same and to provide a method for assaying apolipoprotein C-I using the antibody and a kit used therefor. <P>SOLUTION: The high-purity recombinant apolipoprotein C-I is obtained by transferring an expression plasmid comprising an Lac promoter as a promoter, a DNA sequence encoding thioredoxin and a DNA sequence encoding apolipoprotein C-I to Escherichia coli, expressing a fusion protein of thioredoxin and apolipoprotein C-I in the Escherichia coli, and recovering apolipoprotein C-I from the fusion protein. The antibody having higher specificity to natural apolipoprotein C-I is obtained by preparing the antibody against apolipoprotein C-I by using apolipoprotein C-I as an antigen. Apolipoprotein C-I in a specimen is accurately assayed by using the antibody by an immunological assay method. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to recombinant apolipoprotein. More specifically, a method for producing a recombinant apolipoprotein, a recombinant plasmid expression vector used in the production method, a recombinant apolipoprotein CI obtained by the production method, an antibody against the recombinant apolipoprotein CI, The present invention relates to a method for measuring CI of apolipoprotein using an antibody and a kit used for the method.

Numerous proteins in living organisms have been clinically measured in relation to the presence / absence of disease. In this case, as a method for measuring a protein, an immunoassay method using an antibody against the protein is generally used, and clinical diagnostic drugs are widely supplied based on the principle.
Apolipoprotein CI, which is one of the biological proteins, is a protein having a molecular weight of 6.6 kDa produced mainly in the liver, and has been reported to be involved in the development of hyperlipidemia (Non-patent Document 1). ). Although natural apolipoprotein C-I and antibodies made using it are actually commercially supplied, immunoassay reagents for measuring apolipoprotein C-I are clinical laboratory reagents. Not used as.
With regard to apolipoprotein C-I, in recent years, with the development of mass spectrometers such as SELDI-MS and MALDI-MS and the analysis method of HPLC method, the relationship between the apolipoprotein CI and the pathological condition has been newly known. For example, it has been reported that the level of apolipoprotein CI is related to the possibility of stroke, sepsis, tumor, heart disease, and Crohn's disease using mass spectrometer and HPLC (Patent Literature). 1 and Patent Document 2).
As a clinical test agent, in order to measure apolipoprotein CI, although it is preferable to measure it with an immunological determination method, when this is examined with an immunoassay method, It is pointed out that the results are different from those measured in (Patent Document 2).
Therefore, development of a clinical test drug for measuring apolipoprotein C-I by an immunological determination method is strongly desired.
International Publication No. WO2005 / 017523 (Patent Publication No. 2007502401) Pamphlet of International Publication No. WO2005 / 038461 (Patent Publication No. 2007060775) Arteriosclerosis, Thrombosis, and Vascular Biology, 1999; 19; 472-484

Under such circumstances, as a problem of the immunoassay for apolipoprotein CI, the present inventors have found that a commercially available anti-apolipoprotein C-I antibody has a large non-specific reaction. It was discovered that there was a problem with the antigen used to make the A, ie, natural apoprotein CI. Therefore, an attempt was made to create apolipoprotein CI by genetic engineering techniques. Although the production of recombinant apolipoprotein C-I was examined, it was an unknown technology and was extremely difficult to succeed. For example, according to the study by the present inventors, recombinant apolipoprotein C-I could not be produced using mouse ovary cells close to humans. In addition, because lipoproteins are metabolized in E. coli without using apolipoprotein C-I, recombinant apolipoproteins can be obtained even if E. coli is used as a host cell and normal T7 promoter or Lac promoter is used. C-I could not be expressed.
Accordingly, an object of the present invention is to provide a method for producing recombinant apolipoprotein C-I that can produce an anti-apoprotein C-I antibody having a higher purity and less non-specific reaction than apolipoprotein C-I obtained from nature. Is to provide. Furthermore, it is providing the manufacturing method for manufacturing other recombinant apolipoprotein in addition to recombinant apolipoprotein CI. Another object of the present invention is to provide a recombinant apolipoprotein C-I capable of producing an anti-apoprotein CI antibody with little nonspecific reaction, an antibody against the recombinant apolipoprotein CI, and apolipoprotein C using the antibody. It is to provide a method for measuring -I and a kit used for the method.

  As a result of intensive research aimed at solving the above-mentioned problems, the present inventors have used a protein derived from Escherichia coli as a promoter and Escherichia coli as a host cell, which is a water-soluble protein that can be expressed in Escherichia coli. Recombination capable of producing high-purity recombinant apolipoprotein C-I with less non-specific reaction by expressing it as a fusion protein of a certain thioredoxin and apolipoprotein C-I Apolipoprotein C-I is obtained, and this production method can be widely applied to the production of other recombinant apolipoprotein, and anti-apolipoprotein C-I prepared using this recombinant apolipoprotein C-I as an antigen. The ability to accurately measure natural apolipoprotein C-I by immunoassay using antibodies Heading, which has led to the completion of the present invention.

Therefore, the present invention introduces a recombinant plasmid expression vector having a promoter derived from E. coli, a DNA sequence encoding a water-soluble protein that can be expressed in E. coli, and a DNA sequence encoding apolipoprotein into E. coli. A method for producing a recombinant apolipoprotein, comprising culturing the obtained E. coli, expressing a water-soluble protein that can be expressed in E. coli and apolipoprotein, and obtaining apolipoprotein from the fusion protein About.
The present invention further relates to a recombinant plasmid expression vector having a promoter derived from E. coli, a DNA sequence encoding a water-soluble protein that can be expressed in E. coli, and a DNA sequence encoding apolipoprotein.
The present invention further relates to recombinant apolipoprotein CI or a variant thereof.
The present invention further relates to an antibody against recombinant apolipoprotein C-I obtained by administering recombinant apolipoprotein C-I as an immunogen to a mammal.
Furthermore, the present invention relates to a method for measuring apolipoprotein C-I, which comprises measuring apolipoprotein C-I in a specimen by immunoassay using an antibody against recombinant apolipoprotein C-I.
Furthermore, the present invention relates to a kit for measuring apolipoprotein CI, comprising an antibody against recombinant apolipoprotein CI.

  Using a promoter derived from E. coli as the promoter and E. coli as the host cell, and expressing it as a fusion protein of a water-soluble protein that can be expressed in E. coli and apolipoprotein, a highly pure recombinant apolipoprotein is obtained. It is done. By preparing an antibody against apolipoprotein C-I using the high-purity recombinant apolipoprotein C-I obtained by this method as an antigen, an antibody with high specificity to natural apolipoprotein C-I can be obtained. Obtainable. Using this antibody, apolipoprotein CI in a specimen can be accurately measured by an immunoassay. Therefore, by using the antibody against recombinant apolipoprotein C-I obtained in the present invention as a clinical test agent, it is possible to accurately diagnose the possibility of stroke, sepsis, tumor, heart disease, Crohn's disease, etc.

In the present invention, the recombinant apolipoprotein is a recombinant plasmid expression vector having a promoter derived from E. coli, a DNA sequence encoding a water-soluble protein that can be expressed in E. coli, and a DNA sequence encoding apolipoprotein. And then culturing the introduced Escherichia coli, expressing a water-soluble protein that can be expressed in Escherichia coli and apolipoprotein, and obtaining apolipoprotein from the fusion protein.
Examples of the promoter derived from Escherichia coli used here include lac promoter, tac promoter, trp promoter and the like, and lac promoter, tac promoter, particularly lac promoter is preferable.

The water-soluble protein that can be expressed in E. coli is preferably a protein having a molecular weight of 8000 to 60000, such as thioredoxin, glutathione S-transferase (GST), maltose-binding protein (MBP), or variants thereof. It is done. Variants include proteins that have the same function as the original protein, consisting of amino acid sequences in which one or several amino acid residues are deleted, substituted, or added in the amino acid sequences of these proteins. Here, the same function refers to a function that can be expressed as a fusion protein in E. coli together with apolipoprotein. In the present invention, a protein that is soluble in water derived from E. coli and can be expressed in E. coli is preferred, and thioredoxin or a variant thereof, in particular, thioredoxin derived from E. coli or a variant thereof is preferred.
The amino acid sequences of these water-soluble proteins that can be expressed in Escherichia coli and the DNA sequences encoding them are described in various literatures and are well known.
In the recombinant plasmid expression vector, the DNA sequence encoding a protein that is water-soluble and can be expressed in E. coli is preferably located upstream of the DNA sequence encoding apolipoprotein.

As the apolipoprotein, apolipoprotein A, C, E and the like are preferable, and further, subtypes thereof such as apolipoprotein A-I, C-I, C-II, C-III, E2, E3, E4, etc. Is mentioned. Or these variants may be sufficient. Examples of the mutant include a protein having an amino acid sequence in which one or several amino acid residues are deleted, substituted or added in the amino acid sequence of these apolipoproteins and having the same function as the original protein. Here, the same function refers to the ability to produce an antibody having the same antigenicity as the original apolipoprotein and the same specificity.
Among these apolipoproteins, apolipoprotein CI is particularly preferable. The amino acid sequences of these apolipoproteins and the DNA sequences encoding them are described in various literatures and are well known.

  In the present invention, the recombinant plasmid expression vector preferably further comprises a DNA sequence encoding a purified peptide for purifying the expressed fusion protein. Preferable examples of the purified peptide include peptides capable of affinity purification such as His tag, FLAG, and STREP. For example, the His tag is one in which about 6 to 10 histidine residues are continuously bonded, and is used by being bonded to a fusion protein. Since this His tag interacts with the chelate carrier, the expressed fusion protein can be easily purified. FLAG is a peptide tag consisting of 8 amino acid residues, and can be expressed by binding to a fusion protein, and the fusion protein can be purified using an anti-FLAG antibody. STREP is a peptide tag consisting of eight amino acid residues that binds to streptavidin, and the fusion protein can be purified using reversible binding to streptavidin. Among these purified peptides, the His tag is preferable. The DNA sequence encoding these purified peptides is placed between or between the DNA sequence encoding a protein that is water soluble and can be expressed in E. coli and the DNA sequence encoding apolipoprotein. Used in conjunction.

  In the present invention, the recombinant plasmid expression vector preferably further comprises a DNA sequence encoding a protease cleavage peptide for releasing apolipoprotein from the expressed fusion protein. Examples of the protease-cleaving peptide include peptides that are cleaved by proteases such as thrombin, factor Xa, TEV protease, and PreScission protease. The DNA sequence encoding these protease-cleaving peptides is located between the DNA sequence encoding a protein that is water-soluble and can be expressed in E. coli and the DNA sequence encoding apolipoprotein. It is preferably present immediately upstream. By using a DNA sequence encoding such a protease-cleaving peptide, apolipoprotein can be released from the fusion protein by, for example, causing thrombin to act on the expressed fusion protein.

  Construction of a recombinant plasmid expression vector containing the various DNA sequences described above can be performed, for example, as shown in FIG. Specifically, a plasmid pUC-Trx having a DNA sequence encoding Lac promoter, E. coli-derived thioredoxin, 6His tag and thrombin cleavage peptide in this order from the upstream side, while DNA sequence encoding human apolipoprotein CI Plasmid pTOPO-ApoCI having a fragment containing a DNA sequence encoding Lac promoter, E. coli-derived thioredoxin, 6His tag and thrombin cleaving peptide, and a DNA sequence encoding human apolipoprotein CI. The desired recombinant plasmid expression vector pUC-Trx-ApoCI can be constructed by obtaining the fragments and ligating the fragments. Recombinant plasmid expression vectors for obtaining other various recombinant apolipoproteins can be similarly constructed.

A recombinant plasmid expression vector is introduced into Escherichia coli, and the introduced Escherichia coli is cultured to express a fusion protein of an apolipoprotein that is water-soluble and can be expressed in Escherichia coli. Can be carried out by a usual method. The expressed fusion protein is purified using the above-described purified peptide, and apolipoprotein is released from the purified fusion protein using the above-mentioned protease cleavage peptide to produce the desired recombinant apolipoprotein. Can do.
By the production method of the present invention, high-purity recombinant apolipoprotein is obtained, and by immunizing an animal as an antigen, recombinant apolipoprotein that induces the production of an antibody having high specificity for the corresponding natural apolipoprotein. Protein is obtained. In particular, the production method of the present invention provides recombinant apolipoprotein C-I that induces the production of antibodies with high specificity for natural apolipoprotein C-I.

By administering the recombinant apolipoprotein C-I obtained by the production method of the present invention to a mammal as an immunogen, an antibody against the recombinant apolipoprotein C-I can be obtained. The antibody may be either a polyclonal antibody or a monoclonal antibody.
Polyclonal antibodies can be prepared by immunizing a mammal with recombinant apolipoprotein CI and collecting serum from the immunized animal. A monoclonal antibody can be prepared by a hybridoma obtained by immunizing a mammal using recombinant apolipoprotein C-I as an immunogen and fusing the antibody-producing cells and bone marrow tumor cells produced by the animal.

The hybridoma mixes recombinant apolipoprotein C-I together with Freund's complete, incomplete adjuvant, aluminum hydroxide adjuvant, pertussis adjuvant, etc. to make a sensitive adjuvant solution and divide it into several times. Polyethylene glycol and the like by immunizing animals such as mice and rats, and then antibody-producing cells derived from the spleen etc. of immunized animals and cells that become bone marrow tumor cells (myeloma cells) by the usual method of Kohler and Milstein (Nature.256,495.1975) Can be obtained by selecting a hybridoma that produces an antibody that recognizes apolipoprotein CI from the fused cells.
The hybridoma can be cultured in a medium usually used for cell culture, and the monoclonal antibody can be recovered from the culture supernatant. Alternatively, an animal from which a hybridoma is derived can be treated with pristane in advance, and ascites can be retained by intraperitoneal injection of cells into the animal, and the monoclonal antibody can be recovered from the ascites.

The antibody of the present invention has high specificity for natural apolipoprotein C-I compared to an antibody obtained by administering natural apolipoprotein C-I to a mammal as an immunogen. Apolipoprotein CI in a sample can be detected with high sensitivity and specificity by an immunoassay using the antibody of the present invention.
Here, examples of the target specimen include tissues such as blood, serum, plasma, and bone.
Examples of the immunoassay include enzyme immunoassay (EIA method), immunoturbidimetric assay (TIA method), latex immunoaggregation method (LATEX method), electrochemiluminescence method, and fluorescence method. In addition, immunochromatography and methods using test paper are also effective. These methods are all methods well known to those skilled in the art, and these well known methods can be employed as they are.
For example, as an enzyme immunoassay, a sandwich assay ELISA method will be described below as an example.
The antibody is bound directly or indirectly to a solid phase such as polystyrene, polypropylene, polycarbonate, polyethylene, nylon, polymethacrylate or the like directly or indirectly using physical binding, chemical binding, or affinity. The analyte is then allowed to act to bind apolipoprotein C-I to the antibody bound to the solid phase, the solid phase is washed, and then a corresponding secondary labeled antibody, such as an anti-immunoglobulin labeled antibody, is added for a further 2 Next react. The solid phase is washed again, and a chromogenic substrate is added to react. When HRP is used as the labeling substance, known DAB, TMB, etc. can be used as the substrate, and the labeling substance is not limited to this. For example, not only enzymes, but also distinguishable materials such as gold colloids, europium-labeled metals, various chemical and biological fluorescent materials such as FITC, rhodamine, Texas Red, Alexa and GFP, and radioactive materials such as ³² P and ⁵¹ Cr Is mentioned.

  When carrying out the measurement method of the present invention, a kit for immunoassay of apolipoprotein CI, which comprises an antibody against recombinant apolipoprotein C-I, can be used. In this kit, the antibody may be adsorbed on a solid support, and after apolipoprotein CI in the sample is bound to the antibody, in order to remove components not adsorbed on the solid support, A cleaning solution may be included. As the cleaning liquid, for example, a Tris buffer containing a surfactant can be used. Further, for example, an enzyme reaction stop solution such as potassium hydroxide or sodium hydroxide solution, for example, a buffer solution such as Tris can also be included.

EXAMPLES Hereinafter, although an Example demonstrates this invention still in detail, this invention is not limited at all by these Examples.
Example 1
Construction of recombinant plasmid expression vector Preparation of DNA fragment encoding human apolipoprotein C-I pTOPO containing a DNA sequence encoding human apolipoprotein C-I used as a raw material plasmid in the construction of the recombinant human apolipoprotein C-I expression vector shown in FIG. -ApoCI was constructed.
(1) Acquisition of human apolipoprotein CI gene Total RNA was extracted from human liver cancer cell line HepG2 (ATCC HB 8065) using ISOGEN (Nippon Gene). Subsequently, reverse transcription reaction was performed using a First-Strand cDNA Synthesis Kit (GE Healthcare Bioscience) according to the recommended protocol to obtain single-stranded cDNA derived from poly (A) -containing mRNA.
(2) Cloning of human apolipoprotein CI gene (pTOPO-ApoCI
Construction)
A 5 ′ terminal primer (SEQ ID NO: 1) and a 3 ′ terminal primer (SEQ ID NO: 2) are designed from the sequence of Homo Sapiens Apolipoprotein CI (NCBI NM001645), and PCR using the single-stranded cDNA obtained in (1) above as a template is performed. went. These primers are provided with an NdeI restriction enzyme site for the 5 ′ end primer and a BamHI restriction enzyme site for the 3 ′ end primer for subsequent expression vector construction. That is, using 1 unit of KODplus (Toyobo Co., Ltd.) and 5 μL of the attached 10 × PCR buffer, it becomes 0.25 μM oligonucleotides of SEQ ID NO: 1 and SEQ ID NO: 2, 0.2 mM dNTPs, 1.5 mM MgSO ₄ , 5% DMSO. The reaction solution was prepared as described above, and after heat treatment at 98 ° C. for 1 minute, an extension reaction was performed 30 times at 94 ° C. for 20 seconds, 65 ° C. for 90 seconds, 72 ° C. for 40 seconds, and 74 ° C. for 5 minutes. Subsequently, the obtained reaction product was cloned using Zero Blunt TOPO PCR Cloning Kit (Invitrogen) according to the attached protocol. As a result of sequence analysis using M13 primer M4 and M13 primer RV (Takara Bio Inc.), it was confirmed that the cloned DNA fragment had the sequence shown in SEQ ID NO: 3 and was a human apolipoprotein CI gene. .

B. Construction of Expression Vector As shown in FIG. 1, a plasmid pUC-Trx having a DNA sequence encoding lac promoter derived from E. coli, thioredoxin (Trx), 6His tag and thrombin cleaving peptide was prepared, while pTOPO constructed in A above. -Using ApoCI to obtain from each plasmid a fragment containing a DNA sequence encoding the lac promoter, thioredoxin, 6His tag and thrombin cleaving peptide and a fragment containing a DNA sequence encoding human apolipoprotein CI, The desired recombinant plasmid expression vector pUC-Trx-ApoCI was constructed by ligating the fragments. Similarly, a recombinant plasmid expression vector pET-Trx-ApoCI was constructed from pET-Trx containing a T7 promoter derived from T7 phage.

(1) Construction of a plasmid (pET-Trx) containing a T7 promoter, a thioredoxin-encoding DNA sequence, a 6His tag and a thrombin cleaving peptide From pET24c (Novagen), a DNA sequence encoding thioredoxin (Trx) derived from E. coli, 6His In order to obtain a DNA fragment of the thioredoxin region (366th to 692nd) containing a tag (6His) and a thrombin cleavage peptide (Thrombin cleavage seq.), A thioredoxin 5 ′ end primer to which an NcoI restriction enzyme site was added (SEQ ID NO: 4 ), PCR was performed with a 3′-end primer (SEQ ID NO: 5) to which a 6 × histidine sequence serving as a purification tag was added. Further, a 3 ′ end primer (SEQ ID NO: 6) was added to the 3 ′ end of the resulting product to give a thrombin protease cleavage sequence, an NdeI restriction enzyme site, and a BamHI restriction enzyme site for cloning into an expression vector was added 3 'A terminal primer (SEQ ID NO: 7) was designed and subjected to extension reaction by PCR. That is, using 1 unit of KOD plus (Toyobo Co., Ltd.), 5 μL of the attached 10 × PCR buffer, oligonucleotides of 0.25 μM SEQ ID NO: 4 and SEQ ID NO: 5, 0.2 mM dNTPs, 1.5 mM MgSO ₄ , 5% DMSO A reaction solution was prepared as described above, and after heat treatment at 98 ° C. for 1 minute, an extension reaction was performed at 74 ° C. for 5 minutes by 30 thermal cycles of 94 ° C. for 20 seconds, 65 ° C. for 90 seconds, and 72 ° C. for 40 seconds. Similarly, PCR using the oligonucleotides of SEQ ID NO: 4 and SEQ ID NO: 6, and then SEQ ID NO: 4 and SEQ ID NO: 7 was performed on the 1000-fold diluted solution of the obtained PCR product. The PCR product finally obtained was treated with NcoI and BamHI, and ligated using Ligation-Convenience Kit (Nippon Gene) with similarly treated pET21d (+) containing T7 promoter. PET-Trx containing the T7 promoter, Trx (SEQ ID NO: 8), 6His and thrombin cleavage peptide was constructed.

(2) lac promoter, DNA sequence encoding thioredoxin, 6His tag and
Construction of plasmid (pUC-Trx) containing thrombin cleavage peptide and thioredoxin 5 ′ end primer (SEQ ID NO: 9) provided with HindIII restriction enzyme site, 3 ′ end primer (SEQ ID NO: 10) provided with EcoRI restriction enzyme site PCR was performed using pET-Trx constructed in (1) above as a template. That is, using 1 unit of KOD plus (Toyobo Co., Ltd.), 5 μL of the attached 10 × PCR buffer, oligonucleotides of 0.25 μM SEQ ID NO: 4 and SEQ ID NO: 5, 0.2 mM dNTPs, 1.5 mM MgSO ₄ , 5% DMSO A reaction solution was prepared as described above, and after heat treatment at 98 ° C. for 1 minute, an extension reaction was performed at 74 ° C. for 5 minutes by 30 thermal cycles of 94 ° C. for 20 seconds, 65 ° C. for 90 seconds, and 72 ° C. for 40 seconds. Next, the obtained PCR product was digested with HindIII and EcoRI to obtain a fragment containing a DNA sequence encoding thioredoxin, a 6His tag and a thrombin cleavage peptide, and subjected to a subsequent ligation reaction.
On the other hand, after inserting the oligonucleotide shown in SEQ ID NO: 11 into the NdeI site of pUC19 containing the lac promoter, this was treated with HindIII and EcoRI, and ligated with the PCR product obtained above to construct pUC-Trx did.

(3) Fusion of human apolipoprotein CI and thioredoxin controlled by T7 promoter
Construction of protein expression vector (pET-Trx-ApoCI) The human apolipoprotein CI gene was excised by digesting the pTOPO-ApoCI plasmid of A with NdeI and BamHI, and the NdeI and BamHI restriction enzyme sites of pET-Trx. PET-Trx-ApoCI was constructed by inserting in between. A schematic diagram of pET-Trx-ApoCI is shown as a in the upper right of FIG.
(4) Human apolipoprotein CI and thioredoxin fusion by lac promoter control
Construction of Combined Protein Expression Vector (pUC-Trx-ApoCI) As in (3) above, the human apolipoprotein CI gene was excised by digesting the aforementioned pTOPO-ApoCI plasmid with NdeI and BamHI, and pUC-Trx PUC-Trx-ApoCI was constructed by inserting it between NdeI and BamHI restriction enzyme sites. A schematic diagram of pUC-Trx-ApoCI is shown as b in the lower right of FIG.

Example 2
Production of recombinant human apolipoprotein C-I The recombinant expression plasmid constructed in Example 1 was introduced into Escherichia coli to produce human apolipoprotein C-I.
(1) Contains a recombinant plasmid containing DNA encoding human apolipoprotein CI
Preparation of transformants to be used E. coli was transformed using pET-Trx-ApoCI and pUC-Trx-ApoCI, which are recombinant plasmids having the DNA sequence encoding human apolipoprotein CI constructed in B of Example 1. Transformation was performed by the method. In pET-Trx-ApoCI, B strain λDE3 lysogen BL21 (DE3) or C41 (DE3), which functions as a T7 promoter, is transformed into pET-Trx-ApoCI / BL21 (DE3) and pET-Trx-ApoCI. / C41 (DE3) was prepared. In pUC-Trx-ApoCI, pUC-Trx-ApoCI / JM109 and pUC-Trx-ApoCI / BL21 were prepared by transforming K12 strain JM109 or B strain BL21 not incorporating λDE3.
Each transformant was screened for drug resistance on LB plate medium (polypeptone 1%, yeast extract 0.5%, sodium chloride 1%, glucose 1%, carbenicillin 100 μg / mL). TB medium (polypeptone 1.2%, yeast extract 2.4%, glycerol 0.4%, 17 mM potassium dihydrogen phosphate, 72 mM dipotassium hydrogen phosphate, glucose 2%, carbenicillin 200 μg / mL) IPTG (isopropylthio-β-D-galactopyranoside), which is a promoter inducer, was added to the mid-logarithmic growth phase (OD600 of culture solution = 0.4 to 0.6) to induce expression. The cell pellet obtained by centrifugation of the culture solution was suspended in 50 mM potassium phosphate buffer (pH 7.5) and subjected to ultrasonic crushing. The supernatant obtained by centrifugation of this suspension was used as the soluble fraction. On the other hand, the precipitated portion was resuspended and solubilized in 8M urea and sodium phosphate buffer (pH 7.4). The supernatant portion obtained by centrifugation of this suspension was used as the insoluble fraction.

  Recombinant human apoliposome containing a 6 × histidine sequence serving as a purification tag by a batch method using Ni Sepharose 6 Fast Flow (GE Healthcare Bioscience) for the soluble fraction and the insoluble fraction of each of the above transformants. The thioredoxin fusion protein of protein C-I was detected. Based on the attached protocol, the eluate from Ni Sepharose 6 Fast Flow resin was analyzed by Tricine SDS-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE). The results are shown in FIG. As can be seen from FIG. 2, a 6 × histidine sequence having an estimated molecular weight of 20 KDa only in the insoluble fraction obtained from pUC-Trx-ApoCI / JM109 and pUC-Trx-ApoCI / BL21 having the lac promoter derived from E. coli as an expression vector. A band of a thioredoxin fusion protein of recombinant human apolipoprotein C-I containing the protein was confirmed by Coomassie brilliant blue (CBB) staining. On the other hand, in the combination of the vector and the E. coli strain shown below, no protein band containing recombinant human apolipoprotein CI could be confirmed. That is, pET-Trx-ApoCI / C41 (DE3) having a T7 promoter, pET-Trx-ApoCI / BL21 (DE3), pET-ApoCI / C41 (DE3) not containing thioredoxin, pET-ApoCI / BL21 (DE3), In the case of the combination of pUC-ApoCI / JM109 and pUC-ApoCI / BL21 having lac promoter and not containing thioredoxin, no protein band containing recombinant human apolipoprotein CI could be confirmed.

(2) Production and purification of recombinant human apolipoprotein C-I The above TB medium (polypeptone 1.2%, yeast extract 2.4%, glycerol 0.4%, 17 mM potassium dihydrogen phosphate, 72 mM hydrogen phosphate diphosphate) 30 mL of the pUC-Trx-ApoCI / BL21 culture solution previously cultured overnight at 37 ° C. in potassium, glucose 2%, carbenicillin 200 μg / mL) was inoculated into 3 L TB medium that had been autoclaved, and the incubator (Maruhishi Bioengineering) ) At 37 ° C. with aeration and stirring. After culturing for 6 hours, 3 mL of 1M IPTG (isopropylthio-β-D-galactopyranoside) was added, and the aeration and agitation culture was continued at 37 ° C. for another 4 hours. After completion of the culture, the whole culture was collected by centrifugation and frozen at -20 ° C. The frozen cell pellet was suspended in 300 mL of 50 mM potassium phosphate buffer (pH 7.5) and subjected to ultrasonic crushing. The supernatant obtained by centrifugation of this suspension was used as the soluble fraction. On the other hand, the precipitated portion was resuspended and solubilized in 300 mL of 8M urea and sodium phosphate buffer (pH 7.4). The supernatant portion obtained by centrifugation of this suspension was used as the insoluble fraction.

  In the purification of recombinant human apolipoprotein C-I from the insoluble fraction, it is possible to use a method such as dilution, dialysis, and a desalting column to remove urea as a denaturing agent. The denaturant was removed and the denatured protein was refolded. That is, a HisTrapFF 5 mL affinity column (GE Healthcare) equilibrated with buffer 1 (50 mM sodium phosphate, 0.5 M sodium chloride, 4 M urea pH 7.8) under the control of AKTA purifier chromatography system (GE Healthcare Bioscience). 50 mL of the above insoluble fraction sample was applied to Bioscience), the unbound fraction was separated with buffer 1, and buffer 1 was combined with buffer 2 (50 mM sodium phosphate, 0.5 M sodium chloride, 20 mM imidazole pH 7.4). The column eluate was removed of urea by performing a linear gradient of 100% → 0% during Next, after collecting the fraction eluted with buffer 2, by performing 0 → 100% linear gradient with buffer 3 (50 mM sodium phosphate, 0.5 M sodium chloride, 500 mM imidazole pH 7.4), The target protein was eluted. Each eluted fraction was analyzed by Tricine-SDS-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE). The results are shown in FIG. As can be seen from FIG. 3, in the buffer 2 and (3) elution fractions, the band of recombinant human apolipoprotein C-I thioredoxin fusion protein containing a 6 × histidine sequence having an estimated molecular weight of 20 KDa is coomassie brilliant blue (CBB). ) Confirmed by staining. At this stage, a recombinant human apolipoprotein C-I thioredoxin fusion protein containing a soluble 6x histidine sequence already showing a purity of 80% or more was obtained.

Subsequently, thrombin protease was allowed to act in order to remove the thioredoxin and histidine tag from the purified recombinant protein obtained. That is, 1 U of thrombin protease per 1 mg of recombinant human apolipoprotein C-I thioredoxin fusion protein containing a soluble 6x histidine sequence as a substrate was allowed to act and incubated overnight at room temperature. Immediately after dialysis against buffer 2 (50 mM sodium phosphate, 0.5 M sodium chloride, 20 mM imidazole pH 7.4), it was applied to HisTrapFF 5 mL affinity column and HiTrap Benzamidine FF 5 mL (GE Healthcare Biosciences). Both passing-through fractions were collected. Further, the buffer was replaced with PBS by a desalting column, followed by concentration to obtain purified recombinant human apolipoprotein CI.
The obtained purified recombinant human apolipoprotein CI is analyzed by Tricine-SDS-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE), and the purity is 95% or more by Coomassie Brilliant Blue (CBB) staining. Was confirmed, and Western blotting using a commercially available goat anti-human human apolipoprotein CI antibody was performed by a conventional method to confirm its antigenicity. These results are shown in FIG. As a quantitative determination of the recombinant human apolipoprotein CI, the Lowry method using BSA as a standard substance was performed, and about 3 mg of the recombinant human apolipoprotein CI was obtained from one batch of the 3L culture.

Example 3
Preparation of monoclonal antibody using recombinant human apolipoprotein C-I as immunogen The purified recombinant human apolipoprotein C-I obtained in Example 3 was used as the immunogen and purified natural apolipoprotein C-I derived from human plasma In order to compare the performance of the obtained antibody with the cases described above, monoclonal antibodies were prepared using both of them as antigens. The production of a monoclonal antibody using recombinant human apolipoprotein C-I as an immunogen is described below, but purified natural apolipoprotein C-I derived from human plasma was subjected to the same operation using a separate body of the same mouse. It was.
(1) Immunization of mouse with purified recombinant human apolipoprotein CI Prepared recombinant human apolipoprotein CI with PBS (pH 7.4) so as to be 1 mg / mL. It was mixed well with a band (Wako Pure Chemical Industries) until emulsified. 50 μL of the prepared suspension was intraperitoneally administered to Balb / c 6-week-old female mice (CLEA Japan, Inc.) under diethyl ether anesthesia. Two weeks later, the same amount of recombinant human apolipoprotein C-I was mixed with Freund's incomplete adjuvant (Wako Pure Chemical Industries, Ltd.) to give an emulsified suspension by exactly the same operation as Freund's complete adjuvant. Each mouse was sensitized. Thereafter, the same operation was carried out 2 weeks later, and in the fourth round, recombinant human apolipoprotein CI was prepared as PBS (pH 7.4) so as to be 1 mg / mL as the final immunization, and 50 μL was injected by mouse tail vein injection. Administered.

(2) Establishment of hybridoma Three days after the final immunization, the spleen surgically removed under anesthesia with diethyl ether from mice sensitized with recombinant human apolipoprotein C-I was aseptically dispersed to prepare spleen cells. The fusion was performed according to the method of Köhler and Milstein (Nature. 256, 495.1975), and spleen cells and myeloma cells P3-X63-Ag8-U1 (P3U1) were obtained using polyethylene glycol (PEG 4000) (Merck). Fused. The fusion ratio at the time of recombinant human apolipoprotein CI fusion was the number of spleen cells: myeloma cells P3U1 = (2: 1) to (5: 1). The fused cells were dispersed in 10% FCS (Invitrogen), α-MEM (IRVINE), and HAT (Cosmo Bio) medium and dispensed into a 96-well microtiter culture plate (Sumitomo Bakelite) at 37 ° C, 5%. Cultivation was performed under CO2 conditions.
(3) Preparation of antigen plate and BSA plate As an antigen plate for screening, purified natural human apolipoprotein CI (natural ApoC-I) derived from human plasma was sensitized as an antigen. That is, human apolipoprotein CI (Athens Research & Technology) derived from human plasma is dissolved in PBS (pH 7.4) and added to a 96-well microtiter plate (Nunc) so as to be 1 μg / 100 μL / well. Dispensed. The plate was allowed to stand at 4 ° C. for 2 nights and then washed 3 times with PBS containing 0.05% Tween 20 (Wako Pure Chemical Industries, Ltd.) to suppress nonspecific reaction. 1.5% BSA (SIGMA) And 200 μL of a blocking solution containing 10% saccharose (Wako Pure Chemical Industries, Ltd.) was further placed at 4 ° C. overnight.
Furthermore, in order to investigate non-specific reaction with BSA, a BSA plate was also prepared. That is, 200 μL of a blocking solution containing 1.5% BSA (SIGMA) and 10% saccharose (Wako Pure Chemical Industries, Ltd.) was dispensed onto a 96-well microtiter plate (Nunc) without sensitizing the antigen solution. Furthermore, it stood still at 4 degreeC overnight.

(4) Colony screening After about 2 weeks, colony growth was confirmed and screening was performed. The screening method performed is described below.
The antigen plate prepared in (3) above was washed three times with PBS containing 0.05% Tween 20 (Wako Pure Chemical Industries, Ltd.), and then 100 μL of the culture supernatant of the three hybridomas obtained in (2) above. After further washing, HRP-labeled anti-mouse immunoglobulin antibody (Zymed), which is a secondary antibody, was added and reacted. After washing, 100 μL of 3 mg / mL o-phenylenediamine (OPD) (Nacalai) citric acid coloring solution, which is an HRP coloring substrate, was added for a certain period of time, and then 1N sulfuric acid (Wako Pure Chemical Industries) was further used as a stop solution. 100 μL was added, and the absorbance was measured at a measurement wavelength of 492 nm. The results are shown in Table 1.
As shown in Table 1, when the immunogen was purified recombinant human apolipoprotein CI (recombinant ApoC-I), three types of recombinant ApoC-I monoclonal antibodies were used, and the immunogen was purified from human plasma. When natural apolipoprotein C-I (natural ApoC-I) was used, three types of natural ApoC-I monoclonal antibodies could be obtained, but the recombinant ApoC-I monoclonal antibody was not specific to BSA. It was found that the natural ApoC-I monoclonal antibody had a large non-specific reaction compared to the low reaction.

  The hybridoma (Mouse-Mase hybridoma AMA-1) producing the recombinant ApoC-I antibody (clone 3) obtained above was obtained from the National Institute of Advanced Industrial Science and Technology (AIST) on August 1, 2007. And has been given FERM ABP-10889 as the receipt number.

  By using a promoter derived from E. coli such as Lac promoter as a promoter, using E. coli as a host cell, and expressing it as a fusion protein of an apolipoprotein and a water-soluble protein such as thioredkin that can be expressed in E. coli. Of recombinant apolipoprotein is obtained. By preparing an antibody against apolipoprotein C-I using the high-purity recombinant apolipoprotein C-I obtained by this method as an antigen, an antibody with high specificity to natural apolipoprotein C-I can be obtained. Obtainable. Using this antibody, apolipoprotein C-I in a specimen can be accurately measured by an immunoassay. Therefore, by using an antibody against recombinant apolipoprotein C-I as a clinical test agent, Stroke, sepsis, tumor, heart disease, possibility of Crohn's disease, etc. can be accurately diagnosed.

The construction of an expression vector for expressing a fusion protein of human apolipoprotein C-1 and thioredkin is shown. The detection result of the fusion protein of human apolipoprotein C-1 and thioredkin expressed by the expression vector is shown. The refolding by affinity chromatography and the purification result of the fusion protein of human apolipoprotein C-1 and thioredkin expressed by the expression vector are shown. The results of Tricine-SDS-polyacrylamide electrophoresis and Western blotting of purified recombinant human apolipoprotein CI are shown.

Claims (14)

  A recombinant plasmid expression vector having a promoter derived from E. coli, a DNA sequence encoding a water-soluble protein that can be expressed in E. coli, and a DNA sequence encoding apolipoprotein is introduced into E. coli, and the introduced E. coli is cultured. A method for producing a recombinant apolipoprotein comprising expressing a fusion protein of a water-soluble protein that can be expressed in E. coli and apolipoprotein, and obtaining apolipoprotein from the fusion protein.   The method for producing a recombinant apolipoprotein according to claim 1, wherein the apolipoprotein is apolipoprotein CI or a variant thereof.   The method for producing a recombinant apolipoprotein according to claim 1 or 2, wherein the water-soluble protein that can be expressed in Escherichia coli is thioredoxin or a variant thereof.   The method for producing a recombinant apolipoprotein according to any one of claims 1 to 3, wherein the promoter derived from E. coli is a lac promoter or a tac promoter.   The method for producing a recombinant apolipoprotein according to any one of claims 1 to 4, wherein the recombinant plasmid expression vector further comprises a DNA sequence encoding a purified peptide for purifying the expressed fusion protein.   The method for producing a recombinant apolipoprotein according to any one of claims 1 to 5, wherein the recombinant plasmid expression vector further comprises a DNA sequence encoding a protease cleaving peptide for releasing apolipoprotein from the expressed fusion protein.   A recombinant plasmid expression vector having a promoter derived from E. coli, a DNA sequence encoding a water-soluble protein that can be expressed in E. coli, and a DNA sequence encoding apolipoprotein.   Recombinant apolipoprotein CI or a variant thereof.   Recombinant apolipoprotein CI of Claim 8 obtained by the manufacturing method in any one of Claim 1 to 6, or its variant.   An antibody against recombinant apolipoprotein C-I obtained by administering the recombinant apolipoprotein C-I of claim 8 or 9 to a mammal as an immunogen.   11. The antibody according to claim 10, which has a higher specificity for natural apolipoprotein C-I than an antibody obtained by administering natural apolipoprotein C-I to a mammal as an immunogen.   The antibody according to claim 10 or 11, wherein the antibody is a monoclonal antibody.   A method for measuring apolipoprotein C-I, comprising measuring apolipoprotein C-I in a specimen by immunoassay using the antibody against recombinant apolipoprotein C-I according to any one of claims 10 to 12.   A kit for measuring apolipoprotein CI, comprising an antibody against the recombinant apolipoprotein CI according to any one of claims 10 to 12.