JP2019004705A - Method for producing limulus reagent, and method for measuring endotoxin - Google Patents

Abstract

To provide a method for preparing a limulus reagent containing recombinant C factor, recombinant B factor, and recombinant proclotting enzyme for measuring endotoxin more simply and highly sensibly.SOLUTION: A method for producing a limulus reagent in which a transformant of Trichoplusia ni which is allowed to hold a gene coding C-factor derived from horseshoe crab, a transformant of Trichoplusia ni which is allowed to hold a gene coding B-factor derived from horseshoe crab, and a transformant of Trichoplusia ni which is allowed to hold a gene coding proclotting enzyme derived from horseshoe crab are severally cultured, an obtained cultured product is used as a raw material, and a limulus reagent containing recombinant C-factor, recombinant B-factor, and recombinant proclotting enzyme is prepared.SELECTED DRAWING: None

Description

  The present invention relates to a method for producing a Limulus reagent in which Factor C, Factor B and procoagulase are all recombinant proteins, and a method for measuring endotoxin using the Limulus reagent.

  Endotoxin is a lipopolysaccharide that constitutes the cell wall of Gram-negative bacteria and is a very powerful toxin. For this reason, detection of endotoxin contamination in water, pharmaceuticals, foods and drinks, etc. is very important. The detection of endotoxin is generally performed using a lysate reagent prepared from a blood cell extract component (amebocyte lysate) of American horseshoe crab (Limulus polyphemus).

  This lysate reagent contains factor C and factor B, procoagulase (proclotting enzyme), and coagulogen derived from horseshoe crab. When a lysate reagent is mixed with a sample containing endotoxin, factor C is activated by endotoxin, factor B is activated by this activated factor C, procoagulase is activated by this activated factor B, and a coagulation enzyme ( Clotting enzyme) is generated. Coagulogen in the lysate reagent is hydrolyzed by the coagulation enzyme, resulting in the production of coagulin and the lysate reagent becoming an insoluble gel. By measuring the amount of this gel, the amount of endotoxin can be measured. Alternatively, endotoxin can also be obtained by adding a synthetic substrate in which a coloring dye is bound to the peptide at the digestion site of the coagulation enzyme to the reaction system and measuring the amount of color developed by the coloring dye released from the synthesis substrate by digestion with the coagulation enzyme. Can be measured.

  This lysate reagent must be prepared from American horseshoe crab, which is not preferable from the viewpoint of stable supply and protection of biological resources. Therefore, Factor C, Factor B and procoagulase are synthesized as recombinant proteins using gene recombination techniques, and reagents containing these are used as Limulus reagents (see, for example, Patent Documents 1 and 2). ).

JP 2009-150903 A International Publication No. 2008/004674

  As an expression system for producing a recombinant protein such as factor C, expression systems using Escherichia coli, yeast, insect cells and the like are used. Among these, Sf9 insect cells, which are cultured cells derived from Spodoptera frugiperda, are widely used because they are suitable for mass production and post-expression modifications such as sugar chain modification are suitable for eukaryotic protein expression. ing.

  However, when Sf9 cells are used as hosts, the expression level of factor C and the like is insufficient, and therefore it is difficult to measure and use endotoxin with the culture supernatant obtained by culturing the recombinant cells as it is as a Limulus reagent. If the culture supernatant is used for endotoxin measurement as it is, the enzyme activity of the coagulation enzyme is weak and the endotoxin detection sensitivity is insufficient. For this reason, in order to detect endotoxin with sufficient sensitivity, it was necessary to improve the specific activity by concentrating the recombinant protein from the culture supernatant.

  Therefore, an object of the present invention is to provide a method for producing a Limulus reagent composed of a recombinant protein, and more easily producing a Limulus reagent without impairing endotoxin detection sensitivity.

  The present inventors have intensively studied to achieve the above-mentioned object. As a result, when preparing C, B factors and procoagulase derived from horseshoe crab used as a Limulus reagent as recombinant proteins, the recombinant host is Trichopulcia.・ By using Trichoplusia ni, it was found that even when the culture supernatant was used as it was as a Limulus reagent without being concentrated, a very small amount of 0.001 EU / mL endotoxin could be detected with sufficient sensitivity. The invention has been completed.

That is, the method for producing a Limulus reagent and the method for measuring endotoxin according to the present invention are the following [1] to [6].
[1] A method for producing a Limulus reagent containing recombinant factor C, recombinant factor B and a pre-recombination coagulation enzyme,
(A) a culturing step of culturing a transformant of Trichopulcia ni harboring a gene encoding C factor from horseshoe crab to obtain a culture containing recombinant C factor;
(B) a culture step of culturing a transformant of Trichopulcia ni harboring a gene encoding factor B derived from horseshoe crab to obtain a culture containing recombinant factor B;
(C) culturing a transformant of Trichopulcia ni harboring a gene encoding a procoagulase derived from horseshoe crab to obtain a culture containing the recombinant precoagulase;
(D) Using the culture obtained in the step (a), the culture obtained in the step (b), and the culture obtained in the step (c) as raw materials, the recombinant factor C, A preparation step for preparing a recombinant factor B and a Limulus reagent containing the pre-recombination coagulation enzyme;
A method for producing a Limulus reagent, comprising:
[2] A method for producing a Limulus reagent containing recombinant factor C, recombinant factor B and a pre-recombination coagulase,
Culturing a transformant of Trichopulcia ni harboring a gene encoding factor C derived from horseshoe crab, a gene encoding factor B derived from horseshoe crab, and a gene encoding procoagulase derived from horseshoe crab A culturing step to obtain a culture containing factor C, recombinant factor B, and pre-recombinant precoagulase;
A preparation step of preparing a Limulus reagent containing the recombinant factor C, the recombinant factor B, and the pre-recombinant coagulation enzyme, using the culture obtained in the culture step as a raw material;
A method for producing a Limulus reagent, comprising:
[3] The horseshoe crab-derived C factor is wild-type horseshoe crab C factor or a mutant thereof,
The horseshoe crab-derived factor B is wild-type horseshoe crab factor B or a variant thereof,
The horseshoe crab-derived procoagulation enzyme is a wild-type horseshoe crab precoagulation enzyme or a variant thereof,
The method for producing the Limulus reagent of [1] or [2].
[4] The method for producing a Limulus reagent according to any one of [1] to [3], wherein the culture is a culture supernatant.
[5] A preparation step of preparing a Limulus reagent by the method for producing a Limulus reagent according to any one of [1] to [4],
The test sample is mixed with the Limulus reagent obtained in the preparation step, the substrate of the coagulation enzyme obtained by the conversion of the procoagulase, and the test sample, and based on the enzyme activity of the coagulation enzyme, the endotoxin of the test sample Measuring process for measuring
A method for measuring endotoxin.
[6] The substrate is a synthetic substrate in which a labeling substance is bonded to a peptide that is a substrate of the coagulation enzyme, and the enzyme activity of the coagulation enzyme is released by the degradation of the synthetic substrate by the coagulation enzyme. The method for measuring endotoxin according to the above [5], which is measured based on the labeling substance.

  In the present invention, the productivity of recombinant protein such as factor C can be dramatically improved by using Trichopulcia ni having high protein productivity per unit amount of the culture solution as a recombinant host. Therefore, by using the present invention, endotoxin detection can be performed with sufficient sensitivity even when the culture supernatant of recombinant cells is used as a reagent for direct measurement without performing treatment for improving specific activity such as concentration operation. Can be produced.

In Example 1, it is the dye | stained image of the protein band isolate | separated by SDS-PAGE of the culture supernatant of each recombinant cell. In Example 1, using the Limulus reagent consisting of a recombinant protein obtained by expressing C factor, B factor and procoagulase in Trichopulsia ni cells or Sf9 cells, the test was performed by the luminescent synthetic substrate method. It is the figure which plotted the measured value (RLU) of the emitted light intensity at the time of measuring the endotoxin in a sample for every endotoxin density | concentration in a reaction solution.

  The method for producing a Limulus reagent according to the present invention is a method for producing a Limulus reagent containing recombinant factor C, recombinant factor B, and a pre-recombination coagulation enzyme, as a recombinant host for producing these recombinant proteins. , Using Trichopulcia ni cells. Trichopulcia ni has a much larger cell size than the commonly used SF9 insect cells, and when used as a recombinant host, the amount of recombinant protein produced per unit volume of the culture solution is SF9. Much more than insect cells. In the method for producing a Limulus reagent according to the present invention, the culture supernatant of a recombinant cell is subjected to concentration treatment or the like by using Trichopulcia ni having high protein productivity per unit amount of a culture solution as a recombinant host. A Limulus reagent with sufficient detection sensitivity can be obtained without a treatment for improving the specific activity.

Specifically, the method for producing a Limulus reagent according to the present invention includes the following steps (a) to (d).
(A) a culturing step of culturing a transformant of Trichopulcia ni harboring a gene encoding C factor from horseshoe crab to obtain a culture containing recombinant C factor;
(B) a culture step of culturing a transformant of Trichopulcia ni harboring a gene encoding factor B derived from horseshoe crab to obtain a culture containing recombinant factor B;
(C) culturing a transformant of Trichopulcia ni harboring a gene encoding a procoagulase derived from horseshoe crab to obtain a culture containing the recombinant precoagulase;
(D) Using the culture obtained in the step (a), the culture obtained in the step (b), and the culture obtained in the step (c) as raw materials, the recombinant factor C, A preparation step for preparing a recombinant factor B and a Limulus reagent containing the recombination precoagulation enzyme.

  In the present invention and the specification of the present application, the term “shoe crab” means, for example, horseshoe crab belonging to the genus Tachypleus such as Tachypleus tridentatus and Tachypleus gigas, and Limulus polyphemus. Examples include horseshoe crabs belonging to the genus Limulus, and horseshoe crabs belonging to the genus Carcinoscorpius such as Carcinoscorpius rotundicauda.

  The “gene encoding a horseshoe crab-derived factor C” used in the present invention may be a gene encoding a wild-type protein having the same amino acid sequence as factor C purified from a wild horseshoe crab blood cell extract. It may be a gene encoding a mutant protein (mutant) in which various mutations are introduced into the wild type protein, and other peptides or proteins are fused to the N-terminal or C-terminal of the wild-type protein or mutant. It may be a variant. Similarly, the “gene encoding a horseshoe crab-derived factor B” and the “gene encoding a horseshoe crab-derived procoagulase” used in the present invention are, respectively, factor B purified from a wild horseshoe crab blood cell extract or It may be a gene that encodes a wild-type protein consisting of the same amino acid sequence as the precoagulase, or a gene that encodes a mutant in which various mutations are introduced into the wild-type protein. It may be a modified body in which other peptides or proteins are fused to the N-terminal or C-terminal of the body.

  Any mutants may be used as long as they do not impair each activity. For example, if there is a known one, it may be used, and one obtained by newly modifying the wild type may be used. In the case of a new modification, for example, the expressed C factor, B factor, or the one that can be designed so that the enzyme activity in the active form of the procoagulase is higher than that in the wild type is preferable. For example, an amino acid sequence in which one or several amino acids are deleted, inserted, substituted or added in the wild-type amino acid sequence. Here, “one or several amino acids are deleted, inserted, substituted or added” means deletion, insertion, substitution or addition by a known mutant polypeptide production method such as site-directed mutagenesis. It is intended that as many amino acids as possible (preferably 10 or less, more preferably 7 or less, most preferably 5 or less) are deleted, inserted, substituted or added. In addition, the sequence identity between the wild type and the mutant amino acid sequence is preferably 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more.

  The sequence identity (homology) between amino acid sequences is the alignment obtained by juxtaposing two amino acid sequences side by side with a gap in the portion corresponding to insertion and deletion so that the corresponding amino acids most closely match. It is determined as the ratio of matched amino acids to the entire amino acid sequence excluding the gap in the middle. The sequence identity between amino acid sequences can be determined using various homology search software known in the art.

  The peptide or protein to be fused to wild-type or mutant factor C, factor B, and procoagulase is not particularly limited as long as each activity is not impaired. Examples of the peptide include tags commonly used in the expression and purification of recombinant proteins such as a histidine tag, an HA (hemagglutinin) tag, a Myc tag, and a Flag tag.

  Genes encoding wild type factor C, factor B, and procoagulase are known from literature and databases (for example, EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/embl/)). . Thus, for example, the gene encoding factor B is Biol. Chem. 268, 21384-21388 (1993), the gene encoding factor C is Biol. Chem. 266, 6554-6561 (1991), a gene encoding a procoagulase can be appropriately amplified by a PCR method or the like with reference to the nucleotide sequence information published in International Publication No. 2008/004674 and cloned. . The gene encoding mutant C factor, factor B, and procoagulase may be produced by chemical synthesis based on a base sequence encoding a protein consisting of the target amino acid sequence. It can also be produced by appropriately modifying the gene. In addition, in the genes encoding factor C, factor B, and procoagulase introduced into the transformant used in the present invention, the degenerate codon is changed to one having a high codon usage of the host Trichopulcia ni. It is preferable that

  In the present invention and the present specification, the “gene” is a polynucleotide consisting of DNA or RNA and encoding a protein. Genetic modification can be performed by methods well known to those skilled in the art, such as site-directed mutagenesis, random mutagenesis, and organic synthesis.

  A transformant of Trichopulcia ni harboring a gene encoding C factor derived from horseshoe crab, a transformant of Trichopulcia ni harboring a gene encoding factor B derived from horseshoe crab, and precoagulation derived from horseshoe crab A transformant of Trichopulcia ni harboring a gene encoding the enzyme can be produced by introducing a recombinant vector containing each gene into Trichopulsia ni.

  A recombinant vector containing each gene can be obtained by inserting each gene into a vector capable of expressing the protein of interest in the host Trichopulcia ni cell according to methods well known to those skilled in the art. Each gene is inserted into a vector by digesting a DNA fragment to which an appropriate restriction enzyme recognition sequence has been added to each gene with the corresponding restriction enzyme, and then obtaining the obtained gene fragment into the corresponding restriction enzyme recognition sequence of the vector or It can be performed by inserting into a cloning site and ligating to a vector.

  Examples of vectors that can be used in Trichopulcia ni cells include plasmids derived from baculovirus (pFastBac, pIEX / Bac, pBac, etc.), and chromosomal insertion plasmid vectors (pIZ / V5-His, pIB / V5-His, pIZT). / V5-His, pMIB / V5-His, pIEX, etc.). A commercially available product may also be used. The recombinant vector may be one that causes the protein encoded by the incorporated gene to be transiently expressed in the host cell or one that causes stable expression.

  Each gene is preferably incorporated into a recombinant vector as an expression cassette having a promoter arranged upstream. In the expression cassette, an enhancer, a terminator, a splicing signal, a poly A addition signal, a ribosome binding sequence, and the like can be arranged as necessary. The promoter is not limited as long as it functions in Trichopsisia ni cells, and is derived from the same species as the species from which the protein (factor C, factor B, or procoagulase) encoded by each gene is derived. Promoters derived from different species, or artificially synthesized promoters.

  A recombinant vector containing a gene encoding factor C, factor B, or procoagulase may also contain a selection marker gene for selecting transformed cells and untransformed cells. preferable. Examples of the selection marker gene include drug resistance genes such as a kanamycin resistance gene, a hygromycin resistance gene, and a zeocin resistance gene.

  A transformant of Trichopulcia ni harboring a gene encoding C factor derived from horseshoe crab, a transformant of Trichopulcia ni harboring a gene encoding factor B derived from horseshoe crab, and precoagulation derived from horseshoe crab A transformant of Trichoplusia ni harboring a gene encoding an enzyme can be obtained by introducing a recombinant vector containing each gene into Trichopulsia ni cells which are host cells. Introduction of each recombinant vector into a host cell can be performed by methods well known to those skilled in the art, such as calcium chloride method, electroporation method, polyethylene glycol method, lipofection method, particle gun method and the like.

  A culture containing recombinant C factor can be obtained by culturing a transformant of Trichopulcia ni harboring a gene encoding C factor derived from horseshoe crab. Similarly, a culture containing recombinant B factor is obtained by culturing a transformant of Trichopulcia ni harboring a gene encoding the factor B derived from horseshoe crab, and a coagulant enzyme derived from horseshoe crab is obtained. By culturing a transformant of Trichopulcia ni harboring the gene to be encoded, a culture containing a recombinant precoagulation enzyme can be obtained.

  In the present invention, steps (a), (b) and (c) may be performed independently or simultaneously. In the case of performing simultaneously, for example, a transformant of Trichopulcia ni that carries a gene encoding C factor derived from horseshoe crab, and a transformant of Trichopulsia ni that carries a gene encoding factor B derived from horseshoe crab And a transformant of Trichopulcia ni harboring a gene encoding a procoagulase derived from horseshoe crab in the same medium, recombinant factor C, recombinant factor B, and recombinant A culture containing the procoagulant enzyme is obtained. In addition, a recombinant vector comprising all of a gene encoding a horseshoe crab-derived factor C, a gene encoding a horseshoe crab-derived factor B, and a gene encoding a horseshoe crab-derived procoagulase is constructed. -A transformant having all these three types of genes introduced into D is produced, and the transformant is cultured to obtain recombinant factor C, recombinant factor B, and pre-recombinant coagulase. A culture containing is obtained.

  The “culture” of the transformant used as a raw material for the Limulus reagent according to the present invention may be any of a culture supernatant, a cultured cell, and a cell disrupted product. For example, recombinant factor C, recombinant factor B, and pre-recombinant coagulation enzyme can be obtained by recovering a transformant from the culture by a method well known to those skilled in the art, for example, centrifugation, It can collect | recover from a transformant by performing the ultrasonic crushing process or the extraction process by surfactant or the like. A mixture of the recovered recombinant factor C, recombinant factor B, and coagulation enzyme before recombination as they are may be used as the Limulus reagent according to the present invention, and further purified may be used as the Limulus reagent according to the present invention. As the purification method, for example, ammonium sulfate precipitation, SDS-PAGE, gel filtration chromatography, ion exchange chromatography, affinity chromatography and the like can be carried out alone or in combination as appropriate.

  Recombinant factor C, recombinant factor B, and pre-recombinant coagulation enzyme expressed in each transformant are used to prepare a Limulus reagent in the present invention because at least a part of them is secreted outside the transformant. The "culture containing recombinant C factor", "culture containing recombinant factor B", and "culture containing pre-recombinant precoagulation enzyme" include the culture supernatant of each transformant. preferable. For example, a culture supernatant of a transformant of Trichopulcia ni harboring a gene encoding C factor derived from horseshoe crab, and a transformant of Trichopulcia ni harboring a gene encoding factor B derived from horseshoe crab The Limulus reagent according to the present invention can be prepared by mixing the above culture supernatant with the culture supernatant of a transformant of Trichopulcia ni harboring a gene encoding a coagulase derived from horseshoe crab. A culture supernatant of a transformant having all of a gene encoding a horseshoe crab-derived factor C, a gene encoding a horseshoe crab-derived factor B, and a gene encoding a horseshoe crab-derived procoagulase is obtained according to the present invention. It can be a reagent.

  In the present invention, the recombinant protein is expressed in Trichopulcia ni having high protein productivity per unit amount of the culture solution, so that the culture supernatant obtained by culturing the recombinant cell is concentrated or purified. It can be used as a Limulus reagent as it is without requiring an operation to improve the above.

  The content ratio of the recombinant factor B to the recombinant factor C contained in the Limulus reagent according to the present invention (recombinant factor B / recombinant factor C) is preferably 10/1 to 0.1 / 1 in terms of mass ratio. / 1 to 0.5 / 1 is more preferable. In addition, the content ratio of the pre-recombination coagulation enzyme to the recombinant factor C contained in the Limulus reagent according to the present invention (pre-recombination coagulation enzyme / recombinant factor C) is 10/1 to 0.1 / 1 in mass ratio. Is preferable, and 2/1 to 0.5 / 1 is more preferable.

  The Limulus reagent according to the present invention may appropriately contain other components not derived from the horseshoe crab blood cell extract such as a salt and a pH adjuster.

  Each transformant can be cultured by methods well known to those skilled in the art. The medium used for culturing each transformant is not particularly limited as long as it is generally used for culturing insect cells such as Trichopulcia ni cells and Sf9 cells, and carbon that insect cells can assimilate. Contains sources (glucose, sucrose, lactose, etc.), nitrogen sources (peptone, meat extract, yeast extract, etc.), inorganic salts (phosphate, carbonate, sulfate, etc.), etc. Any medium can be used as long as the medium is a natural medium and a synthetic medium. The culture of each transformant may be any of shaking culture, stirring culture, stationary culture, etc. The culture temperature and culture time are generally the same as the culture conditions for culturing insect cells. It can be carried out.

  When the transformant to be cultured has resistance to an antibiotic (neomycin, zeocin, etc.), it is preferable to add the antibiotic to the medium.

  The Limulus reagent according to the present invention can be used in a luminescent synthetic substrate method (luminescence method). The luminescent synthetic substrate method (luminescent method) is a detection system using a luminescent synthetic substrate, which will be described later, as a substrate of active factor C, active factor B, or a coagulase produced by binding endotoxin to factor C. . In the luminescent synthetic substrate method (luminescent method), first, the luminescent synthetic substrate is hydrolyzed by the protease activity of active factor C, active factor B, or coagulation enzyme, and the luminescent substrate described later is released. Subsequently, the luminescent substrate emits light by allowing a luminescent enzyme described later to act on the luminescent substrate. Since the intensity of luminescence correlates with the concentration of endotoxin, the endotoxin concentration can be calculated by measuring the luminescence intensity.

  That is, the method for measuring endotoxin according to the present invention comprises a preparation step for preparing a Limulus reagent by the method for producing a Limulus reagent, a Limulus reagent obtained in the preparation step, and a coagulation obtained by conversion of the procoagulase. A measurement step of mixing and reacting an enzyme substrate and a test sample, and measuring endotoxin of the test sample based on the enzyme activity of the coagulation enzyme. When endotoxin is contained in the test sample, recombinant factor C is activated by endotoxin, recombinant factor B is activated by activated recombinant factor C, and recombinant precoagulase is activated by activated recombinant factor B. The substrate is decomposed by the protease activity of the resulting clotting enzyme. Thus, when endotoxin is contained in the test sample, the substrate of the clotting enzyme is degraded, and therefore, endotoxin in the test sample can be measured using the degradation amount of the substrate as an index.

  As the substrate, a synthetic substrate obtained by binding a labeling substance to a peptide serving as a substrate for a coagulation enzyme can be used. Such a synthetic substrate is decomposed by a clotting enzyme converted from a pre-recombination clotting enzyme, thereby releasing a labeling substance. The enzyme activity of the coagulation enzyme is measured based on the released labeling substance. The more endotoxin in the test sample, the higher the enzyme activity of the coagulation enzyme converted from the precoagulation enzyme, and the more the labeling substance released.

  As the labeling substance, a luminescent substrate is preferable because it is relatively easy to distinguish a semi-quantitative measurement by distinguishing a substrate before digestion with a coagulation enzyme and a labeling substance released by digestion. A luminescent substrate is a substrate of a luminescent enzyme, and is a substance that bioluminescents by the action of the luminescent enzyme. Examples of the luminescent substrate include luciferin. The kind of luminescent substrate may be individual and may be a combination of 2 or more types. As a labeling substance contained in a substrate before digestion by a coagulation enzyme used in the present invention, since an amino group can form an amide bond with a carboxyl group of a peptide, it is easy to bind a peptide to a luminescent substrate. Luciferin is preferred.

  The peptide serving as a substrate for the coagulation enzyme may be any peptide having an amino acid sequence that is cleaved by the protease activity of the coagulation enzyme, with a labeling substance bonded to the C-terminal by an amide bond. The number of amino acid residues and the amino acid sequence of the peptide are not limited, but the number of amino acid residues is preferably 2 to 10 from the viewpoints of specificity, synthesis cost, ease of handling, and the like. Moreover, the kind of the said peptide may be individual, and 2 or more types of combinations may be sufficient as it.

  Specifically, examples of the peptide having a coagulation enzyme recognition sequence include Gly-Val-Ile-Gly-Arg- (SEQ ID NO: 1), Val-Leu-Gly-Arg- (SEQ ID NO: 2), Leu-Arg- Arg- (SEQ ID NO: 3), Ile-Glu-Gly-Arg- (SEQ ID NO: 4), Leu-Gly-Arg- (SEQ ID NO: 5), Val-Ser-Gly-Arg- (SEQ ID NO: 6), Val- Gly-Arg- (SEQ ID NO: 7) and the like can be mentioned. The N-terminus of the peptide may be protected with a protecting group. Any protecting group that can be used in this field can be used without limitation. Specific examples include N-succinyl group, tert-butoxycarbonyl group, benzoyl group, p-toluenesulfonyl group and the like.

The synthetic substrate used in the present invention may be a commercially available one or a synthesized one. As a commercially available synthetic substrate (luminescent synthetic substrate) whose labeling substance is a luminescent substance, for example, a luminescent synthetic substrate (benzoyl--) attached to “Proteasome-Glo ^™ Assay Systems” commercially available from Promega. Leu-Arg-Arg-aminoluciferin). Examples of the synthesis method include the method described in JP-T-2005-530485 (International Publication No. 2003/066611).

  The luminescent enzyme that emits light from the luminescent substrate released from the luminescent synthetic substrate may be any substance that catalyzes the bioluminescence of the released luminescent substrate and generates light, and examples thereof include luciferase and aequorin. The luminescent enzyme may be a natural protein or a recombinant protein. The luminescent enzyme may be a commercially available product or a purified product. The luminescent enzyme may be used alone or in combination of two or more.

  When the luminescent substrate is luciferin, the luminescent enzyme is preferably luciferase. When the luminescent substrate is aminoluciferin, beetle-derived luciferase is preferable as the luminescent enzyme. As the beetle-derived luciferase, beetle-derived luciferases such as North American fireflies, Genji fireflies, Heike fireflies, Japanese fireflies, Japanese fireflies, wild fireflies, bots, light beetles, railroad insects, and the like are preferable.

  The luciferase may be a wild type or a mutant type. The amino acid sequence of wild-type luciferase and the gene encoding it are known, and can be known by appropriately referring to the database. For example, the base sequence of the North American firefly luciferase gene (cDNA) is registered in a database (for example, EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/embl/)) as an accession number: M15077. .

  Examples of the amino acid sequence different from the wild type amino acid sequence include an amino acid sequence in which one or several amino acids are deleted, inserted, substituted or added in the wild type amino acid sequence. Here, “one or several amino acids are deleted, inserted, substituted or added” means deletion, insertion, substitution or addition by a known mutant polypeptide production method such as site-directed mutagenesis. It is intended that as many amino acids as possible (preferably 10 or less, more preferably 7 or less, most preferably 5 or less) are deleted, inserted, substituted or added. Further, the sequence identity between the wild-type and mutant amino acids is preferably 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more.

  Examples of test samples include pharmaceuticals such as injections, infusions, and dialysates; clinical samples such as blood (plasma) and urine; foods; water such as clean water, sewage, river water, and marine water; soil and air; It is done.

  When a luminescent synthetic substrate is used as the substrate, the endotoxin of the test sample is measured by, for example, first adding and mixing a Limulus reagent to the test sample to prepare a reaction solution, and the reaction solution at 20 to 40 ° C. for 5 to 20 Let stand for a minute. Next, a luminescent synthetic substrate is added to the reaction solution, mixed, and allowed to stand at 20 to 40 ° C. for 2 to 10 minutes. Thereafter, a luminescent enzyme is added to and mixed with the reaction solution, and the luminescence intensity of the reaction solution is measured using a bioluminescence measuring device. A commercially available bioluminescence measuring device can be used. Examples of the bioluminescence measuring device include “Lumitester (registered trademark) C1000” (manufactured by Kikkoman Corporation).

  The concentration of the luminescent synthetic substrate is preferably 0.1 to 100 μM, more preferably 1 to 20 μM as the concentration after adding the substrate reagent to the reaction solution. If the concentration of the luminescent synthetic substrate is equal to or higher than the lower limit value, sufficient detection sensitivity can be obtained, and if it is equal to or lower than the upper limit value, the material cost can be suppressed.

In addition, in a luminescent reaction using luciferin as a luminescent synthetic substrate and luciferase as a luminescent enzyme, adenosine triphosphate (ATP) and a divalent metal ion are required. For this reason, before adding a luminescent enzyme with a luminescent enzyme, ATP and a bivalent metal ion are mixed with the reaction liquid in an appropriate amount. The concentration of ATP is preferably 10 ⁻⁷ to 10 ⁻³ M, more preferably 10 ⁻⁶ to 10 ⁻⁴ M, after adding the luminescent enzyme to the reaction solution. If the ATP concentration is not less than the lower limit, ATP can be prevented from being insufficient in the enzyme reaction. On the other hand, if the ATP concentration is not more than the upper limit, the material cost can be suppressed, and inhibition of the enzyme reaction by excessive ATP can be prevented. I can prevent it.

  The concentration of the luminescent enzyme is preferably 1 ng / μL to 10 μg / μL, more preferably 10 ng / μL to 100 ng / μL, as the concentration after adding the luminescent enzyme to the reaction solution. If the concentration of the luminescent enzyme is not less than the lower limit value, sufficient detection sensitivity can be obtained, and if it is not more than the upper limit value, the material cost can be suppressed.

  EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.

[Example 1]
Factor C, factor B, and procoagulase were expressed in Trichopulcia ni cells or Sf9 cells, and the sensitivity of the resulting Limulus reagent consisting of the recombinant protein was compared.

<Preparation of Limulus reagent>
Genes encoding factor C, factor B and procoagulase were amplified by PCR from a cDNA library prepared from tachypreus gigas hematopoietic tissue. The base sequence of the amplified gene is accession number: AF467804 for the gene encoding factor C, accession number: D14701 for the gene encoding factor B, and accession number: for the gene encoding procoagulase. The protein coding region of the base sequence registered as M58366 was included.

  Each amplified gene was recombined and cloned into an insect cell expression vector phr512Z to obtain an expression vector. The insect cell expression vector phr512Z is based on the pIEx-5 vector (manufactured by Novagen), the gene encoding ampicillin in the vector is removed, and the pIEx-4 vector (Novagen) is placed where the gene is present. Chromosome recombination designed so that insect cells exhibit zeocin resistance by inserting a DNA obtained by recombining a zeocin resistance gene derived from a pIZT vector (manufactured by Life Technologies Japan) under the IE1 promoter Type expression vector. Here, the expression vector obtained by recombination of the gene encoding factor C is “C51Z”, the expression vector obtained by recombination of the gene encoding factor B is “B51Z”, and the expression vector obtained by recombination of the gene encoding procoagulase Is referred to as “CE51Z”.

  C51Z, B51Z, and CE51Z were introduced into Trichopulcia ni cells by the lipofectin method using Cellfectin (registered trademark) II (manufactured by Life Technologies Japan) to obtain transgenic cells. For culture of Trichopulcia ni cells, medium supplemented with SF 900 III SFM (manufactured by Life Technologies Japan) so that fetal bovine serum is 10% by volume and Zeocin (manufactured by Life Technologies Japan) is 600 ppm. Was used. Each recombinant cell was cultured for 3 weeks to obtain zeocin resistant cells. Here, a zeocin resistant cell expressing factor C is referred to as “C51Z recombinant Trichopulcia ni cell”, a zeocin resistant cell expressing factor B as “B51Z recombinant Trichopulcia ni cell”, and a zeocin expressing procoagulase. Resistant cells were referred to as “CE51Z recombinant Trichopulcia ni cells”. In addition, as a control, each gene was introduced into Sf9 cells in the same manner instead of Trichopulcia ni cells, and a zeocin resistant cell expressing C factor “C51Z recombinant Sf9 cell”, a zeocin resistant cell expressing factor B “ B51Z recombinant Sf9 cells ”and zeocin resistant cells“ CE51Z recombinant Sf9 cells ”expressing procoagulase were obtained.

Each recombinant cell was cultured at 27 ° C. in 50 μL of a culture medium placed in a 250 mL flask, and the culture was terminated when the number of cells reached 5 × 10 ⁶ cells / mL. 10 μL of the culture supernatant of each recombinant cell was applied to each lane and subjected to SDS-PAGE, and the separated protein band was stained. FIG. 1 shows a stained image of the SDS-PAGE band of each culture supernatant. In the figure, “Factor C”, “Factor B”, and “proClotting Enzyme” indicate the lanes of the culture supernatant in which recombinant factor C, recombinant factor B, and recombinant coagulation enzyme are expressed, respectively. Of each lane, “sf9” is a culture supernatant lane of a transformant whose host is Sf9 cell, and “High Five” is a culture supernatant lane of a transformant whose host is Trichopulcia ni cell. , Respectively. As shown in FIG. 1, all of recombinant factor C, recombinant factor B, and pre-recombination coagulase are clearly more apparent in the recombinant protein of Trichopulcia ni cell than in the transformant of Sf9 cell. By using a large amount of Trichopulcia ni cells as a host, the productivity of the recombinant protein per culture broth was greatly improved.

  10 μL of each culture supernatant of C51Z recombinant Trichopulcia ni cells, B51Z recombinant Trichopulcia ni cells, and CE51Z recombinant Trichopulsia ni cells was mixed with Limulus reagent (Trichopulcia ni preparation). did. Similarly, 10 μL of each culture supernatant of C51Z recombinant Sf9 cells, B51Z recombinant Sf9 cells, and CE51Z recombinant Sf9 cells was mixed and used as a Limulus reagent (Sf9 preparation).

<Preparation of luciferase>
Luciferase was obtained by the method described in Japanese Patent Application Laid-Open No. 2007-97577. Specifically, first, a gene encoding a brightened mutant North American firefly luciferase with a histidine tag added to the C-terminus was recombined into an expression vector pET28a (manufactured by Merck) for E. coli. Subsequently, the vector was introduced into Escherichia coli to express a brightened mutant North American firefly luciferase with a histidine tag added to the C-terminus. Next, Escherichia coli was sonicated to prepare a crude extract containing the brightened mutant North American firefly luciferase. Next, the brightened mutant firefly luciferase was purified from the crude extract using Ni Sepharose excel (GE Healthcare). The purified brightened mutant firefly luciferase was dissolved in a solution (pH 8.0) containing 10-5 M ATP, 1 mM magnesium acetate, and 50 mM Tris-Cl to obtain a 250 ng / μL luciferase solution.

<Endotoxin measurement>
For the test sample, endotoxin (endotoxin derived from Escherichia coli O111: B4) (manufactured by Seikagaku Corporation) was diluted with pyrogen-free water and prepared in 5 stages in the range of 10 ⁻⁵ to 10 ⁻¹ EU / mL. Was used. In addition, pyrogen-free water containing no endotoxin was used for the blank.

  As the luminescent synthetic substrate, benzoyl-Leu-Gly-Arg-aminoluciferin (manufactured by AAT Bioquest) was used. The luminescent synthetic substrate was dissolved in a solution (pH 8.0) containing 1 mM magnesium acetate and 50 mM Tris-Cl so as to be 75 μM, and this was used as a substrate solution.

First, 50 μL of Limulus reagent was dispensed into a reaction test tube. Next, 50 μL of the sample was added to the Limulus reagent and mixed for several seconds using a vortex mixer to obtain a reaction solution. The reaction solution was allowed to stand for 10 minutes in a 37 ° C. incubator.
Next, 50 μL of the substrate solution was added to the reaction solution, and the reaction solution was allowed to stand for 5 minutes in a 37 ° C. incubator.
Subsequently, the reaction solution was transferred to a bioluminescence measurement test tube “Lumitube” (manufactured by Kikkoman Corporation), and 50 μL of luciferase solution was added. This was set in a bioluminescence measuring apparatus “Lumitester (registered trademark) C1000” (manufactured by Kikkoman Corporation), and the luminescence intensity was measured.

  FIG. 2 shows a graph in which the results of measurement using each Limulus reagent are plotted with the endotoxin concentration (logarithmic value) on the horizontal axis and the measured value of luminescence intensity (RLU, logarithmic value) on the vertical axis. In the figure, white circles are the results of measurement using Limulus reagent (Trichopulcia ni preparation), and black circles are the result of measurement using Limulus reagent (Sf9 preparation). As shown in FIG. 2, when the culture supernatant of a transformant using a conventional Sf9 cell as a host is used as it is as a Limulus reagent without being concentrated, the luminescence intensity is insufficient at 0.001 EU / mL or less. The endotoxin measurement sensitivity was low. On the other hand, when the culture supernatant of a transformant using Trichopulcia ni cells as a host is used as it is as a Limulus reagent without being concentrated, the luminescence intensity is sufficient even at 0.001 EU / mL or less, A low concentration of endotoxin of 0.00001 EU / mL could also be measured. In particular, the emission intensity at 0.1 EU / mL is 295,680 RLU when the Limulus reagent (Sf9 preparation) is used, and 2,950,000 RLU when the Limulus reagent (Trichopulcia ni preparation) is used. The endotoxin measurement activity was about 10 times higher with the Limulus reagent (Trichopulcia ni preparation) than with the Limulus reagent (Sf9 preparation). Trichopulcia ni cells were larger in size than Sf9 cells, and the productivity of recombinant protein per culture broth was high. Therefore, it was estimated that endotoxin measurement sensitivity was improved. As described above, the culture supernatant of the transformant of Trichoplusia ni that expresses the recombinant protein of factor C, factor B and procoagulase has sufficient sensitivity without requiring treatment for improving specific activity such as concentration. It was confirmed that it can be used as a Limulus reagent.

Claims (6)

A method for producing a Limulus reagent comprising recombinant factor C, recombinant factor B and a pre-recombinant coagulation enzyme,
(A) culturing a transformant of Trichoplusia ni carrying a gene encoding C factor derived from horseshoe crab to obtain a culture containing recombinant C factor;
(B) a culture step of culturing a transformant of Trichopulcia ni harboring a gene encoding factor B derived from horseshoe crab to obtain a culture containing recombinant factor B;
(C) culturing a transformant of Trichopulcia ni harboring a gene encoding a procoagulase derived from horseshoe crab to obtain a culture containing the recombinant precoagulase;
(D) Using the culture obtained in the step (a), the culture obtained in the step (b), and the culture obtained in the step (c) as raw materials, the recombinant factor C, A preparation step for preparing a recombinant factor B and a Limulus reagent containing the pre-recombination coagulation enzyme;
A method for producing a Limulus reagent, comprising: A method for producing a Limulus reagent comprising recombinant factor C, recombinant factor B and a pre-recombinant coagulation enzyme,
Culturing a transformant of Trichopulcia ni harboring a gene encoding factor C derived from horseshoe crab, a gene encoding factor B derived from horseshoe crab, and a gene encoding procoagulase derived from horseshoe crab A culturing step to obtain a culture containing factor C, recombinant factor B, and pre-recombinant precoagulase;
A preparation step of preparing a Limulus reagent containing the recombinant factor C, the recombinant factor B, and the pre-recombinant coagulation enzyme, using the culture obtained in the culture step as a raw material;
A method for producing a Limulus reagent, comprising: The horseshoe crab-derived factor C is wild-type horseshoe crab factor C or a variant thereof,
The horseshoe crab-derived factor B is wild-type horseshoe crab factor B or a variant thereof,
The horseshoe crab-derived procoagulation enzyme is a wild-type horseshoe crab precoagulation enzyme or a variant thereof,
A method for producing the Limulus reagent according to claim 1 or 2.   The method for producing a Limulus reagent according to any one of claims 1 to 3, wherein the culture is a culture supernatant. A preparation step of preparing a Limulus reagent by the method for producing a Limulus reagent according to any one of claims 1 to 4,
The test sample is mixed with the Limulus reagent obtained in the preparation step, the substrate of the coagulation enzyme obtained by the conversion of the procoagulase, and the test sample, and based on the enzyme activity of the coagulation enzyme, the endotoxin of the test sample Measuring process for measuring
A method for measuring endotoxin.   The labeling substance, wherein the substrate is a synthetic substrate in which a labeling substance is bound to a peptide that is a substrate of the coagulation enzyme, and the enzyme activity of the coagulation enzyme is released by the degradation of the synthetic substrate by the coagulation enzyme The method for measuring endotoxin according to claim 5, which is measured based on the above.