JP2009201403A - POLYNUCLEOTIDE ENCODING HUMAN Fc RECEPTOR, AND METHOD FOR PRODUCING HUMAN Fc RECEPTOR UTILIZING THE SAME - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To create a microorganism producing human Fc receptor FcγRI in an industrial scale, and to provide the FcγRI by carrying out the production of the human FcγRI by utilizing the new Fc receptor production system. <P>SOLUTION: The human FcγRI can be expressed in large amount without carrying out the operation for solubilization or the like of the human FcγRI by preparing a polynucleotide in which a codon of the polynucleotide encoding the human FcγRI is converted from a human type to a Bacillus bacterium type, and transforming the plasmid vector into which the polynucleotide is inserted into the Bacillus bacterium. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for expressing a large amount of FcγRI, which is one of human type Fc receptors, by using a bacterium belonging to the genus Bacillus, without performing an operation such as solubilization.

  Fc receptors are a group of molecules that bind to the Fc region of immunoglobulin molecules. Fc receptors are classified according to the type of immunoglobulin to which they bind, and include Fcγ receptors that bind to the Fc region of IgG, Fcε receptors that bind to the Fc region of IgE, Fcα receptors that bind to the Fc region of IgA, etc. Patent Document 1). Each receptor is further classified according to the difference in structure. In the case of an Fcγ receptor, the presence of FcγRI, FcγRII, and FcγRIII has been reported (Non-patent Document 1).

FcγRI, which is one of the Fcγ receptors, is expressed in monocytes and macrophages, and is inducibly expressed in neutrophils by γ interferon (Non-patent Document 1). FcγRI has high binding affinity for IgG, and its equilibrium dissociation constant (Kd) is 10 ⁻⁸ M or less (Non-patent Document 2). FcγRI is divided into an extracellular region, a transmembrane region, and an intracytoplasmic region, and binding to IgG occurs in the Fc region of IgG and the extracellular region of FcγRI, and then a signal is transmitted to the cytoplasm. FcγRI is composed of two types of subunits, an α chain with a molecular weight of about 42000 that is directly involved in binding to IgG, and a γ chain. The γ chain covalently binds at the boundary between the cell membrane and the extracellular region, thereby (Non-Patent Document 3).

  The amino acid sequence of human FcγRI and the gene sequence (SEQ ID NO: 1) are published in public databases such as ExPASy (Primary accession number: P12314). In addition, the functional domain in the structure of FcγRI, the signal peptide sequence for penetrating the cell membrane, and the position of the transmembrane region are also published, and FIG. 1 shows a schematic diagram of the structure of human FcγRI. The amino acid numbers in the figure correspond to the amino acid numbers described in SEQ ID NO: 1. That is, from methionine (Met) of amino acid number 1 of SEQ ID NO: 1 to valine (Val) of 289, extracellular region, leucine (Leu) of amino acid number 290 of SEQ ID NO: 1 to threonine (Thr) of 374 penetrates the cell membrane. Regions and intracellular regions.

  In recent years, the unexpected immunosuppressive biological properties of Fc receptors have attracted attention as pharmaceuticals, particularly in the areas of autoimmune disease or autoimmune syndrome, transplant rejection and malignant lymphoproliferation. Reference 2). Further, the antibody adsorption ability, which is a function of FcγRI, can also be used as a protein responsible for the capture function of various antibody purification chromatography gels.

  The amino acid sequence and gene base sequence of the FcγRIα chain (Non-patent Document 4) were clarified by Janet et al., And then expression using E. coli (Patent Document 1) or animal cells has been reported by genetic recombination techniques. However, in the expression system using E. coli, the expression amount of the extracellular region protein of FcγRI is extremely low, and the expressed protein is expressed in the cell, so that the expressed protein often becomes an insoluble inclusion body. Inclusion body protein can be prepared as an active protein by an operation such as solubilization, but it requires a complicated operation. Moreover, in the system using animal cells, an expression level higher than that of Escherichia coli has been reported (Non-patent Document 3), but it takes a long time to culture and the productivity is not high.

JP-T-2004-530419 J. et al. V. Ravetch et al., Annu. Rev. Immunol. , 9, 457, 1991 Toshiyuki Takai, Jpn. J. et al. Clin. Immunol. , 28, 318, 2005 A. Paetz et al., Biochem. Biophys. Res. Commun. , 338, 1811, 2005 J. et al. M.M. Allen et al., Science, 243, 378, 1989 Nucleic Acid Res. , 30, e43, 2002 Protein Expr Purif. 47, 441-445, 2006 Masa Nagao et al., Biosci. Biotech. Biochem. 61, 670, 1997

  An object of the present invention is to produce a microorganism that produces human Fc receptor FcγRI with high productivity on an industrial production scale. Furthermore, human FcγRI production is performed using a novel Fc receptor production system to provide the human FcγRI.

  As a result of intensive studies on the above problems, the inventors of the present application have converted the human type Fc receptor FcγRI gene codon from a human type into a Bacillus genus bacterial type, and then inserted the converted polynucleotide into an expression plasmid vector. By using a bacterium belonging to the genus Bacillus transformed by the inserted plasmid vector, active FcγRI that does not require solubilization and the like can be directly produced, and the expression level is also dramatically increased. Found to improve.

That is, the present invention includes the following inventions:
(1) The polynucleotide sequence encoding the human type Fc receptor FcγRI shown in SEQ ID NO: 1 is characterized in that the codon of at least the 64th to 867th polynucleotide is converted from a human type to a Bacillus bacterium type A polynucleotide encoding the human type Fc receptor FcγRI.
(2) The polynucleotide encoding the human Fc receptor FcγRI according to (1), wherein the polynucleotide is the polynucleotide of SEQ ID NO: 3.
(3) A plasmid vector into which the polynucleotide according to (1) or (2) is inserted.
(4) A transformant obtained by transforming the plasmid vector according to (3) into a bacterium belonging to the genus Bacillus.
(5) A method for producing a human-type Fc receptor FcγRI polypeptide comprising the step of culturing the transformant according to (4).

  Hereinafter, the present invention will be described in detail.

  The polynucleotide of the present invention comprises at least the 64th to 867th polynucleotides of the polynucleotide sequence encoding FcγRI, which is one of the human Fc receptors shown in SEQ ID NO: 1 (the amino acid number 22 of SEQ ID NO: 1). A polynucleotide encoding a human Fc receptor FcγRI comprising a polynucleotide obtained by converting a codon of alanine (Ala) to 269 valine (Val) encoding a codon from a human type to a Bacillus bacterium type Preferably, the human Fc receptor, wherein the polynucleotide obtained by converting the codons of the 64th to 867th polynucleotides from human to Bacillus is a polynucleotide of SEQ ID NO: 3 Polynucleotides encoding FcγRI It is.

  The polynucleotide obtained by converting the codon of the polynucleotide encoding FcγRI of the present invention from the human type to the Bacillus genus bacterial type is a polynucleotide of at least the 64th to 867th nucleotides of SEQ ID NO: 1 among the genes encoding the human type FcγRI. The rare codon in the Bacillus bacterium present in (1) is converted to a codon that is frequently used in the translation mechanism of the Bacillus bacterium, with the encoded amino acid being the same. The rare codon means a codon that is used less frequently in the host. The frequency of codon usage in the host can be inferred from the analysis result of the base sequence of the genomic gene and the like. For example, as a rare codon in Brevibacterium choshinensis, a kind of bacteria belonging to the genus Bacillus, the amino acid serine (Ser) codon is used. UCA, leucine (Leu) codon CUA, arginine (Arg) codon CGG, AGA, AGG, isoleucine (Ile) codon AUA. Information on codon usage frequency can also be obtained from a public database (http://www.kazusa.or.jp/codon/). Conversion from a rare codon to a frequently used codon is possible by converting the corresponding base sequence, and the base sequence can be converted by a known mutagenesis method such as Site-directed mutationage method. The method is a DNAWorks method (Non-patent document 5) or a Synthetic Gene Designer method (Non-patent document 6) in which a synthetic oligonucleotide and PCR are combined. In the above method, a full-length gene can be prepared by synthesizing an oligonucleotide group consisting of several tens of bases based on the amino acid sequence encoding the polypeptide and assembling the synthetic oligonucleotide by the PCR method. Incidentally, the codon conversion of the human FcγRI-encoding polynucleotide from the human type to the Bacillus genus bacterial type is carried out by removing all rare codons of the human type FcγRI gene sequence (SEQ ID NO: 1) from the human type as described in Examples. It may be converted into a Bacillus bacterium type (SEQ ID NO: 2), or a part of rare codons, for example, a rare codon at positions 64 to 867 in the polynucleotide sequence shown in SEQ ID NO: 1 may be changed from a human type to a Bacillus bacterium type. You may convert (sequence number 3).

  Further, the polynucleotide encoding human FcγRI encodes methionine for initiating transcription on the 5 ′ end side of the polynucleotide obtained by converting the codon of the polynucleotide encoding human FcγRI from the human type to the Bacillus genus bacterial type. May be added, or an oligonucleotide encoding a signal peptide sequence may be added to the 5′-terminal side of the polynucleotide described above. The signal peptide described here is a polypeptide for allowing a protein expressed in the cytoplasm to pass through the cell membrane and be secreted outside the cell membrane, and is usually present on the N-terminal side of the protein and after passing through the cell membrane. Cleaved by specific protease enzymes. Examples of the signal peptide include peptides of amino acid numbers 1 to 15 or 1 to 20 of SEQ ID NO: 1.

  In the present invention, for the purpose of easily purifying human FcγRI, an oligonucleotide encoding a peptide serving as a tag may be added to the polynucleotide described above. Examples of the tag peptide include polyhistidine tag (His-tag), Mick tag (C-myc tag) and the like. The position to be added may be on either the N-terminal side or the C-terminal side as long as the biological activity of the above-mentioned polypeptide is not impaired. Addition of an oligonucleotide encoding a tag peptide to the oligonucleotide described above can be made by genetic engineering by a method well known to those skilled in the art.

  The gene recombinant plasmid vector into which the human type FcγRI polynucleotide in which the codon has been converted from the human type to the Bacillus bacterium type according to the present invention is inserted into the above-described human type FcγRI polynucleotide at an appropriate position of the known expression plasmid vector. By genetic engineering, a recombinant plasmid vector capable of expressing human FcγRI can be obtained. Examples of known expression plasmid vectors include pUB110, pC194, pE194, pWVO1, etc. used for transformation of Bacillus bacteria. The appropriate position mentioned here means a position that does not destroy the replication function of the plasmid vector, a desired antibiotic marker, or a region related to transmissibility. Then, human FcγRI can be expressed by culturing a transformant obtained by transforming the aforementioned recombinant plasmid vector into a bacterium belonging to the genus Bacillus.

  The host cell used for transformation in the present invention may be any bacterium belonging to the genus Bacillus that is the target of codon conversion. Examples include megaterium, Bacillus mecerans, Bacillus coagulans, Bacillus lentus, and the like. In particular, Brevibacillus choshinensis (Non-patent Document 7), in which a spore formation-related gene is destroyed and the intracellular protease gene jmp and extracellular protease gene emp are also preferably used as the host cell. Moreover, the Bacillus bacterium mutant induced | guided | derived by carrying out the mutation process of the bacteria which belong to the said Bacillus genus can also be utilized. Mutation treatment may be performed using a mutating agent well known to those skilled in the art, such as nitrosoguanidine, ethyl methanesulfonate, ultraviolet light, and radiation.

  The procedures and methods for the introduction and expression of foreign genes into Bacillus bacteria of the present invention include those conventionally used in the field of genetic engineering in addition to the methods described in the Examples. Examples of the method include electroporation method and Tris-PEG method.

  The bacterium belonging to the genus Bacillus used for producing human-type FcγRI of the present invention can be grown in a known medium suitable for culturing selected host cells. Any medium can be used as the medium used in the present invention as long as bacteria can grow and produce human FcγRI. The carbon source includes molasses, glucose, fructose, maltose, sucrose, starch, Lactose, glycerol, acetic acid, etc. are used for the nitrogen source, natural ingredients such as corn steep liquor, peptone, yeast extract, meat extract, soybean meal, etc., and amino acids such as ammonium acetate, aspartic acid, glycine, etc. Phosphate and sodium chloride such as monosodium acid, disodium phosphate, monopotassium phosphate, and dipotassium phosphate include magnesium chloride, magnesium sulfate, ferrous sulfate, ferric sulfate, and chloride. Ferrous, ferric chloride, iron citrate, iron iron sulfate, calcium chloride dihydrate, calcium sulfate, zinc sulfate, copper sulfate Copper chloride, manganese sulfate, manganese chloride, and yeast extract as vitamins, biotin, nicotinic acid, thiamine, riboflavin, inositol, pyridoxine, or the like can be used. In a preferred embodiment, the medium may contain a selection agent based on the construction of the expression plasmid vector in order to selectively allow growth of Bacillus bacteria containing the expression plasmid vector. For example, neomycin is added to the medium for the growth of cells expressing the neomycin resistance gene. In addition to carbon, nitrogen and inorganic salt sources, a suitable nutrient source may be added to the medium. If desired, one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate, and dithiothreitol may be included. The culture temperature for Bacillus bacterium growth is about 20 to 40 ° C, preferably 25 to 35 ° C, more preferably about 30 ° C. The pH of the medium is about 5 to 10, preferably 7.0.

  When a promoter derived from the cell wall protein of the host fungus is used for the expression plasmid vector of the present invention, since the growth of the fungus works actively after entering the stationary phase, the turbidity (absorbance at 600 nm) of the culture solution is measured, After the transition from the logarithmic growth phase to the stationary phase, the protein can be secreted and expressed in the culture solution by subsequent culturing. The culture time is 24 to 96 hours, preferably 40 to 50 hours, but the optimum culture time varies depending on conditions such as medium components, culture temperature, and aeration volume, so the expression level and activity of the expressed protein are measured. Is preferably determined.

  In order to obtain the human FcγRI of the present invention from the culture solution, an extraction method may be appropriately selected depending on the expression form. When expressed in the culture supernatant, the cells are separated by centrifugation, and human-type FcγRI may be extracted from the resulting culture supernatant. When expressed in the cytoplasm, the bacterial cells are collected by centrifugation, and the bacterial cells are disrupted by adding an enzyme treatment agent, a surfactant or the like, and human FcγRI can be extracted. In order to separate and purify human FcγRI from the extracted protein, liquid chromatography can be used. Examples of liquid chromatography include ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, affinity chromatography and the like. A high-purity human FcγRI can be prepared by performing a purification operation by combining these chromatographies.

  By immobilizing the purified human FcγRI obtained according to the present invention on the stationary phase, the antibody can be adsorbed and separated from the liquid phase. Examples of the stationary phase include polysaccharides such as cellulose and agarose, glass, ceramics, and plastic materials such as polypropylene, vinyl chloride, and polystyrene. It can also be used as a chromatography gel for antibodies by filling a column with human FcγRI immobilized on a solid phase. Moreover, by using human serum as a specimen, highly specific immunoassay is possible, and it can be used as various diagnostic materials.

  The human type FcγRI can be obtained by converting the codon of the gene encoding the human type Fc receptor FcγRI from a human type to a bacterium belonging to the genus Bacillus, and transforming a plasmid into which the polynucleotide has been inserted into a bacterium belonging to the genus Bacillus. It can be expressed in large quantities without manipulation such as solubilization. This expression system is useful for the production of human FcγRI on an industrial production scale.

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.
Example 1 Design of DNA sequence encoding human Fc receptor Codons were converted to Brevibacillus genus by DNAworks method (Non-patent Document 5) based on the amino acid sequence of human Fc receptor FcγRI described in SEQ ID NO: 1. did. FIG. 2 shows the result of comparing the base sequence of human FcγRI (SEQ ID NO: 2) whose codons were converted by this method with the base sequence of human FcγRI (SEQ ID NO: 1) before codon conversion. As shown in FIG. 2, the DNA sequence was converted without changing the amino acid sequence, and the similarity between the DNA sequences was 75%.
Example 2 Preparation of DNA sequence encoding human Fc receptor A DNA sequence in which the codon of the human FcγRI gene was converted from human to Brevibacillus was prepared by the following method.
(1) 52 types of oligonucleotides for synthesizing a human FcγRI gene codon from a human type to a Brevibacillus genus bacterial type were synthesized. The synthesized oligonucleotides are shown in SEQ ID NOs: 4 to 55.
(2) In order to prepare DNA encoding full-length human FcγRI from the oligonucleotide synthesized in (1), two-step PCR was performed.
(2-1) The reaction solution of the first stage PCR is as shown in Table 1, and the reaction conditions are 94 ° C for 5 minutes after heat treatment, 94 ° C for 30 seconds first step, 62 ° C for 30 seconds second. Step, the third step of 72 ° C. for 1 minute is performed 25 cycles, followed by the fourth step of 72 ° C. for 7 minutes. The DNA mix in Table 1 is a solution in which a fixed amount of each of 52 types of synthesized 50 pmol / μL oligonucleotides was sampled and mixed.

（２−２）二段階目のＰＣＲは一段階目のＰＣＲの反応液を用いて、表２の反応液組成で行なった。ＰＣＲプライマーの配列は、配列番号４（５’−ＡＴｇＴｇｇＴＴＣＴＴｇＡＣＡＡＣＴＣＴＣＣＴｇＣＴＴＴｇｇｇＴＣＣＣ−３’）と配列番号５５（５’−ＡｇＴＡｇＣｇＣＣＴＴｇＣｇｇＴＴＣＴＴＴＡＣｇＡＴｇＣＡＣｇＣＣＣＴＣＣ−３’）のオリゴヌクレオチドを用いた。反応条件は９４℃・５分の熱処理後、９４℃・３０秒間の第一ステップ、６５℃・３０秒間の第二ステップ、７２℃・１分間の第三ステップを２５サイクル行ない、最後に、７２℃・７分の第四ステップである。

(2-2) The second-stage PCR was performed with the reaction liquid composition shown in Table 2 using the first-stage PCR reaction liquid. As the sequence of the PCR primer, the oligonucleotide of SEQ ID NO: 4 (5′-ATgTggTTCTTgACAACTCTCCCTgCTTTggggTCCC-3 ′) and SEQ ID NO: 55 (5′-AgTAgCgCCTTgCggTTCTTTTACgATgCACgCCCTCC-3 ′) was used. The reaction conditions were as follows: heat treatment at 94 ° C. for 5 minutes, 25 cycles of the first step at 94 ° C. for 30 seconds, the second step at 65 ° C. for 30 seconds, and the third step at 72 ° C. for 1 minute. The fourth step at 7 ° C.

反応終了後０．９％のアガロース電気泳動で確認したところ、設計通りのサイズのＤＮＡバンド（約１１００塩基対）を確認することができた。
（３）目的バンドをアガロースゲルから抽出（ＱＩＡｑｕｉｃｋ Ｇｅｌ ｅｘｔｒａｃｔｉｏｎ ｋｉｔ（商品名）：キアゲン社製）後、抽出ＤＮＡの５’末端をリン酸化し、制限酵素ＳｍａＩで消化したｐＵＣ１９プラスミドベクターに挿入し、５０μｇ／ｍＬの抗生物質カルベシニリンを添加したＬＢ寒天培地により大腸菌ＪＭ１０９株（タカラバイオ社製）を形質転換した。これをｐＢＢＦｃＲとした。図３に構造を示す。
実施例３ ヒト型ＦｃγＲＩ細胞外領域発現プラスミドベクターの作製
ヒト型ＦｃγＲＩの細胞外領域を発現させるために、以下に示す方法でヒト型ＦｃγＲＩ発現プラスミドベクターの作製を行なった。
（１）ｐＢＢＦｃＲをテンプレートにし配列番号５６（５’−ＡＣＡＴｇ［ＣＣＡＴｇｇ］ＣＴＴＴＣｇＣＴｇＣＡｇｇＡＴＣＣｇＣＴｇＴＡＡＴＣＡＣＴＣＴｇＣＡｇＣＣＡＣ−３’：角かっこ内の塩基は制限酵素ＮｃｏＩサイト）と配列番号５７（５’−ｇＣ［ＴＣＴＡｇＡ］ＣＴＡＡＴｇｇＴｇＡＴｇｇＴｇＡＴｇｇＴｇｇＡＣＴｇｇＡｇＴＡｇｇＣＡｇＴＴｇ−３’：角かっこ内の塩基は制限酵素ＸｂａＩサイト）のオリゴヌクレオチドをプライマーにしてＰＣＲを行ない、細胞外領域をコードするＤＮＡを増幅した。なお、ヒト型ＦｃγＲＩの調製および定量を行なうために、発現タンパク質のＣ末端側にポリヒスチジンタグが付加されるようにＰＣＲプライマーを設計した（配列番号５７）。反応条件は９４℃・５分の熱処理後、９４℃・３０秒間の第一ステップ、６５℃・３０秒間の第二ステップ、７２℃・１分間の第三ステップを２５サイクル行ない、最後に、７２℃・７分の第四ステップである。反応液組成を表３に示す。

When the reaction was confirmed by 0.9% agarose electrophoresis, a DNA band having a size as designed (about 1100 base pairs) could be confirmed.
(3) After extracting the target band from an agarose gel (QIAquick Gel extraction kit (trade name): Qiagen), the 5 ′ end of the extracted DNA is phosphorylated and inserted into a pUC19 plasmid vector digested with the restriction enzyme SmaI. Escherichia coli JM109 strain (manufactured by Takara Bio Inc.) was transformed with an LB agar medium supplemented with 50 μg / mL of antibiotic carbecinillin. This was designated as pBBFcR. FIG. 3 shows the structure.
Example 3 Preparation of Human FcγRI Extracellular Region Expression Plasmid Vector In order to express the human FcγRI extracellular region, a human FcγRI expression plasmid vector was prepared by the following method.
(1) SEQ ID NO: 56 (5′-ACATg [CCATgg] CTTTTCgCTgCAggATCCgCTgTAATCACTCTgCAgCCAC-3 ′: the base in brackets is the restriction enzyme NcoI site) and SEQ ID NO: 57 (5′-gC [TCTAgA] CTAATggTgTgTgTgTgTgTgTgTgTgTg : The base in square brackets was subjected to PCR using the oligonucleotide of the restriction enzyme XbaI site) as a primer to amplify the DNA encoding the extracellular region. In order to prepare and quantify human FcγRI, a PCR primer was designed such that a polyhistidine tag was added to the C-terminal side of the expressed protein (SEQ ID NO: 57). The reaction conditions were 94 ° C and 5 minutes heat treatment, followed by 25 cycles of the first step of 94 ° C and 30 seconds, the second step of 65 ° C and 30 seconds, and the third step of 72 ° C and 1 minute. This is the fourth step at 7 ° C. Table 3 shows the reaction solution composition.

反応終了後、０．９％のアガロース電気泳動で確認したところ、設計通りのサイズのＤＮＡバンドを確認することができた。
（２）目的バンドをアガロースゲルから抽出（ＱＩＡｑｕｉｃｋ Ｇｅｌ ｅｘｔｒａｃｔｉｏｎ ｋｉｔ（商品名）：キアゲン社製）後、抽出ＤＮＡを制限酵素ＮｃｏＩとＸｂａＩにより消化し、これらの制限酵素により事前に消化したｐＮＣＭＯ２（タカラバイオ社製）とライゲーションし、５０μｇ／ｍＬの抗生物質カルベシニリンを添加したＬＢ寒天培地により大腸菌ＪＭ１０９株を形質転換し調製した。これをｐＮＣＢＢＦｃＲとした。図４にｐＮＣＭＯ２プラスミドベクターのマルチクローニングサイト周辺の塩基配列、図５に上記に記載したプラスミドベクターｐＮＣＢＢＦｃＲの作製方法の概略を示す。
（３）並行して、ヒト型ＦｃγＲＩの細胞外領域をコードするヒト型コドンのＤＮＡを用いた発現ベクターも作製した。挿入断片の作製はＰＣＲで行ない、Ｈｕｍａｎ ｃＤＮＡ ｃｌｏｎｅ ＴＣ１１９８４１プラスミドベクター（Ｏｒｉｇｅｎｅ社製）をテンプレートとし、配列番号５８（５’−ＣｇＣ［ｇｇＡＴＣＣ］ｇＣＡｇＴｇＡＴＣＡＣＴＴＴｇＣＡｇＣＣＴＣＣＡＴｇｇｇ−３’：角かっこ内の塩基は制限酵素ＢａｍＨＩサイト）と配列番号５９（５’−ｇＣ［ＴＣＴＡｇＡ］ＣＴＡＡＴｇｇＴｇＡＴｇｇＴｇＡＴｇｇＴｇｇＡＣＡｇｇＡｇＴＴｇｇＴＡＡＣＴｇｇ−３’：角かっこ内の塩基は制限酵素ＸｂａＩサイト）のオリゴヌクレオチドをＰＣＲプライマーとして使用した。なお、ヒト型ＦｃγＲＩの調製および定量を行なうために、発現タンパク質のＣ末端側にポリヒスチジンタグが付加されるようにＰＣＲプライマーを設計した（配列番号５９）。ＰＣＲは９４℃・５分の熱処理後、９４℃・３０秒間の第一ステップ、６５℃・３０秒間の第二ステップ、７２℃・１分間の第三ステップを２５サイクル行ない、最後に、７２℃・７分の第四ステップである。反応液組成を表４に示す。

When the reaction was confirmed by 0.9% agarose electrophoresis, a DNA band of the designed size could be confirmed.
(2) After extracting the target band from an agarose gel (QIAquick Gel extraction kit (trade name): Qiagen), the extracted DNA was digested with restriction enzymes NcoI and XbaI, and pNCMO2 (Takara) previously digested with these restriction enzymes. And the Escherichia coli JM109 strain was transformed and prepared using an LB agar medium supplemented with 50 μg / mL of the antibiotic carbesinillin. This was designated as pNCBBBFcR. FIG. 4 shows the base sequence around the multicloning site of the pNCMO2 plasmid vector, and FIG. 5 shows an outline of the method for preparing the plasmid vector pNCBBFcR described above.
(3) In parallel, an expression vector using human codon DNA encoding the extracellular region of human FcγRI was also prepared. An insert fragment was prepared by PCR, and a human cDNA clone TC119841 plasmid vector (manufactured by Origen) was used as a template. ) And SEQ ID NO: 59 (5′-gC [TCTAgA] CTAATggTgATggTgATggTggACAggAgTTggTAACTgg-3 ′: the base in brackets is the restriction enzyme XbaI site) was used as a PCR primer. In order to prepare and quantify human FcγRI, a PCR primer was designed so that a polyhistidine tag was added to the C-terminal side of the expressed protein (SEQ ID NO: 59). In PCR, after heat treatment at 94 ° C. for 5 minutes, the first step at 94 ° C. for 30 seconds, the second step at 65 ° C. for 30 seconds, and the third step at 72 ° C. for 1 minute are performed for 25 cycles.・ The fourth step of 7 minutes. Table 4 shows the reaction solution composition.

反応終了後０．９％のアガロース電気泳動で確認したところ、設計通りのサイズのＤＮＡバンドを確認することができた。
（４）目的バンドをアガロースゲルから抽出（ＱＩＡｑｕｉｃｋ Ｇｅｌ ｅｘｔｒａｃｔｉｏｎ ｋｉｔ（商品名）：キアゲン社製）後、抽出ＤＮＡを制限酵素ＢａｍＨＩとＸｂａＩにより消化し、これらの制限酵素により事前に消化したｐＮＣＭＯ２（タカラバイオ社製）とライゲーションし（Ｌｉｇａｔｉｏｎ Ｋｉｔ Ｖｅｒ．２（商品名）：タカラバイオ社製）、５０μｇ／ｍＬの抗生物質カルベシニリンを添加したＬＢ寒天培地により大腸菌ＪＭ１０９株を形質転換し調製した。これをｐＮＣＨＵＦｃＲとした。図６に、上記に記載したプラスミドベクターｐＮＣＨＵＦｃＲの作製方法の概略を示す。
実施例４ 配列の確認
実施例３において作製したｐＮＣＢＢＦｃＲ およびｐＮＣＨＵＦｃＲプラスミドベクターに挿入したＤＮＡの配列をチェーンターミネータ法に基づくＢｉｇ Ｄｙｅ Ｔｅｒｍｉｎａｔｏｒ ｖ３．１ Ｃｙｃｌｅ Ｓｅｑｕｅｎｃｉｎｇ ｋｉｔ（商品名）（ＰＥアプライドバイオシステム社）を用いてサイクルシークエンス反応に供し、全自動ＤＮＡシークエンサーＡＢＩ Ｐｒｉｓｍ ３１０ ＤＮＡ ａｎａｌｙｚｅｒ（商品名）（ＰＥアプライドバイオシステム社）にて解析した。なお、配列番号６０（５’−ＣｇＣＴＴｇＣＡｇｇＡＴＴＣｇｇ−３’）と６１（５’−ＣＡＡＴｇＴＡＡＴＴｇＴＴＣＣＣＴＡＣＣＴｇＣ−３’）に示すオリゴヌクレオチドをシークエンス用プライマーとして使用した。

When the reaction was confirmed by 0.9% agarose electrophoresis, a DNA band having the size as designed could be confirmed.
(4) After extracting the target band from an agarose gel (QIAquick Gel extraction kit (trade name): Qiagen), the extracted DNA was digested with restriction enzymes BamHI and XbaI, and pNCMO2 (Takara) previously digested with these restriction enzymes. (Manufactured by Bio Inc.) and ligated (Ligation Kit Ver. 2 (trade name): manufactured by Takara Bio Inc.), and Escherichia coli JM109 strain was transformed and prepared using an LB agar medium supplemented with 50 μg / mL of the antibiotic carbesinillin. This was designated as pNCHUFcR. FIG. 6 shows an outline of a method for preparing the plasmid vector pNCHUFcR described above.
Example 4 Confirmation of Sequence A Big Dye Terminator v3.1 Cycle Sequencing Kit (trade name) (PE Applied Biosystems) based on the chain terminator method was used to insert the DNA sequences inserted into the pNCBBFcR and pNCHUFcR plasmid vectors prepared in Example 3. The sample was subjected to a cycle sequence reaction and analyzed with a fully automatic DNA sequencer ABI Prism 310 DNA analyzer (trade name) (PE Applied Biosystems). In addition, the oligonucleotide shown to sequence number 60 (5'-CgCTTgCAggATTCgg-3 ') and 61 (5'-CAATgTAATTgTTCCCTACCTgC-3') was used as a primer for a sequence.

As a result of the analysis, it was confirmed that the base sequences of the DNAs inserted into pNCBBFcR and pNCHUFcR were as designed. The amino acid sequences of human FcγRI expressed by pNCBBFcR and pNCHURcR are shown in SEQ ID NOs: 62 and 63, respectively. Each sequence includes a signal sequence on the pNCMO2 plasmid vector.
Example 5 Culture of Transformant The pNCBBFcR plasmid vector was transformed into Brevibacillus choshinensis SP3 strain (manufactured by Takara Bio Inc.) using TM agar medium (Table 5) supplemented with 10 μg / mL antibiotic neomycin.

３７℃で１８時間培養後、出現した任意のコロニーを選択し１０μｇ／ｍＬの抗生物質ネオマイシンを添加したＴＭ液体培地１ｍＬを含む試験管に接種した。表６にＴＭ液体培地の組成を示す。

After culturing at 37 ° C. for 18 hours, an arbitrary colony that appeared was selected and inoculated into a test tube containing 1 mL of TM liquid medium supplemented with 10 μg / mL antibiotic neomycin. Table 6 shows the composition of the TM liquid medium.

激しく撹拌しながら３０℃で４８時間培養後、遠心分離操作（１００００ｒｐｍ、１０分間）により菌体と培養上清に分離した。ｐＢＢＨＵＦｃＲプラスミドベクターについても同様に操作し、任意にコロニーから培養上清を調製した。
実施例６ 形質転換体の抗体結合活性評価
実施例５で調製した形質転換体の培養上清をＥＬＩＳＡ反応により抗体結合活性を評価した。
（１）９６穴のＥＬＩＳＡプレート（Ｎｕｎｃ社製）に５０μｇ/ｍＬから段階的に希釈したガンマグロブリン製剤（化学及血清療法研究所製）を各ウェルに１００μＬずつ添加し、４℃で１８時間静置することにより固定した。
（２）ＴＢＳ緩衝液（０．２％（ｗ／ｖ）Ｔｗｅｅｎ ２０、１５０ｍＭ ＮａＣｌを含むＴｒｉｓ−ＨＣｌ緩衝液（ｐＨ８．０））で洗浄後、Ｓｔａｒｔｉｎｇ Ｂｌｏｃｋ Ｂｌｏｃｋｉｎｇ Ｂｕｆｆｅｒｓ（ＰＩＥＲＣＥ社製）によりブロッキング操作を施した。
（３）同様に、ＴＢＳ緩衝液で洗浄後、実施例５で調製した任意のコロニー由来の培養上清を１００μＬ添加し、固定化した抗体であるヒトガンマグロブリンと反応させた（３０℃、２時間）。反応終了後、ＴＢＳ緩衝液で洗浄し、Ｈｉｓ−ｐｒｏｂｅ（Ｈ−１５）ＨＲＰ抗体（Ｓａｎｔａ Ｃｒｕｚ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ社製）を添加した。
（４）反応終了後、ＴＢＳ緩衝液で洗浄し、ＴＭＢ Ｐｅｒｏｘｉｄａｓｅ Ｓｕｂｓｔｒａｔｅ（ＫＰＬ社製）を添加し４５０ｎｍの吸光度を測定した。

The cells were cultured at 30 ° C. for 48 hours with vigorous stirring, and then separated into bacterial cells and culture supernatant by centrifugation (10000 rpm, 10 minutes). The pBBHUFcR plasmid vector was similarly operated, and a culture supernatant was arbitrarily prepared from the colony.
Example 6 Evaluation of Antibody Binding Activity of Transformant Antibody binding activity of the culture supernatant of the transformant prepared in Example 5 was evaluated by ELISA reaction.
(1) To a 96-well ELISA plate (manufactured by Nunc), add 100 μL of a gamma globulin preparation (manufactured by Chemo-Serum Therapy Laboratories) stepwise from 50 μg / mL to each well, and leave at 4 ° C. for 18 hours. It was fixed by placing.
(2) After washing with TBS buffer (Tris-HCl buffer (pH 8.0) containing 0.2% (w / v) Tween 20, 150 mM NaCl), blocking operation is performed with Starting Block Blocking Buffers (manufactured by PIERCE). Was given.
(3) Similarly, after washing with TBS buffer, 100 μL of the culture supernatant derived from any colony prepared in Example 5 was added and reacted with human gamma globulin, which was immobilized (30 ° C., 2 time). After completion of the reaction, the reaction mixture was washed with TBS buffer, and His-probe (H-15) HRP antibody (manufactured by Santa Cruz Biotechnology) was added.
(4) After completion of the reaction, the reaction mixture was washed with TBS buffer, TMB Peroxidase Substrate (manufactured by KPL) was added, and the absorbance at 450 nm was measured.

The results are shown in FIG. As shown in FIG. 7, it was found that the antibody binding activity of the culture supernatant derived from Bacillus bacteria transformed with pNCBBFcR was significantly higher than that of pNCHUFcR. That is, when human-type FcγRI is expressed in a host Bacillus bacterium, a gene in which the codon of the base sequence encoding the protein is converted from a human type to a Bacillus bacterium type is higher expressed in the Bacillus bacterium host. . In addition, human type FcγRI expressed using the transformant prepared in Example 5 was obtained because an active protein having antibody binding property was obtained without any manipulation such as solubilization. The protein production method using the transformant prepared in Example 5 can be said to be useful for production of the protein as compared with the conventional system using E. coli or animal cells.
Example 7 Changes in culture time of Bacillus bacteria transformed with pNCBBFcR Among the arbitrary colonies evaluated in Example 5, the clone with the highest antibody binding activity was selected and cultured in a flask. That is, a highly active clone was inoculated into a 500 mL baffled flask to which 200 mL of TM liquid medium (Table 6) added with 10 μg / mL antibiotic neomycin was added, and cultured at 30 ° C. with stirring at 150 rpm. The culture solution was sampled for an appropriate time, and a culture supernatant was prepared in the same manner as in Example 6. The antibody binding activity was evaluated in the same manner as in Example 6. The results are shown in FIG. As shown in FIG. 8, it was found that human FcγRI was produced from about 24 hours in culture and most produced in 53 hours of culture.
Example 8 Detection of Fc receptor by Western blotting method The culture supernatant prepared in Example 7 was subjected to SDS-PAGE electrophoresis, and detection by Western blotting was performed.
(1) 10 μL of the culture supernatant and 10 μL of a sample buffer containing mercaptoethanol were mixed, and after heat treatment at 98 ° C. for 5 minutes, electrophoresis using a polyacrylamide gel with a 10 to 20% gradient was performed.
(2) After CBB staining, the product was transferred to a PVDF membrane previously blocked with 4% skin milk (150 mA, 120 minutes).
(3) An anti-CD64 (also known as Fc receptor FcγRI) antibody (goat polyclonal antibody) (manufactured by R & D systems) diluted 2000-fold with a phosphate buffer containing 0.05% (w / v) Tween20, The reaction was carried out at 30 ° C. for 2 hours.
(4) After completion of the reaction, the resultant was washed with a phosphate buffer containing 0.05% (w / v) Tween 20, and anti-goat IgG (H + L) antibody-HRP labeled as a secondary antibody (ZYMED Laboratories) Was diluted 2000 times and added to react.
(5) After washing with a phosphate buffer containing 0.05% (w / v) Tween 20, add 2 mL of ECL plus solution (GE Healthcare Biosciences) per electrophoresis gel, and The film was exposed to light after standing for 5 minutes.

The development results are shown in FIG. As shown in FIG. 9, human-type FcγRI was detected from the preparation solution sampled between 24 hours and 100 hours after culturing.

It is a figure which shows the structure of human type | mold Fc receptor FcγRI. It is the figure which compared the base sequence between the human type | mold codon of a human type | mold FcγRI, and a Bacillus genus bacteria type codon. In the figure, Bacillus FcγRI is the base sequence of human FcγRI (SEQ ID NO: 2) converted to a Bacillus bacterial codon, and HumanFcγRI is the base sequence of human FcγRI (SEQ ID NO: 1) before codon conversion. It is a figure which shows the structure of plasmid vector pBBFcR. It is a figure which shows the base sequence of plasmid vector pNCMO2 multicloning site. It is a figure which shows the preparation methods of plasmid vector pNCBBFcR. It is a figure which shows the preparation methods of plasmid vector pNCHUFcR. It is the figure which compared the antibody binding activity of the human type FcγRI expressed using the transformant (derived from human type codon or derived from Bacillus genus bacterial type codon) prepared in Example 5. It is a figure which shows the production of human type FcγRI by Bacillus bacteria. It is a figure which shows the detection result of human-type FcγRI by Western blotting. The lane numbers in the figure are: 1 for 24 hours after culture, 2 for 33 hours, 3 for 48 hours, 4 for 58 hours, and 5 for 100 hours after anti-CD64. It is the result detected by the antibody.

Claims (5)

A human type wherein the codons of at least the 64th to 867th polynucleotides of the polynucleotide sequence encoding the human type Fc receptor FcγRI shown in SEQ ID NO: 1 are changed from a human type to a Bacillus genus bacterial type A polynucleotide encoding the Fc receptor FcγRI. The polynucleotide encoding the human Fc receptor FcγRI according to claim 1, wherein the polynucleotide is the polynucleotide of SEQ ID NO: 3. A plasmid vector into which the polynucleotide according to claim 1 or 2 is inserted. A transformant obtained by transforming the plasmid vector according to claim 3 into a bacterium belonging to the genus Bacillus. A method for producing a human type Fc receptor FcγRI polypeptide, comprising a step of culturing the transformant according to claim 4.

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