JP2010166905A - New shine-dalgarno sequence and method for producing protein using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a control factor for producing active proteins in large quantities in the production of higher organism-derived proteins using genetically engineered means, and a method for producing the proteins using the factor. <P>SOLUTION: Protein production is performed using Shine-Dalgarno (SD) sequence including a part of genus Bacillus bacterium-derived neutral protease gene and a recombinant microorganism obtained by transforming an expression plasmid including the sequence into host cells. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a Shine-Dalgarno sequence useful for expressing a large amount of protein, and a method for producing a protein by genetic engineering using the sequence.

  To date, techniques for utilizing various microorganisms have been developed in order to efficiently produce large quantities of industrially useful substances. In particular, a system using E. coli has developed a strong promoter system, precise control system, and various host systems, and it is possible to control the expression of genes of higher organisms such as plants and animals besides genes derived from microorganisms. It has become. However, since Escherichia coli has an outer membrane outside the cell wall, generally secreted proteins accumulate in the periplasmic layer existing between the inner membrane and are not released into the culture medium. Therefore, it is necessary to crush the cells physically or chemically to extract the target protein. However, during the extraction operation, the target protein loses its activity due to mechanical or enzymatic destruction. There was a problem.

  In recent years, a technique for secreting and producing a soluble and active protein outside the bacterial body of E. coli via the periplasmic layer has been developed (Patent Document 1). However, the amount of protein to be produced is small, and there is a problem in economical efficiency when applied to industrial production.

  In addition, Escherichia coli has a problem that it is often obtained as an insoluble substance having no activity when expressing proteins derived from higher organisms, and it is necessary to reconstruct it into a protein having an active structure by an operation called refolding. there were. Therefore, application to industrial production has been further difficult.

  In recent years, a technique for obtaining a heterologous protein in an active form by secretory expression in other microorganisms has been developed. As one such expression system, a method using a Bacillus bacterium having a high protein secretion ability as a host has been reported (Non-patent Document 1). As important regulators for regulating the protein expression level in the cells of the genus Bacillus, a promoter, a Shine-Dalgarno (SD) sequence, a signal peptide, and a base sequence called a terminator are known.

  A promoter is a specific short base sequence involved in the initiation of mRNA synthesis. In the case of bacteria, the promoter is found about 10 bases upstream (-10 region) and about 35 bases upstream (-35 region) from the transcription start point. It consists of two short sequences.

  The SD sequence is a sequence that is found upstream of the initiation codon in prokaryotic mRNA, and is a part that becomes a binding site for ribosome, and is said to be involved in the initiation of protein synthesis in the ribosome.

  The signal peptide is a peptide that directs protein localization and membrane transport in the cytoplasm, and is recognized and cleaved into a region where a positively charged amino acid near the N-terminal is localized, a hydrophobic core region, and a signal peptidase. A peptide consisting of a region and having 20 to 50 amino acid residues. The peptide is thought to play a central role in producing extracellular proteins from Bacillus bacteria.

  The terminator is a base sequence connected to the end of the structural gene, and is considered to be a sequence involved in the completion of mRNA synthesis.

  As an example of the control factor, a promoter, SD sequence, signal peptide and terminator sequence derived from a neutral protease of the genus Bacillus are disclosed in Non-patent Document 2 and Patent Documents 2 to 5, and antibodies are expressed in Bacillus bacteria. A possible promoter and secretory signal sequence are disclosed in US Pat.

  All of the regulatory factors disclosed in the above literature have been reported as powerful expression factors, but they do not always work well depending on the target protein, and the active form is difficult to secrete particularly for proteins derived from higher organisms. was there. For example, SD sequences and signal peptides of proteins that are generally highly expressed in Bacillus bacteria such as proteases do not necessarily act strongly on all proteins. For each host type and protein to be expressed, a promoter, SD sequence, It is necessary to select an optimal one that is compatible with the signal peptide and the terminator. In recent years, the demand for industrial production of active proteins derived from various higher organisms has increased, and provision of novel control factors suitable for this has been required.

JP-A-8-322586 JP-A-2-265491 JP-A-2-265492 JP-A-2-268687 JP-A-2-268688 Japanese Patent Laid-Open No. 7-075581 JP-A-5-184363 Japanese Patent Laid-Open No. 10-309198

Wolfgang Schuman, Adv. Appl. Microbiol. 62, 137 (2007) Kubo and Imanaka, J.A. Gen. Microbiol. 134, 1883 (1988) Hanahan, J. et al. Mol. Biol. 16, 557 (1983) Devnau and Avidoff-Abelson, J.A. Mol. Biol, 56, 209 (1971)

  An object of the present invention is to provide a control factor for producing a large amount of an active protein in the production of a protein derived from a higher organism using a genetic engineering technique, and a method for producing a protein using the factor. is there.

  As a result of detailed examination of the gene for the neutral protease derived from the genus Bacillus, the inventors of the present invention found that a part of the sequence published as a sequence having no special function in the prior literature was Shine-Dalgarno. It was found that the (SD) sequence has a powerful function, and the present invention was completed.

That is, the present invention includes the following inventions:
The first invention is a Shine-Dalgarno (SD) sequence including a sequence consisting of at least AAAGGAGGA.

  The second invention is a gene recombinant obtained by transforming an expression plasmid containing the Shine-Dalgarno sequence described in the first invention into a host.

  The third invention is a method for producing a protein using the gene recombinant according to the second invention.

  A fourth invention is a method for producing a protein according to the third invention, wherein the protein is an antibody or an antibody fragment.

  Hereinafter, the present invention will be described in detail.

  The Shine-Dalgarno (SD) sequence of the present invention only needs to contain at least AAAGGAGGA, and an example is the sequence shown in SEQ ID NO: 29. In addition, as a method for obtaining DNA encoding the SD sequence of the present invention, pUBTZ2 (a shuttle vector between Escherichia coli and Bacillus subtilis in which a neutral protease gene derived from Bacillus stearothermophilus is cloned, Patent Literature From 7), a method of amplifying the DNA fragment by PCR reaction and a method of directly obtaining the DNA by chemical synthesis can be exemplified.

  The host of the present invention is not particularly limited as long as it is a bacterium belonging to the genus Bacillus, and among these, Bacillus subtilis having a high protein secretion ability and no pathogenicity is preferable.

  In the expression plasmid used in the present invention, the promoter may be any promoter as long as it functions in bacteria belonging to the genus Bacillus. For example, the promoter disclosed in Patent Document 4 is a promoter having -35 region consisting of TTTTCC and -10 region consisting of TATTGT. It is done. In addition, in order to obtain a promoter and intervening DNA, a method of directly obtaining the DNA by known chemical synthesis, a method of obtaining a DNA from a naturally occurring DNA with a restriction enzyme, or a nucleic acid amplification by PCR reaction from existing DNA It is obtained by a method obtained by

  In the expression plasmid used in the present invention, the signal peptide used for secretory expression is not particularly limited as long as it functions in bacteria belonging to the genus Bacillus. As an example, it is represented by MKRKMKLRSFGVAAGLA (SEQ ID NO: 30) disclosed in claim 4 of Patent Document 3. And a peptide consisting of MKRKMKMKMLASFGLAAGLAAQVFLPYNALA (SEQ ID NO: 31) disclosed in Non-Patent Document 2. Examples of the method for obtaining the DNA encoding the signal peptide include a method for obtaining the DNA by nucleic acid amplification from an existing gene by PCR reaction, and a method for obtaining the DNA directly by a chemical synthesis method.

  In the present invention, the protein for secretory expression is not particularly limited, but a protein that is secreted and produced outside the cell, such as amylase, protease, and antibody, is preferable. The method for obtaining the gene encoding the protein is not particularly limited, and an example of the case where an antibody is used as a target protein is an anti-estradiol antibody gene acquisition method disclosed in Patent Document 8. In addition, a method disclosed in Patent Document 8 for cutting out a cloned gene from a plasmid, or a method for amplifying the gene fragment by a PCR reaction or the like can also be used.

  In the expression plasmid used in the present invention, the terminator located at the end point of the structural gene group of protein expression, which is another constituent element, may be any one that functions in the bacterium belonging to the genus Bacillus. The sequence including CATCAGGTGGGGGATTTTTTCCTCCCACTGATG (SEQ ID NO: 32) disclosed in 3 is exemplified.

  In the protein production of the present invention, the SD sequence of the present invention is arranged downstream of an arbitrary promoter, and a DNA sequence having a gene encoding a desired protein is inserted downstream of the DNA sequence encoding an arbitrary signal peptide. An expression plasmid in which an arbitrary terminator is arranged downstream thereof is prepared, and protein production is carried out by introducing the plasmid into a bacterium belonging to the genus Bacillus. That is, generally, from the 5 ′ end side, a promoter, an SD sequence, a signal peptide, a target protein gene, and a terminator are arranged in this order to prepare an expression plasmid.

  The conditions used for expression culture may be appropriately selected from the conditions normally set for culture of Bacillus bacteria. The culture temperature is preferably 20 to 40 ° C, particularly preferably 25 to 30 ° C. The pH during the culture is preferably 6 to 8. As a medium used for the culture, a medium in which Bacillus bacteria can grow and the target protein can be secreted and expressed may be appropriately selected. The culture time can be arbitrarily set as long as it is suitable for secretory expression of the target protein, but it is usually set between several hours and 100 hours.

  Examples of the method for quantifying the expressed protein include a method for colorimetric quantification by staining with a dye or an immunological method after separating the protein by general SDS-PAGE, and an ELISA method, or expressing an enzyme or the like In some cases, a method for measuring the activity of the enzyme can also be used. In particular, when an antibody, a receptor, or the like is produced, the activity quantification method by the ELISA method is simple, which is preferable.

  The combination of proteins in the ELISA method is not particularly limited as long as the target protein can be quantified. For example, a sandwich method in which an antigen against the target antibody, target antibody, and enzyme-labeled anti-target antibody are stacked in this order can be mentioned. . Here, examples of the enzyme-labeled anti-target antibody include anti-target antibodies labeled with an enzyme such as alkaline phosphatase or horseradish peroxidase.

  In addition, the detection method of ELISA is not particularly limited, but specific coloring reagents, fluorescent reagents or chemiluminescent reagents for enzymes used for labeling are commercially available, and they are appropriately selected and used depending on the enzyme used for labeling. can do. For example, when horseradish peroxidase is used, there is a method for colorimetric quantification by oxidizing a chromogenic substrate with peroxidase and hydrogen peroxide. After coloring with a reagent, colorimetric determination can be performed with a commercially available measuring apparatus such as a microtiter plate reader MPR4i (trade name, manufactured by Tosoh Corporation).

  The present invention uses a part of a sequence published as a Shine-Dalgarno (SD) sequence as a Shine-Dalgarno (SD) sequence in a prior protease-derived neutral protease gene. Provided is a method for producing a protein capable of secreting and producing a desired higher organism-derived protein in bacteria. The protein obtained by the present invention is secreted in an active form outside the cells. This eliminates the need for a cell disruption step for protein extraction and the reconstitution of the protein into an active form with a denaturing agent, and a desired protein can be obtained efficiently. In addition, compared to the Shine-Dalgarno (SD) sequence disclosed in Non-Patent Document 2, secretory production is possible even with a short DNA sequence in the promoter portion and the signal sequence portion. Expression is expected.

Process drawing of plasmid pUCHS1L preparation. In the figure, VH + CH is a gene fragment of the VH region and CH1 region of the antibody H chain, stop is a stop codon, VL + CL is a gene fragment of the VL region and CL region of the antibody L chain, and Pr is a promoter region derived from a thermostable neutral protease. , SD represents the Shine-Dalgarno (SD) sequence of the present invention (SEQ ID NO: 29), SP represents a signal peptide region derived from a heat-resistant neutral protease, Amp ^r represents an ampicillin resistance gene region, EcoRI, NheI, KpnI , PstI and SphI indicate the cleavage sites of the corresponding restriction enzymes. Process drawing of plasmid pUCSHS1L preparation. The abbreviations in the figure are the same as those in FIG. Process drawing of plasmid pUBCH1LΔt preparation. Of the abbreviations in the figure, Km ^r indicates that the kanamycin resistance gene region, others equivalent to FIG. Process drawing of plasmid pUBCH1L preparation. Of the abbreviations in the figure, Km ^r is a kanamycin resistance gene region, TT indicates respectively that the terminator sequence from thermostable neutral protease, others equivalent to FIG. Schematic of plasmid pUBCH2L. The abbreviations in the figure are the same as those in FIG. Schematic of plasmid pUBCH3L. Of the abbreviations in the figure, Sup represents an additional sequence of 13 bases (SEQ ID NO: 33). Others conform to FIG. After culturing Bacillus subtilis DB117 strain (black circle), DB104 strain (white circle), and MT2 strain (white triangle) transformed with plasmid pUBCH2L at 25 ° C., 30 ° C., or 37 ° C. for 24 hours, The result of measuring antibody titer is shown. Each symbol in the figure indicates an average value when 24 identical experiments are performed under each condition. Moreover, a broken line shows a standard deviation. The result of having measured the antibody titer in the culture supernatant after culture | cultivating the Bacillus subtilis DB117 stock | strain transformed by plasmid pUBCH1L, pUBCH2L, or pUBCH3L at 30 degreeC for 24 hours is shown. The bar | burr in a figure shows the average value of the result of having culture | cultivated each transformant 24 times. Moreover, the vertical line shown on the bar indicates the standard deviation.

  Examples of the present invention are shown below, but the present invention is not limited thereto.

Example 1 Construction of expression vector pUBCH1L (1) Synthesis of insert sequence (base sequence including signal sequence from promoter) (1-1) SEQ ID NO: 1 to 12 (SEQ ID NO: 1 is the 1st to 40th bases of SEQ ID NO: 13) SEQ ID NO: 2 is the complementary strand of the 35th to 79th bases of SEQ ID NO: 13, SEQ ID NO: 3 is the 73rd to 115th bases of SEQ ID NO: 13, and SEQ ID NO: 4 is the 108th to 152nd bases of SEQ ID NO: 13. SEQ ID NO: 5 is the nucleotide from the 144th to 188th base of SEQ ID NO: 13, SEQ ID NO: 6 is the complementary strand of the 179th to 223th base of SEQ ID NO: 13, and SEQ ID NO: 7 is the 217 of SEQ ID NO: 13. SEQ ID NO: 8 is the complementary strand of the 254th to 298th base of SEQ ID NO: 13, SEQ ID NO: 9 is the 291th to 335th base of SEQ ID NO: 13 SEQ ID NO: 10 is the complementary strand of the 327th to 371th bases of SEQ ID NO: 13, SEQ ID NO: 11 is the 366th to 410th bases of SEQ ID NO: 13, and SEQ ID NO: 12 is the 400th to 435th bases of SEQ ID NO: 13. The complementary strands were mixed with oligonucleotides indicated respectively, and DNA was amplified by performing the first PCR using DNA polymerase (trade name, PrimeSTAR HS DNA Polymerase) manufactured by Takara Bio Inc. After the PCR reaction was heated at 94 ° C. for 5 minutes, the cycle of heating at 94 ° C. (30 seconds), annealing at 57 ° C. (30 seconds) and extension reaction at 72 degrees (45 seconds) was repeated 25 times. And heating at 72 ° C. for 7 minutes.
(1-2) The reaction solution after the first PCR reaction was purified with a PCR purification kit manufactured by Qiagen to obtain a 50 μL DNA solution.
(1-3) The second PCR reaction was performed under the same conditions as the first time, using 1 μL of the DNA solution obtained in (1-2) as a template and the oligonucleotides of SEQ ID NOS: 1 and 12 as primers. .
(1-4) A DNA fragment of about 400 bp was excised from the reaction solution after the second PCR reaction by agarose gel electrophoresis, purified with a gel extraction kit manufactured by Qiagen, and inserted into the insert sequence (5 ′ end) An EcoRI site and a SacI site are included in the vicinity, a neutral protease promoter sequence, the Shine-Dalgarno (SD) sequence of the present invention (bases from SEQ ID NOs: 29, 325 to 339), and a neutral protease signal sequence (sequence) No. 31 base sequence encoding the peptide of No. 31 and the 340th to 429th bases) and the NheI site in the vicinity of the 3 ′ end).
(1-5) As oligonucleotides for the second PCR reaction, SEQ ID NOs: 14 and 15 (SEQ ID NO: 14 is the 1st to 34th bases of SEQ ID NO: 16, and SEQ ID NO: 15 is the 396th to 433th positions of SEQ ID NO: 16. The same procedure as in (1-3) to (1-4) was performed except that the complementary strand of the base was used, and the insert sequence shown in SEQ ID NO: 16 (containing a KpnI site near the 5 ′ end) Neutral protease promoter sequence, SD sequence of the present invention (SEQ ID NO: 29, base from 319 to 333), and neutral protease signal sequence (base sequence encoding peptide of SEQ ID NO: 31, 334 to 423) Base) and a PstI site in the vicinity of the 3 ′ end.

(2) Synthesis of insert sequence (base sequence including signal sequence from SD sequence) (2-1) As oligonucleotides for the first PCR reaction, SEQ ID NO: 10, 11, 15, 17 (SEQ ID NO: 17 is SEQ ID NO: 18 The same procedure as in (1-1) to (1-4) was performed, except that SEQ ID NOS: 15 and 17 were used as oligonucleotides for the second PCR reaction. The insert sequence shown in SEQ ID NO: 18 (containing a KpnI site in the vicinity of the 5 ′ end, the SD sequence of the present invention (SEQ ID NO: 29, bases 13 to 27) in the center and a neutral protease signal sequence (peptide of SEQ ID NO: 31) And a PstI site in the vicinity of the 3 ′ end).
(2-2) As the oligonucleotide for the second PCR reaction, except that SEQ ID NOS: 15 and 19 (SEQ ID NO: 19 corresponds to the 1st to 36th bases of SEQ ID NO: 20), (2-1) and The same operation was performed, and the insert sequence shown in SEQ ID NO: 20 (containing a KpnI site and a 13 base addition sequence (SEQ ID NO: 33, bases from the 13th to 25th positions) near the 5 ′ end, and the SD sequence of the present invention in the center) (Includes SEQ ID NO: 29, 26th to 40th bases) and neutral protease signal sequence (base sequence encoding peptide of SEQ ID NO: 31; 41st to 130th bases) and PstI site near the 3 ′ end) Got.

(3) Amplification of antibody H chain gene fragment (VH to CH1 region) Anti-estradiol antibody expression plasmid prepared by the method disclosed in Patent Document 8 was used as a template, and oligonucleotides shown in SEQ ID NOs: 21 and 22 were used as primers. Antibody H chain gene fragment (VH to CH1 region) was amplified by PCR reaction. After the PCR reaction was heated at 94 ° C. for 5 minutes, the cycle of heating at 94 ° C. (30 seconds), annealing at 57 ° C. (30 seconds) and extension reaction at 72 degrees (45 seconds) was repeated 25 times. And heating at 72 ° C. for 7 minutes. The amplified gene was purified with a Qiagen PCR purification kit.

(4) Amplification of antibody L chain gene fragment (VL to CL region) PCR was performed in the same manner as in (3) except that the oligonucleotides shown in SEQ ID NOs: 23 and 24 were used as primers. An amplified fragment of VL to CL region) was obtained.

(5) Amplification of terminator A PCR was performed in the same manner as in (3) except that the plasmid pUBTZ2 disclosed in Patent Document 7 was used as a template and the oligonucleotides shown in SEQ ID NOs: 25 and 26 were used as primers. An amplified fragment of the terminator (SEQ ID NO: 34, nucleotides 23 to 53 were the same as SEQ ID NO: 32) was obtained.

(6) Construction of expression vector (6-1) The antibody heavy chain gene fragment (VH to CH1 region) prepared in (3) was restricted with restriction enzymes EcoRI and KpnI, and the light chain antibody gene fragment (VL) prepared in (4) To CL region) were cleaved with restriction enzymes PstI and SphI, and the insert sequence shown in SEQ ID NO: 16 prepared in (1-5) was cleaved with KpnI and PstI, respectively, and then purified with a Qiagen PCR purification kit.
(6-2) Plasmid pUC118 (manufactured by Takara Bio Inc.) was digested with EcoRI and SphI, further dephosphorylated by alkaline phosphatase treatment, and then subjected to agarose gel (gel concentration 1%) electrophoresis. It cut out and refine | purified using the gel extraction kit by a company.
(6-3) The antibody H-chain gene fragment treated with the restriction enzyme prepared in (6-1), the insert sequence shown in SEQ ID NO: 16 and the antibody L-chain gene fragment prepared in (6-1) are added to the fragment of (6-2), and T4 DNA ligase Was used to synthesize plasmid pUCHS1L (FIG. 1).
(6-4) Plasmid pUCHS1L (FIG. 1) was digested with EcoRI and NheI, further dephosphorylated by alkaline phosphatase treatment, and then subjected to agarose gel (gel concentration: 1%) electrophoresis to obtain a fragment of about 4.8 kb. It cut out and refine | purified using the gel extraction kit by Qiagen.
(6-5) The insert sequence shown in SEQ ID NO: 13 previously cut with EcoRI and NheI and purified with Qiagen PCR purification kit was ligated to the fragment obtained in (6-4) using T4 DNA ligase, pUCSHS1L (FIG. 2) was synthesized.
(6-6) Plasmid pUCSHS1L (FIG. 2) is cleaved with restriction enzymes SacI and HindIII, subjected to agarose gel (gel concentration 1%) electrophoresis, and a fragment of about 2 kb is excised and purified using a gel extraction kit manufactured by Qiagen. From the 5 ′ end side, neutral protease promoter, SD sequence of the present invention (SEQ ID NO: 29), neutral protease signal sequence, antibody H chain gene fragment (VH to CH1 region), neutral protease promoter, A DNA fragment composed of an SD sequence (SEQ ID NO: 29), a neutral protease signal sequence, and an L chain antibody gene fragment (VL to CL region) was obtained.
(6-7) pUBTZ2 disclosed in Patent Document 7 was digested with SnaBI and HindIII, dephosphorylated with alkaline phosphatase, and then cut out from 1% agarose gel electrophoresis to prepare a fragment of about 5 kbp.
(6-8) Two oligo DNAs (SEQ ID NOs: 27 and 28) previously phosphorylated by T4 DNA kinase treatment are ligated with T4 DNA ligase to the fragment obtained in (6-7) to synthesize plasmid pUBC1 (FIG. 3). did.
(6-9) Plasmid pUBC1 (FIG. 3) was digested with restriction enzymes SacI and HindIII, further dephosphorylated by alkaline phosphatase treatment, and then subjected to agarose gel (gel concentration: 1%) electrophoresis to obtain a fragment of about 5 kb. It cut out and refine | purified using the gel extraction kit by Qiagen.
(6-10) The fragment obtained in (6-9) and the 5 ′ terminal side prepared in (6-6) from the promoter neutral protease promoter, the SD sequence of the present invention (SEQ ID NO: 29), Protease signal sequence, antibody H chain gene fragment (VH to CH1 region), neutral protease promoter, SD sequence of the present invention (SEQ ID NO: 29), neutral protease signal sequence, L chain antibody gene fragment (VL to CL region) The constructed DNA fragment was ligated using T4 DNA ligase to synthesize plasmid pUBH1LΔt (FIG. 3).
(6-11) Plasmid pUBH1LΔt (FIG. 3) was cleaved with restriction enzymes SphI and HindIII and dephosphorylated by alkaline phosphatase treatment, and then subjected to agarose gel (gel concentration: 1%) electrophoresis to obtain a fragment of about 7 kb. It cut out and refine | purified using the gel extraction kit by Qiagen. The terminator fragment synthesized in (5) was ligated to this fragment using T4 DNA ligase to synthesize plasmid pUBCH1L (FIG. 4).

Example 2 Construction of Expression Vector pUBCH2L The same procedure as in (6) of Example 1 was carried out except that SEQ ID NO: 18 prepared in (2-1) was used instead of the insert sequence of SEQ ID NO: 16, and plasmid pUBCH2L (FIG. 5) was synthesized. The difference between the plasmid and the plasmid pUBCH1L prepared in Example 1 is that the former antibody H-chain gene and L-chain gene fragment is composed of a stop codon, the SD sequence of the present invention (SEQ ID NO: 29), and a neutral protease signal sequence. The latter is different in that it is constituted by a stop codon, a neutral protease promoter, the SD sequence of the present invention (SEQ ID NO: 29), and a neutral protease signal sequence.

Example 3 Construction of Expression Vector pUBCH3L The same procedure as in (6) of Example 1 was carried out except that SEQ ID NO: 20 prepared in (2-2) was used instead of the insert sequence of SEQ ID NO: 16, and plasmid pUBCH3L (FIG. 6) was synthesized. The difference between this plasmid and the plasmid pUBCH1L prepared in Example 1 is that the former fragment connecting the antibody H chain gene and L chain gene is a stop codon, an additional sequence of 13 bases (AAATACAAAGAAT, SEQ ID NO: 33), SD of the present invention. It consists of a sequence (SEQ ID NO: 29) and a neutral protease signal sequence, whereas the latter consists of a stop codon, a neutral protease promoter, the SD sequence of the present invention (SEQ ID NO: 29), and a neutral protease signal sequence. Is different (FIG. 6).

Example 4 Transformation into Bacillus subtilis (1) Competent cells of Escherichia coli JM103 were prepared according to the method of Hanahan (Non-patent Document 3) and transformed with the expression vectors pUBCH1L, pUBCH2L or pUBCH3L. These transformed E. coli cells were cultured overnight in LB medium containing 100 μg / mL ampicillin. From this culture solution, 30 μL of an aqueous solution of each plasmid was prepared using a Qiagen plasmid extraction kit.
(2) Competent cells of Bacillus subtilis MT2 strain, DB104 strain or DB117 strain were prepared according to a known method (Non-patent Document 4). Add 5 μL of each plasmid aqueous solution prepared in (1) to 100 μL of the cell suspension, shake gently for 1 hour at 37 ° C., add 900 μL of LB medium, and vigorously for 1.5 hours at 37 ° C. Cultured with shaking. (3) The cells are collected from the culture solution of (2) by centrifugation, resuspended in 100 μL of physiological saline, and 10 μL or 90 μL is applied to an LB plate medium containing 50 μg / mL kanamycin at 37 ° C. The culture was stationary overnight. From the grown kanamycin-resistant colonies, those having the target plasmid were selected and the target transformant Bacillus subtilis strain was prepared.

Example 5 Effect of strain and temperature on expression of antibody gene (1) Among the transformants prepared in Example 4, B. subtilis MT2, DB104 and DB117 strains transformed with plasmid pUBCH2L were treated with 2PY medium (polypeptone 40 g / L). , Bacto yeast extract 5 g / L, Glucose 20 g / L, MgSO ₄ · 7H ₂ O 0.1 g / L, FeSO ₄ · 7H ₂ O 0.01 g / L, MnSO ₄ · 4H ₂ O 0.01 g / L, ZnSO _{4 · 7H 2 O 0.001g / L} ) 96 were placed 0.75mL Anafukaana plate (BM6030S deep plate Rectangular V bottom was inoculated into BM equipment Corporation), temperature 25 ° C., 30 ° C. or 37 The culture was carried out at a shaking speed of 1200 rpm for 24 hours using a bioshaker MBR-022UP (trade name, manufactured by Taitec Co., Ltd.).

(2) The culture solution of (1) was centrifuged to remove the cells, and the antibody titer in the supernatant was measured by ELISA according to the following method.
(2-1) 100 μL of culture supernatant was added to a 96-well plate previously bound with estradiol-labeled bovine serum albumin and then blocked with a perfect block (trade name, manufactured by Pierce), and the antigen was incubated at 37 ° C. for 1 hour. After performing the antibody reaction, the solution was removed.
(2-2) Horseradish peroxidase-modified anti-mouse antibody antibody solution was added and an antigen-antibody reaction was performed at 37 ° C. for 1 hour, and then the solution was removed again.
(2-3) Color development reagent of horseradish peroxidase (TMB 2-Component Microwell Peroxidase Substrate Kit (trade name), manufactured by Funakoshi Co., Ltd.) was added 50 μL, and the color was developed until an appropriate color tone was obtained. 50 μL of 10% phosphoric acid The reaction was stopped with an aqueous solution, and the absorbance at 450 nm was measured. The same reaction was performed using 2PY medium instead of the culture supernatant as a blank, the absorbance at 450 nm was measured, and the antibody titer was determined by subtracting the absorbance of the blank from the absorbance at 450 nm of the specimen.

  The result of ELISA is shown in FIG. Although none of the strains could be confirmed in the culture supernatant when cultured at 37 ° C., the antibody titer was clearly confirmed when cultured at 25 ° C. or 30 ° C. It was shown that an antibody retaining an antigen-binding activity can be obtained by transforming an expression plasmid containing the SD sequence of the invention into Bacillus subtilis. The DB104 strain and DB117 strain produced the most antibody at 30 ° C., but the MT2 strain had lower productivity at 30 ° C. than other strains.

Example 6 Effect of Expression of Antibody Gene on Difference in Plasmid Composition (1) Bacillus subtilis DB117 strain transformed with expression plasmid pUBCH1L, pUBCH2L or pUBCH3L was cultured at a culture temperature of 30 ° C. in the same manner as in Example 5 and centrifuged. Separated to obtain a culture supernatant.

  (2) The antibody titer in the culture supernatant was measured in the same manner as in Example 5.

  The results are shown in FIG. The expression plasmid used is a sequence in which the antibody H chain gene and L chain gene are linked. In pUBCH1L, it contains a neutral protease-derived promoter, the SD sequence of the present invention (SEQ ID NO: 29) and a neutral protease-derived signal sequence, and in pUBCH2L It contains only the SD sequence of the invention (SEQ ID NO: 29) and a signal sequence derived from neutral protease, and in pUBCH3L, an additional sequence of 13 bases (SEQ ID NO: 33), the SD sequence of the present invention (SEQ ID NO: 29) and a signal derived from neutral protease Although it differs in that it includes sequences, there was no effect on antibody productivity due to these differences. FIG. 8 also shows the measurement results of a standard antibody (about 10 μg / mL) prepared from the culture supernatant of an anti-estradiol antibody-producing hybridoma (Patent Document 8). From the comparison of the color development amount, the antibody production amount by the three recombinant Bacillus subtilis was estimated to be about 10 μg / mL.

Claims (4)

Shine-Dalgarno (SD) sequence comprising a sequence consisting of at least AAAGGGAGA. A genetic recombinant obtained by transforming an expression plasmid containing the Shine-Dalgarno sequence according to claim 1 into a host. A method for producing a protein using the gene recombinant according to claim 2. The method for producing a protein according to claim 3, wherein the protein is an antibody or an antibody fragment.

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