JP4996934B2 - Production method of recombinant lysozyme - Google Patents

Description

  The present invention relates to a method for producing lysozyme.

  Antibiotics are generally administered as a therapeutic agent for infectious diseases. However, the widespread use of antibiotics promotes the emergence of resistant bacteria, and the vicious circle is repeated, constantly pressing for the need to develop new antibiotics.

  Further, in recent years, a total amount of antibiotics more than double that of the human body has been used as a treatment or feed additive for livestock and farmed fish. Therefore, antibiotic-resistant bacteria are generated in the animal body, and there is a concern that these resistant bacteria enter the human body. When resistant bacteria propagate in the body and exhibit pathogenicity, antibiotics do not work, so it is very dangerous and is a major problem in nosocomial infections. Therefore, it has become necessary to establish treatments that do not rely on antibiotics for infectious diseases.

  On the other hand, since lysozyme is a natural protein, it does not cause side effects such as immune reaction even when administered to animals. In addition, lysozyme directly attacks and dissolves peptidoglycan, which is a main constituent of bacterial cell walls, and does not promote the development of drug-resistant bacteria. These advantages suggest that lysozyme is one of the best alternatives to replace antibiotics.

  Human lysozyme (hereinafter referred to as “HLY”) has been reported to have a direct antiviral effect on HIV. Accordingly, lysozyme derived from animals other than HLY (for example, porcine lysozyme (hereinafter referred to as “PLY”)) can be expected to be used as an antiviral agent against retroviruses.

  In the conventional production of lysozyme, for example, for PLY, natural PLY has been collected and purified from body fluids such as pig milk. However, since the production method is enormous in cost, it has not been put to practical use because it is too expensive as a livestock preparation.

  In addition, since lysozyme dissolves bacterial cell walls, it cannot be produced by bacterial protein expression systems such as Escherichia coli and Bacillus, and there has been no report that lysozyme was mass-produced.

  In view of the above situation, an object of the present invention is to provide a method for producing lysozyme capable of producing lysozyme in large quantities.

  As a result of diligent research to solve the above problems, it was found that lysozyme can be secreted and produced in a culture solution by introducing a gene encoding lysozyme into yeast or insect cells and culturing the resulting transformant. The present invention has been completed.

  The present invention includes the following.

(1) A method for producing lysozyme, comprising a step of culturing yeast or insect cells having a gene encoding lysozyme, wherein lysozyme is secreted and produced by the culture.
(2) The method according to (1), wherein the lysozyme is a lysozyme derived from a mammal.
(3) The method according to (2), wherein the mammal is selected from the group consisting of pig, human and cow.
(4) The method according to any one of (1) to (3), wherein the insect cell is a cell derived from sugar beet.
(5) The method according to any one of (1) to (4), wherein the culture is performed under an osmotic pressure of 213 mOsm / kg to 309 mOsm / kg.
(6) The method according to any one of (1) to (5), wherein the culture is suspension culture.

  According to the lysozyme manufacturing method of the present invention, lysozyme can be mass-produced safely and inexpensively.

Hereinafter, the present invention will be described in detail.
The method for producing lysozyme according to the present invention is a method for culturing yeast or insect cells having a gene encoding lysozyme (hereinafter referred to as “lysozyme gene”) and secreting and producing lysozyme in the culture solution. According to the method for producing lysozyme according to the present invention, lysozyme can be mass-produced.

  In the present invention, the lysozyme gene includes a lysozyme gene derived from any animal species or plant species, and a lysozyme gene derived from a mammal is preferred. Examples of lysozyme genes derived from mammals include PLY gene (base sequence: SEQ ID NO: 1, corresponding amino acid sequence: SEQ ID NO: 2), HLY gene (base sequence: SEQ ID NO: 3, corresponding amino acid sequence: SEQ ID NO: 4). And bovine-derived lysozyme (hereinafter referred to as “BLY”) gene (base sequence: SEQ ID NO: 5, corresponding amino acid sequence: SEQ ID NO: 6).

  FIG. 1 shows an alignment at the base sequence level between the PLY gene and the HLY gene. In FIG. 1, the upper base sequence is the PLY gene, while the lower base sequence is the HLY gene. FIG. 2 shows alignment at the base sequence level between the PLY gene and the BLY gene. In FIG. 2, the upper base sequence is the PLY gene, while the lower base sequence is the BLY gene. 1 and 2, the 360th base is changed from C to T in the base sequence of the PLY gene.

  As shown in FIGS. 1 and 2, the PLY gene has 80.3% identity with the HLY gene and 77.6% identity with the BLY gene, and these lysozyme genes have high homology.

  In the present invention, the lysozyme gene means a DNA consisting of the base sequences of the respective lysozyme genes including the base sequences of the above-mentioned PLY gene (SEQ ID NO: 1), HLY gene (SEQ ID NO: 3) and BLY gene (SEQ ID NO: 5). To do. In the lysozyme gene, one or several (for example, 1 to 10, preferably 1 to 5, particularly preferably 1 to 3) bases are substituted, deleted or deleted in the base sequence of each lysozyme gene. DNA that consists of an added base sequence and encodes a protein having lysozyme activity (i.e., antibacterial activity), and a DNA that consists of a base sequence complementary to the DNA consisting of the base sequence of each lysozyme gene, under stringent conditions DNA encoding a protein that hybridizes and has lysozyme activity, and the base sequence of each lysozyme gene, 80% or more, preferably 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, In particular, DNA encoding a protein consisting of a base sequence that is 99% or more identical and having lysozyme activity is also included. For example, in consideration of gene stability, in the base sequence of the PLY gene (SEQ ID NO: 1), from the base sequence (SEQ ID NO: 7) in which the 358th to 360th three bases “gac” are changed to “gat” The resulting DNA is also included in the PLY gene. The DNA consisting of the base sequence shown in SEQ ID NO: 7 encodes PLY consisting of the amino acid sequence shown in SEQ ID NO: 2, similarly to the base sequence shown in SEQ ID NO: 1.

  On the other hand, lysozyme is a protein comprising the amino acid sequence of each lysozyme including the amino acid sequences of PLY (SEQ ID NO: 2), HLY (SEQ ID NO: 4) and BLY (SEQ ID NO: 6). Accordingly, the lysozyme gene includes DNA encoding a protein comprising the amino acid sequence of each lysozyme, and 1 or several (for example, 1 to 10, preferably 1 to 5, particularly preferably 1) in the amino acid sequence of each lysozyme. DNA encoding a protein having a lysozyme activity, and a lysozyme amino acid sequence consisting of an amino acid sequence in which (~ 3) amino acids are substituted, deleted or added, and 80% or more, preferably 90% or more, 95 %, 96% or more, 97% or more, 98% or more, particularly preferably 99% or more of the same amino acid sequence, and DNA encoding a protein having lysozyme activity is also included.

  The above-mentioned lysozyme activity (antibacterial activity) can be evaluated using, for example, the hydrolysis activity of the bacteria (for example, Micrococcus lysodeikticus) on the cell wall as an index.

  The stringent conditions described above are, for example, when using probe DNA labeled with phosphorus 32, 5 × SSC (0.75M NaCl, 0.75M sodium citrate), 5 × Denhardt's reagent (0.1% Ficoll, 0.1% In a hybridization solution consisting of 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin) and 0.1% sodium dodecyl sulfate (SDS), the temperature is 45 ° C. to 65 ° C., preferably 55 ° C. to 65 ° C. In the washing step, the temperature is 45 ° C. to 55 ° C. in the washing solution consisting of 2 × SSC and 0.1% SDS, more preferably 45 ° C. to 55 ° C. in the washing solution consisting of 0.1 × SSC and 0.1% SDS. It is. When using an enzyme-labeled probe DNA using the kit of AlkPhos direct labeling module (Amersham Biotech), a hybridization solution having the composition described in the manual of the kit (including 0.5 M NaCl and 4% blocking reagent) The temperature is 55 to 75 ° C. In the washing step, the temperature is 55 ° C. to 75 ° C. in the primary washing solution (containing 2M urea) described in the manual of the kit and the room temperature in the secondary washing solution. In addition, other detection methods may be used, and in that case, standard conditions of the detection method may be used.

  The lysozyme gene can be obtained, for example, by PCR using the genomic DNA or cDNA of the animal species or plant species from which it was derived as a template and using appropriate primers designed for the lysozyme gene.

  On the other hand, examples of yeast used as a host in the present invention include Saccharomyces cerevisiae (for example, cell lines such as kk-4 and A2-1-1A strains), Schizosaccharomyces pombe pombe (Schizosaccharomyces pombe). (For example, cell lines such as NCYC1913 and NCYC2036) and Pichia pastoris (for example, cell lines such as KM71 and SMD1168). Examples of insect cells that serve as hosts include silkworm-derived cells (for example, cell lines such as BmN4 cells), sugar beet-derived cells (for example, Sf-9 cells and expresSF + cells (hereinafter referred to as `` SF + cells '')). And cell lines derived from nettle guava (for example, cell lines such as MG1 cells and High Five cells).

  In the present invention, first, a lysozyme gene is introduced into a yeast or insect cell to produce a yeast or insect cell (ie, transformant) having the lysozyme gene.

  The form of the lysozyme gene to be introduced into yeast or insect cells may be, for example, a DNA containing a lysozyme gene or an expression vector containing a lysozyme gene. An expression vector containing a lysozyme gene can be obtained by inserting the lysozyme gene into a multicloning site or the like of an appropriate vector. Suitable vectors include, for example, YEp vector, YCp vector, pPIC3.5K vector, and the like when the host is yeast, whereas when the host is an insect cell, for example, baculovirus vector (Silk nuclei). Polyhedrosis virus (BmNPV) vector, Autographa californica nuclear polyhedrosis virus (AcNPV) vector, etc.). In addition, it is preferable that control vectors, such as a promoter, an enhancer, and a terminator, are suitably included in the vector.

  The method for introducing a DNA or expression vector containing a lysozyme gene into yeast is not particularly limited as long as it is a method for introducing DNA into yeast, and examples thereof include an electroporation method, a spheroplast method, and a lithium acetate method.

  Examples of methods for introducing DNA or expression vectors containing lysozyme genes into insect cells include the calcium phosphate method, lipofection method, electroporation method and the like.

  In the present invention, the obtained transformant is then cultured, the lysozyme gene is expressed, and lysozyme protein is produced. The culture conditions may be any conditions as long as the lysozyme gene is expressed.For example, when the host is yeast, the temperature is 20 to 35 ° C. (preferably 25 to 30 ° C.), pH 1.4 to 9.0 (preferably pH For example, from 72 to 168 hours (preferably 72 to 120 hours). When the host is a silkworm-derived cell, the culture conditions include, for example, a temperature of 20 to 30 ° C. (preferably 25 to 28 ° C.), pH 6.0 to 7.0 (preferably pH 6.2 to 6.5), 72 hours to 240 hours (preferably 96 to 168 hours). Furthermore, when the host is a mushroom-derived cell or a nettle-derived oyster-derived cell, the culture conditions include, for example, a temperature of 20-30 ° C. (preferably 25-28 ° C.), pH 6.0-7.0 (preferably pH 6.2). -6.5) and 72 hours to 240 hours (preferably 96 to 168 hours). Moreover, it is preferable that culture | cultivation is performed by suspension culture.

  Furthermore, by making the osmotic pressure of the culture solution (medium) suitable, the amount of lysozyme secreted and produced from the transformant can be increased. The osmotic pressure of the culture solution is, for example, 194 mOsm / kg to 321 mOsm / kg, preferably 213 mOsm / kg to 309 mOsm / kg. The osmotic pressure can be adjusted by, for example, increasing or decreasing the inorganic concentration, adding saccharides, or diluting with pure water.

  In the present invention, lysozyme is secreted and produced from a transformant having a lysozyme gene. Therefore, after culturing, the culture solution may be used as it is, or the lysozyme can be isolated and purified by separating the culture solution from the transformant and subjecting it to extraction or purification. The extraction or purification method may be a method generally used when extracting or purifying proteins, and examples thereof include filtration, organic solvent extraction, chromatography, and electrophoresis.

  According to the lysozyme manufacturing method of the present invention described above, lysozyme can be mass-produced safely and inexpensively. Especially when using sugar beet-derived cells (for example, Sf-9 cells, SF + cells) and culturing under low osmotic pressure (213-309mOsm / kg), the productivity is high, and the cost can be drastically reduced. Widespread use can be expected in the world.

  In addition, for example, when a pig suffers from an infection, if it is treated by administering PLY instead of antibiotics, the development of antibiotic-resistant bacteria can be prevented beforehand. In addition, there is no transfer of residual antibiotics from pork and processed products to the human body.

  Conventionally, egg white lysozyme has been used as a food preservative and cold medicine, and induction of allergic symptoms such as atopy has been a problem. Therefore, there is a possibility of solving by substituting egg white lysozyme using lysozyme such as PLY, HLY and BLY produced by the method for producing lysozyme according to the present invention.

  Furthermore, according to the method for producing lysozyme according to the present invention, HLY can be produced, and not only in the livestock field but also widely used in the pharmaceutical field and food field. In addition, the knowledge about the optimal osmotic pressure of insect cell culture solution is considered to contribute to the improvement of secretion amount in the production of various types of proteins as well as lysozyme.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to these Examples.

[Example 1] Secretory production of recombinant PLY (hereinafter referred to as "rPLY") in various cells
An attempt was made to artificially synthesize the PLY gene, express the gene in yeast and insect cells, and secrete rPLY into the culture medium.

  First, based on the known PLY gene (DDBJ (http://www.ddbj.nig.ac.jp/Welcome-j.html), LOCUS: SSU44435) (SEQ ID NO: 1), the PLY shown in SEQ ID NO: 2 The nucleotide sequence encoding the amino acid sequence of was synthesized artificially from a short-chain DNA oligomer using the SPR method and PCR.

  Specifically, a gene having a full length of 484 base pairs shown in SEQ ID NO: 8 (including the base sequence shown in SEQ ID NO: 7) is converted into nine single strands having a length of 70 mer to 80 mer shown in FIG. Using DNA oligomers, artificial synthesis was performed by SPR method and PCR method.

  Next, the obtained PLY gene was cloned into the pUC19 vector and the nucleotide sequence was confirmed, and then the PLY gene was used for the YEp vector (for baker's yeast) and the baculovirus vector (BmNPV: for silkworm-derived cells, AcNPV: for sugar beet-derived cells) ).

  Further, the vector containing the obtained PLY gene was transformed into baker's yeast (Saccharomyces cerevisiae) by the lithium chloride method. Silkworm-derived cells (BmN4 cells) and sugar beet-derived cells (Sf-9 cells, SF + cells) were transformed with a vector containing the PLY gene obtained by the lipofection method.

  After transformation, each transformant was cultured for 7 days, and then the culture solution was collected, and the antibacterial activity of PLY in the culture solution against the cell wall of Micrococcus lysodeticus (manufactured by Sigma) was measured. The results are shown in Table 1. Table 1 shows the secretory production of rPLY in various cell lines.

  As shown in Table 1, when the antibacterial activity of each culture solution was measured, all showed high activity, suggesting the presence of antibacterial substances. The antibacterial substance in the culture solution of SF + cells with the highest activity was purified with a cation exchange column, and mass spectrometry was performed. As a result, it was 14808.83 Da, almost equal to the molecular weight predicted from the amino acid sequence of PLY (SEQ ID NO: 2). Matched. Moreover, when the N-terminal amino acid sequence of this protein was analyzed, the first 5 residues were completely identical, and from this, the same PLY (ie, rPLY) as the natural type was secreted in the culture medium. Proven.

  When the purified rPLY was accurately weighed and the antibacterial activity against Micrococcus lysodydictus cell wall (manufactured by Sigma) was measured, it was found that 1 unit of antibacterial activity corresponds to 0.0438 mg / L. Based on this, the amount of rPLY secretion was calculated from the antibacterial activity (see Table 1).

  When suspension culture was attempted in silkworm-derived BmN4 cells, sugar beet-derived Sf-9 cells, and SF + cells, the amount of rPLY produced significantly increased compared to static culture. In particular, it has been found that productivity is significantly higher when using sugar beet-derived Sf-9 cells and SF + cells than when using other cell lines.

[Example 2] Effect of culture solution osmotic pressure on rPLY secretion production amount In this example, the effect of culture solution osmotic pressure on rPLY secretion production amount was examined. In the same manner as in Example 1, a culture solution with low osmotic pressure was prepared from the transformant of SF + cells into which the PLY gene was introduced, and the optimum osmotic pressure was examined. The results are shown in Table 2.

  As shown in Table 2, it was found that the production of rPLY was significantly higher at an osmotic pressure of 220 to 309 mOsm / kg as compared to a commercially available culture solution (osmotic pressure of 321 mOsm / kg). In particular, at an optimal osmotic pressure of 296 mOsm / kg, the amount of secretory production reached about 1.5 times that of a commercially available culture solution.

FIG. 1 shows an alignment at the base sequence level between the PLY gene and the HLY gene. FIG. 2 shows an alignment at the base sequence level between the PLY gene and the BLY gene. FIG. 3 shows the single-stranded DNA oligomer used in Example 1 for the artificial synthesis of the PLY gene.

Claims (5)

Include the following (a) ~ (c) of any one cell osmotic from Mamestra having genes encoding lysozyme protein 213mOsm / kg~309mOsm / kg culturing under, lysozyme secretion by the culture produced, the production method of lysozyme.
(a) Lysozyme protein consisting of the amino acid sequence set forth in SEQ ID NO: 2
(b) A lysozyme protein comprising an amino acid sequence in which one or several amino acids are substituted, deleted or added in the amino acid sequence shown in SEQ ID NO: 2 and having lysozyme activity
(c) A lysozyme protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 2 and having lysozyme activity The method according to claim 1, wherein the gene is a gene encoding a lysozyme protein consisting of the amino acid sequence set forth in SEQ ID NO: 2. The method according to claim 1 or 2, wherein the gene is DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1 or 7. The method according to any one of claims 1 to 3, wherein the mushroom-derived cells are Sf-9 cells or expresSF + cells. The method according to any one of claims 1 to 4, wherein the culture is suspension culture.

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