JP3529423B2 - Method for producing antimicrobial peptide - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [0001] BACKGROUND OF THE INVENTION The present invention relates to an antimicrobial peptide
Recombinant vector into which a DNA sequence to be inserted is
Transformant carrying the recombinant vector of
Related to a method for producing an antimicrobial peptide using a transformant
It is. [0002] 2. Description of the Related Art Antibacterial activity against various microorganisms has been known.
Many inventions are known for peptides with
You. For example, effective against Gram-positive and Gram-negative bacteria
Phosphonotripeptide (JP-A-57-106689)
Report), phosphonodipeptide derivatives (JP-A-58-135)
No. 94), cyclic peptide derivatives (JP-A-58-21)
No. 3744), a pesticide exhibiting antibacterial and antiviral effects.
Peptide (JP-A-59-51247), effective for yeast
Polypeptides (JP-A-60-130599),
Glycopeptide derivatives effective against Gram-positive bacteria
No. 172998, JP-A-61-251699
Report, JP-A-63-44598), for gram-positive bacteria
Effective oligopeptides (JP-A-62-27988)
Report), peptide antibiotics (Japanese Patent Application Laid-Open No. 62-51697)
Official Gazette, JP-A-63-17897), and other products from North America
Antimicrobial peptide extracted from horseshoe crab blood cells
2-53799), isolated from bee's haemolymph.
Antibacterial peptide (Japanese Patent Application Laid-Open No. 2-5000084)
is there. On the other hand, lactoferrin is used for tears, saliva,
A natural iron-binding protein contained in blood, milk, etc.
Harmful to E. coli, Candida, Clostridium, etc.
It is known to exhibit antibacterial action against microorganisms.
Journal of Pediatrics
trics), vol. 94, page 1, 1979]. [0004] The inventors of the present invention have added lactoferrin.
It has a specific amino acid sequence from the hydrolyzate,
Irrespective of the iron-binding ability of phosphorus, compared to lactoferrin
Antimicrobial peptides with high antibacterial properties
[Journal of Daily Science
f Dairy Science), vol. 74, p. 4137, 19
1991]. [0005] The above antimicrobial peptide
Has previously been chemically synthesized or lactoferrin hydrolyzed
It was produced by separation from dismantled products (Japanese Patent Laid-Open No. 5-78)
392, JP 5-92994, JP 5
-148295, JP-A-5-148296
And JP-A-5-148297). However, the antimicrobial properties of these methods
Peptide production is expensive, cumbersome, and low
Therefore, these peptides can be used inexpensively, conveniently, and in large quantities.
It was desired to manufacture. [0007] Human lactofe, a natural protein,
For phosphorus, its cDNA sequence and eukaryotic cell expression
Lactoferrin and its functional equivalents using the system
Discloses a production method using genetic recombination technology
(Japanese Unexamined Patent Publication No. Hei 4-505101) shows that the produced
Toferin was detected using an anti-human lactoferrin antibody.
Western immunoblot analysis and relative molecular weight determination
Very small, as confirmed by
There is no production on a scale. [0008] The present invention has been made in view of the circumstances described above.
Made antimicrobial peptides cheaper and simpler
New genetic engineering materials for mass production and
Provided is a method for producing an antimicrobial peptide using these materials.
It is intended to be. [0009] SUMMARY OF THE INVENTION The present invention has been made to solve the above problems.
As a solution to
1. A DNA sequence encoding the amino acid sequence described in 1.
A combined recombinant vector is provided. [0010] The present invention also provides at least a vector in a vector.
A DNA sequence encoding the amino acid sequence of SEQ ID NO: 1
Provide a transformant carrying the inserted recombinant vector
Offer. [0011] Further, the present invention provides a vector comprising at least
DNA sequence encoding the amino acid sequence of SEQ ID NO: 1
A transformant carrying the recombinant vector into which
Cultured, and a peptide comprising the amino acid sequence of SEQ ID NO: 1
For producing an antimicrobial peptide, comprising collecting
Also provide. Hereinafter, the structure and preferred embodiments of the present invention will be described.
And will be described in detail. In the following description, antimicrobial
Lactoferricins by combining active peptides
("Lactofericin" is owned by Morinaga Milk Products Co., Ltd.
Registered trademark). Also amino
Acids and peptides are
-Abbreviations adopted by the Biochemical Naming Committee (IUPAC-IUB CBN)
It is displayed based on the law, for example, using the following abbreviations. Ala: L-alanine residue Arg: L-arginine residue Asn: L-asparagine residue Asp: L-aspartic acid residue Cys: L-cysteine residue Gln: L-glutamine residue Glu: L-glutamic acid residue Gly: L-glycine residue His: L-histidine residue Ile: L-isoleucine residue Leu: L-leucine residue Lys: L-lysine residue Met: L-methionine residue Phe: L-phenylalanine residue Pro: L-proline residue Ser: L-serine residue Thr: L-threonine residue Trp: L-tryptophan residue Tyr: L-tyrosine residue Val: L-valine residue First, the recombinant vector of the present invention comprises: (1) Synthesis of DNA sequences encoding lactoferricins
Success (2) Expression vector for the DNA sequence synthesized in step (1)
Can be constructed by the procedure of insertion into Next
These steps will be described. (1) Synthesis of DNA sequences encoding lactoferricins
Success If there are several codons corresponding to the same amino acid,
(The type of host microorganism)
It is known that there is a difference in
Vol. 1541-1553, 1983]. There
In the present invention, lactoferricins are produced.
Select codons that are commonly used for
The corresponding DNA sequence was synthesized by a conventional method. This is
Amino acid sequence corresponding to such a DNA sequence
It contains the amino acid sequence of SEQ ID NO: 1,
Sequence of the antimicrobial peptide (lactoferricins)
And the iron-binding domain of lactoferrin.
It is not particularly limited except that it is characterized by
Absent. However, the length excluding the restriction enzyme cleavage sites at both ends of the sequence
More desirably, the length is up to 180 base pairs. Ma
In addition, lactoferricins are suitable at their N-terminus or C-terminus.
When the protein P is produced in a fusion form,
Encodes lactoferricins and protein P, respectively
Proteases (thrombin,
Recognition cleavage site by factor Xa) or chemical substance
Is added beforehand. (2) Expression vector for the DNA sequence synthesized in (1) above
Insert into A vector for inserting the DNA sequence synthesized in the step (1)
Are species that produce lactoferricins (such as
Depending on the type of microorganisms to be used. these
The vectors include known plasmids pGEX2T, pK
OM1, pRSVNot, etc. are exemplified, and
Various known vectors including various vectors derived by
Can be used as appropriate. Often these
Since the vector is an E. coli shuttle vector,
The construction of the recombinant vector is performed by using the D synthesized in the above step (1).
The NA sequence is ligated to the vector in vitro and then once
It can be performed by introducing into E. coli. Thus, the base sequence of the inserted DNA sequence
Confirmation and amplification / preparation of the constructed recombinant vector
Becomes easier. The construction of the vector for Escherichia coli is described in (1).
The DNA sequence synthesized in the step
By ligation alone to the vector selected for
It can be carried out. The expression vector to be used is
The promoter involved in the expression of the incorporated DNA sequence
Controlled or always working
However, considering the expression level of lactoferricins,
The promoter involved in the expression of the incorporated DNA sequence
What is controlled is more desirable. In addition,
Incorporated so that tofericins are secreted into the medium
Substance derived from DNA sequence has signal sequence
It may be something. The molecular weight of the substance produced is small
In some cases, the expression product forms a fusion with the appropriate protein
Shaped ones can also be used. Next, the recombinant vector of the present invention was used.
A method for producing lactoferricins will be described. Recombination
Production of lactoferricins using a vector (1) Preparation of transformant carrying recombinant vector (2) The transformant prepared in the above step (1) is
Confirmation of producing ferricins (3) Producing lactoferricins in the step (2)
Culture of transformants confirmed to be (4) Lactate from the culture solution obtained in the step (3)
This is performed by purifying ferricins. Then, for each
The procedure will be described. (1) Preparation of transformant carrying recombinant vector The method of introducing the recombinant vector depends on the species to be introduced.
(The kind of host microorganism). example
If Escherichia coli is used as the host,
If the yeast method is used, the standard protoplast method is used when yeast is used.
[DNA Cloning, Vol. 2, 25
Page, IRL Press, 1985]
Is the lithium method.If animal cells are used, calcium phosphate
Um method [Molecular Cloni
ng), Second Edition, Volume 3, Chapter 16, Page 33,
Ludo Spring Harbor Laboratory Press (C
old Spring Harbor Laboratory Press), 1989]
Or electroporation [Molecular chromatography
Cloning, 2nd Edition, Volume 3, Volume 1
Chapter 6, page 54, Cold Spring Harbor
・ Laboratory Press (Cold Spring Harbor Laborato
ry Press), 1989]. set
Selection of transformants carrying the replacement vector was performed using
Drug resistance based on the selectable marker gene carried by the vector
Whether a trait such as sex or no amino acid requirement is acquired
Check with a supplemented medium or a medium without amino acids.
Can be performed. (2) The transformant prepared in step (1) is
Confirmation of production of lysines Lactoferricins of the transformant prepared in the above (1)
Confirmation of production can be performed using the culture supernatant of this transformant or cell-free
The extract is prepared using, for example, a commercially available anti-lactoferrin antibody.
ELISA Method [Biochemical Experiment Method 15 / Introduction to Immunology, No. 1]
50 pages, Society Press, 1983], Wess
Tan blotting method [Molecular cloning
(Molecular Cloning), Second Edition, Volume 3, Chapter 18, Chapter
60 pages, Cold Spring Harbor Labora
Tree Press (Cold Spring Harbor Laboratory Pres)
s), 1989].
Wear. Also, since the substance produced has antibacterial activity,
Antibacterial activity in the culture supernatant or cell extract of this transformant
It can also be confirmed by measuring the activity
You. (3) Producing lactoferricins in the step (2)
Culture of transformants confirmed to be The cultivation of the transformant depends on the species of the transformant (such as the host microorganism).
Type) and the most suitable for each depending on the vector used
Method. For example, for expression of an integrated DNA sequence
Use a vector in which the promoter involved always works,
If the species is Escherichia coli, the drug used to select the transformants
2 × TY medium containing [1.6% bactotryptone,
From 1.0% yeast extract and 0.5% sodium chloride
Become. Molecular Clonin
g), 2nd edition, Volume 3, Appendix A, Page 3, Cold
Spring Harbor Laboratory Press (Cold S
pring Harbor Laboratory Press), 1989]
And usually cultured at 37 ° C. until the stationary growth phase,
Production of lactoferricins is performed. A vector in which a similar promoter always works
Used, the species is baker's yeast Saccharomyces cerevisiae
D (Saccharomyces cerevisiae)
Minimal synthetic medium without amino acids or nucleic acids used for selection
[0.67% Bact East Nitrogen Base (A
Consists of 2% glucose (no amino acid). Methodos Inn
・ Methods in Enzymology, 19th
Volume 4, Page 14, Academic Press
Press), 1991] at 30 ° C until the stationary growth phase.
Culture of lactoferricin
Is When the species is animal cells, 10% calf blood
Dar containing the drug used for the selection of the purified and transformants
Becco Modified Eagle Minimal Medium [DMEM; Viology
(Virology), Vol. 8, p. 396, 1959], etc.
By culturing at 37 ° C. for 3 to 4 days
Production of tofericins is performed. In addition, incorporated
Promoters involved in DNA sequence expression are controlled
When using a vector, release its control and insert
To express the input DNA sequence,
A substance that changes the culture temperature or releases the control depending on the quality
Quality needs to be added. For example, lambda phage right
If it is a promoter, its induction (release of control)
To produce ctofericins, the initial temperature at 30 ° C
Culture until stationary growth phase, then raise the culture temperature to 42 ° C.
Culture for another 90 minutes.
If it is a robot, the guidance (release of control)
37 ° C (for yeast)
At 30 ° C) until the initial stationary growth phase, and then the final concentration
0.1-1.0 mM isopropyl β-D-thiogala
Cuctoside (IPTG) must be added and cultured for another 4 hours.
It is necessary. (4) Lactoferfate from the culture solution obtained in the step (3)
Purification of ricins This method also uses the vector used to prepare the transformant and
It is necessary to use a method suitable for each depending on the host. An example
For example, a lactoform with a secretory signal sequence added to the N-terminus
Large intestine transfected with recombinant vector that produces ferricins
When purifying lactoferricins from bacteria, culture medium
Centrifuge to collect the cells, suspend in sucrose solution and osmotic
Lactoferricins in the periplasm of cells
Followed by gel filtration or reverse phase chromatography.
Lactoferricins by fractionation and purification
Is purified. S. cerevisiae transfected with a similar vector
Or when purifying lactoferricins from animal cells
Contains lactoferricins by centrifuging the culture
Obtain the culture supernatant, followed by gel filtration or reverse
By fractionation and purification by phase chromatography, etc.
Purify the ctofericins. Next, at the N-terminal or C-terminal,
Appropriate proteins through the recognition or cleavage site of
Lactoferi produced in the form of a fusion with white matter P
When purifying synths, the culture was centrifuged and collected.
Then, crush the cells using ultrasonic waves or a homogenizer,
After centrifugation again, the fusion protein of lactoferricins and protein P
Obtain a cell-free extract containing white, followed by gel filtration or
Affinity using affinity for protein P
-After isolating the fusion protein using a column, the fusion protein is
Recognition of added protease or chemical substance
Using proteases or chemicals corresponding to the cleavage site
Cleavage and reverse phase chromatography of lactoferricins
Purification of lactoferricins by fractionation and purification
Production. In addition, regardless of the character of the vector, Escherichia coli
Lactoferricins were produced in large quantities by using
In some cases, the produced protein aggregates in the cells to form granules.
May form. In this case, centrifuge the culture
After collecting the cells, the cells are removed using ultrasound or a homogenizer.
Crushed and centrifuged again to obtain granules, 6M guanidine hydrochloride
Gin or 8 M urea is added to solubilize the protein.
Guanidine hydrochloride or
Performing processes such as gel filtration after reducing the concentration of urea
Can purify lactoferricins. Now, the present invention will be described in further detail with reference to Test Examples.
I will explain. Test example 1 In this test, the expression of lactoferricins was (1) constitutive
And (2) when controlled by IPTG
The efficiency of expression and the effect on the host
Went for. Similar to the embodiment 8 described later, the embodiment described later
Animal cell-based recombinant vector pRSVLF constructed in Example 7
Was carried out utilizing the lac operator. You
That is, in the case of (1), hamster CH is used as a host.
Using O cells, add the cells by calcium phosphate method
After introducing the recombinant vector, the cells after the introduction are cultured for 48 hours.
And lactoferricins were expressed. In case of (2)
Introduced p3'SS in advance, and the control solution by IPTG
Uses CHO cells that express lacI repressor
The vector was introduced into this cell by the calcium phosphate method.
, And the cells after the introduction are cultured for 36 hours.
G and then culturing for another 12 hours.
Were expressed. In each of the above (1) and (2)
The expression level of lactoferricins was determined by the ninhydrin method [ana
Analytical Biochem
istry), Vol. 49, p. 95, 1972].
Measured and compared. Regarding the effect on the host,
Except for the different host cells as described,
Identical to the total cells used for transduction
The ratio of transduced cells expressing elicins (transduction efficiency)
Compared. The results of this test are shown in Table 1.
You. As is evident from Table 1, the ease of animal cell lines
Expression of tofericins leads to saturation of host cell growth
The condition (2), in which control starts after the condition is reached,
Transformants are easily obtained without adverse effects
It was also found that the expression level of lactoferricins was high.
Was. [0025] [Table 1] Now, the present invention will be described in further detail with reference to Examples.
The present invention is limited to the following examples.
Not something. In addition, the source of the reagent is clearly specified.
All products not manufactured by Nacalai Tesque, Inc.
When there were multiple types, the special grade was used. [0027] EXAMPLES Example 1 (Escherichia coli (Escherichia coli)
cherichia coli))
Construction) (1) Synthesis of DNA sequences encoding lactoferricins
Success Select and use codons commonly used in E. coli,
The amino acid of SEQ ID NO: 1 which is part of the ctoferrin sequence
An amino acid sequence represented by SEQ ID NO: 5 containing an acid sequence;
Factor Xa, a protease added upstream of
Amino acid sequence described in SEQ ID NO: 7)
A DNA sequence consisting of the nucleic acid sequence of SEQ ID NO: 8
Done. Note that this DNA sequence also has a small number in SEQ ID NO: 8.
As shown by the letter symbols, the restriction enzyme BamHI
A sequence that is more cleaved and a termination
Don and sequences generated by cleavage with the restriction enzyme HincII
Was added. This DNA sequence was obtained from an automatic DNA synthesizer (A
Made by Pride Biosystems. Model 381A)
Using the phosphoramidite method [tetrahedron / reta
ー (Tetrahedron Letter), Vol. 21, p. 719,
1980]. For synthesis, the sequence number
No. 9 having E-1 to 2 and F-1 to
The synthesis is performed by dividing into two oligonucleotides of 2
Oligonucleotide CPG resin (Applied
From Biosystems) and elimination of protecting groups
Followed the company's manual. Each oligonucleotide
Separation and purification of oligonucleotide purification cartridge
(Applied Biosystems) was used. (2) For insertion of the DNA sequence synthesized in the above step (1)
Of recombinant beku The oligonucleotide according to SEQ ID NO: 9 synthesized in the above step (1)
Of the four nucleotides, E-2 and F-1 were each 20 pmo.
l at the 5 'end of T4 polynucleotide kinase (Toyo
Spinning Co., Ltd.) and follow the company's protocol.
Separately phosphorylated, then heated at 95 ° C for 5 minutes to T
Denatured 4 Kinase, 1/10 volume of 3M sodium acetate
And ethanol was precipitated once. Then to E-2
Is an equimolar addition of an aqueous solution of E-1 to F-2 and to F-1.
Mix, heat at 95 ° C for 15 minutes, slowly cool
Has been updated. The obtained two annealing bodies and the IPT
Tac promoter that can be induced (released) by G
Having an ampicillin resistance gene as a marker
Of the known plasmid pGEX2T (Pharmacia)
 BamHI and SmaI sites were cut (using
All restriction enzymes are manufactured by Toyobo Co., Ltd.), DNA ligation
・ Connected in vitro using a kit (Takara Shuzo)
Thus, a recombinant vector pGEXLF was constructed (see FIG. 1).
See). This was transformed into E. coli JM109 strain (manufactured by Toyobo Co., Ltd.)
Calcium iodide method [Molecu cloning (Molecu
lar Cloning), 2nd Edition, Volume 1, Page 74, Call
De Spring Harbor Laboratory Press (Co
ld Spring Harbor Laboratory Press), 1989]
And 50 μg / ml ampicillin (Sigma)
Containing LB agar medium [1% bactotryptone (Difco
0.5% yeast extract (manufactured by Difco), 0.5%
Sodium chloride, 1.5% agar (Difco)]
And transformants were selected. As a result, 1 × 10 5 μg of DNA^Five
Colonies were obtained. Of these, five claw
(Named LFJME1-5 strains)
DNA sequence and vector encoding ferricins
It is determined that the base sequence at the connection
Quenase version 2.0 DNA sequencing
Kit [(Sequenase Version 2.0 DNA Sequencing
 Kit; United States Biochemical (Unite
d States Biochemical). Example 2 (using the recombinant vector created in Example 1)
Production of lactoferricins) (1) A form holding the recombinant vector created in Example 1
Creating transmutants The LFJME1 strain obtained in Example 1 was
Cultivate overnight at 37 ° C in LB medium containing Syrin,
Potassium denaturation method [Molecular cloning (Molecular
Cloning), 2nd edition, Volume 1, Chapter 1, Page 25,
Old Spring Harbor Laboratory Press
 (Cold Spring Harbor Laboratory Press), 1989
Year], the recombinant vector pGEXLF is amplified and prepared.
did. This vector was prepared using the standard calcium chloride method.
Introduced into E. coli HB101 strain (manufactured by Toyobo Co., Ltd.)
Characterization using LB agar medium containing μg / ml ampicillin
As a result of selecting the transformants, 1 × 10⁸
Colonies were obtained. Of these, 15 clones (LFHB
The strains were named 1 to 15 strains. ) For the alkali modification method
Plasmid DNA from Tris acetate buffer
0.7% agarose gel electrophoresis using
Molecular Cloning, Second Edition, Second Edition
Volume 1, Chapter 6, Page 9, Cold Spring Ha
Bar Laboratory Press (Cold Spring Harbor L
aboratory Press), 1989]
did. (2) The transformant prepared in the above step (1) is
Confirmation of producing ferricins LFHB1-
Of the 15 strains, LFHB1-8 strains were
Using 2 × TY medium containing 0 μg / ml ampicillin, 3
After culturing at 7 ° C. until the initial stationary growth phase, the final concentration is 0.1 m
M for 4 hours after adding IPTG, centrifuging and collecting.
Bacteria and PBS buffer [150 mM sodium chloride,
16 mM disodium hydrogen phosphate, 4 mM dihydrogen phosphate
Sodium (pH 7.3)]
Was centrifuged again after sonication, resulting in glutathione-S-
Transferase (GST) -lactoferricins
Granules expected to contain the synthesized protein were obtained. The granules were added to 6M guanidine hydrochloride-10m
Add M ethylenediaminetetraacetic acid and stir at room temperature for 2 hours
To solubilize the protein and dilute it 5 times with PBS buffer.
Was. Human lactoferricins are anti-human lactoferrin
Rabbits are known to bind immunologically to the body
Anti-human lactoferrin polyclonal antibody IgG fraction
(Manufactured by Kappel) and ELISA [Biochemical Experiment Method 15]
・ Introduction to Immunology, 150th page, Society Publishing Center, 1
983] to the above-mentioned granule lysate.
GST-lactoferricins fusion protein is contained.
And confirmed. (3) Producing lactoferricins in the step (2)
Culture of transformants confirmed to be The GST-lactoferricins fusion protein is produced in the above (2).
Of transformants LFHB1-8 strains confirmed to produce
The LFHB1 strain contained 50 μg / ml ampicillin.
Pre-culture at 37 ° C with 2xTY medium, and use the same culture solution
Add 1% volume to 2 liters of medium of composition and aerate in 5 liter culture tank
While culturing at 37 ° C until the initial stationary growth phase.
0.1 mM IPTG was added, and the cells were further cultured for 4 hours. (4) Lactoferfate from the culture solution obtained in the step (3)
Purification of ricins The culture solution obtained in the step (3) is centrifuged to collect bacteria,
Ultrasonic disruption of cells suspended in 00 ml of PBS buffer
After adding Triton X-100 having a final concentration of 1%,
Centrifugation was performed at 2,000 × g for 20 minutes to obtain granules. this
Add 20 ml of 6 M guanidine hydrochloride-10 mM ethi
Add diamine diacetic acid and stir at room temperature for 2 hours to add protein
Solubilized, then diluted 50-fold with PBS buffer
Was. The granule solubilized solution is subjected to aseptic filtration with a pore size of 0.45 μm.
Filter using a membrane, and add 20 ml each with PBS buffer.
Equilibrated FPLC column for gel filtration Superde75 HR26 / 60
0 (manufactured by Pharmacia, 10 mm x 300 mm)
Flow PBS buffer at a flow rate of 2.6 ml / min.
The eluted fraction after 60 minutes is collected and centrifugal concentrator centrip
It was centrifuged and concentrated using Repou 10 (manufactured by Amicon).
Next, a final concentration of 50 mM Tris (pH 8.0) -100 m
M sodium chloride-in the presence of 1 mM calcium chloride,
0.5% of Factor Xa (New In
Grand Biolabs) 1.0 mg / ml
GST-lactoferricin fusion protein is incubated overnight at room temperature.
White was cut, and the obtained lactoferricins were converted to silica-based T
Using SK gel ODS-120T (manufactured by Tosoh Corporation) as a carrier
Fractionation and purification by reversed-phase high-performance liquid chromatography
Electrophoretically uniform fraction (about 20 mg of protein)
Obtained. Example 3 (Recombinant vector using E. coli-based vector
Build) (1) Synthesis of DNA sequences encoding lactoferricins
Success Select and use codons commonly used in E. coli
Of the ferricins, part of the human lactoferrin sequence
SEQ ID NO: 6 including the amino acid sequence of SEQ ID NO: 1
Amino acid sequence and protea to be added upstream
Cleavage site of factor Xa (SEQ ID NO: 7)
Described in SEQ ID NO: 10).
The DNA sequence was synthesized. Also, this DNA sequence contains 5 '
The sequence formed by cleavage with BalI at the end is terminated at the 3 'end.
Add a stop codon and a sequence cut with BamHI.
Was. This DNA sequence is identical to that of Example 1 (1).
It was chemically synthesized using a moving DNA synthesizer. Synthetic distribution
L-1 to 4 having the nucleotide sequence described in SEQ ID NO: 11 and M
-8 divided into eight oligonucleotides
Purification was carried out in the same manner as in Example 1 (1). (2) For insertion of the DNA sequence synthesized in the above step (1)
Of recombinant vector by The oligo of SEQ ID NO: 11 synthesized in the step (1)
Of the eight nucleotides, L-2, L-3, L-4, M-
The 5 'end of each of 20 pmol of 1, M-2 and M-3 is
Phosphoric acid was separately prepared in the same manner as in item (2) of Example 1.
After that, ethanol precipitation was performed. Then L-2 has M
-2, L-3 of M-3, L-4 of M-4,
An equimolar amount of the aqueous solution of L-1 was added to and mixed with M-1.
Annealing was performed in the same manner as in item (2) of Example 1. Obtained
The four annealed products (double-stranded DNA fragments) were L-1
/ M-1 and L-2 / M-2, and L-3 / M-3 and L
-4 / M-4, each of which was used in Example 1.
DNA ligation kit (Takara)
(Manufactured by Shozo Co., Ltd.). End of reaction
After that, each reaction solution was subjected to polyacrylamide gel electrophoresis (gel
(6% concentration), ethidium bromide staining method
Of the target size (93 salt)
(Base pair +20 bases and 70 base pairs +20 bases)
Cut out part and use electro-elution method
Two target DNA fragments were recovered. Collected sample
Phenol extraction and ethanol precipitation
The target DNA was purified. The two kinds of obtained DNA fragments and the DNA fragment of Example 1 were used.
Ba of the same plasmid pGEX2T used in section (2)
Cut the lI and BamHI sites and cut out as above,
The 465 base pair DNA fragment released by electrophoresis
(All the restriction enzymes used were Toyobo Co., Ltd.
In vitro) in the same manner as in item (2) of Example 1.
After ligation, the recombinant vector pGEXLNL was constructed (see FIG.
2). This was transformed into E. coli in the same manner as in Example 1, (2).
Introduced into JM109 strain (manufactured by Toyobo Co., Ltd.)
Three clones of knee (named LNLE1-3 strains)
Was. ), The nucleotide sequence was confirmed. Example 4 (using the recombinant vector created in Example 3)
Production of lactoferricins) (1) A form holding the recombinant vector prepared in Example 3
Creating transmutants LN obtained in Example 3 in the same manner as in item (1) of Example 2
Amplification and preparation of recombinant vector pGEXLNL from LE1 strain
Made. This vector DNA was prepared in the same manner as in item (1) of Example 2.
Similarly introduced into E. coli HB101 (Toyobo)
Thus, a transformant was obtained. Of these, 10 clones
(Named LNLHB1 to 10 strains)
Preparation of plasmid DNA in the same manner as in item (1) of Example 2
And its presence was confirmed by electrophoresis. (2) The transformant prepared in the above step (1) is
Confirmation of producing ferricins LNLHB1 whose presence of the plasmid was confirmed in (1) above
Example 2 for 1 to 6 LNLHB strains out of 10 strains
They convert lactoferricins in the same manner as in item (2) of
I confirmed that it would produce. Hereinafter, the lactolysis was performed in the same manner as in Example 2.
About 35 mg of ferricins were obtained. Example 5 (Construction of recombinant vector using yeast-based vector)
Construction) (1) Synthesis of DNA sequences encoding lactoferricins
Success Using codons commonly used in yeast, human
Amino acid sequence of SEQ ID NO: 1 which is a part of the
SEQ ID NO: 5 and the upstream sequence thereof
Recognition of factor Xa, a protease to be added to
Encodes a cleavage site (amino acid sequence described in SEQ ID NO: 7)
The DNA sequence described in SEQ ID NO: 12 was synthesized. Also this D
In the NA sequence, a 5'-terminal digested with BamHI was used.
The sequence was cut at the 3 'end with a stop codon and SalI
Added sequences that can be used. The synthesis is shown in SEQ ID NO: 13
Four of S-1 and T-1-2 having a base sequence
This was performed by dividing the oligonucleotide into oligonucleotides.
It carried out by the method similar to (1). (2) For insertion of the DNA sequence synthesized in the above step (1)
Of recombinant vector by The oligo of SEQ ID NO: 13 synthesized in the above step (1)
Of 4 nucleotides, S-2 and T-1 were each 20 pm
The 5 ′ end of ol was treated in the same manner as in item (2) of Example 1.
More phosphorylation and ethanol precipitation, T-2 in S-2
In T-1, an equimolar amount of an aqueous solution of S-1 is added and mixed,
Annealing is performed in the same manner as in item (2) of Example 1.
Was. The sugar source is not expressed in a glucose medium,
GAL1 pro expressed in lactose medium (deregulated)
It has a motor and a uracil production gene as a marker.
The BglII and SalI sites of the plasmid pKOM2 containing
Cleavage (all used restriction enzymes are manufactured by Toyobo Co., Ltd.)
Plasmid DNA and the two annealins obtained above
And DNA ligation kit (Takara Shuzo)
Ligated in vitro using the recombinant vector p
KOMLF was constructed (see FIG. 3). This was transformed into E. coli JM109 strain (Toyobo Co., Ltd.)
The same calcium chloride method as in item (2) of Example 1 was applied to
LB cold containing 50 μg / ml ampicillin
Transformants were selected using a natural medium. As a result, DN
3 × 10 per μg of A^FourColonies were obtained. Of these, five clones (LFJM
The strain was named Y1-5 strain. ) For lactoferricin
The base sequence of the connecting part with the
The same as in item (2) of Example 1
Nase Version 2.0 DNA Sequencing Kit
Confirmed using Example 6 (using the recombinant vector created in Example 5)
Production of lactoferricins) (1) A form holding the recombinant vector prepared in Example 5
Creating transmutants From the LFJMY1 strain obtained in Example 3,
The recombinant vector pKOML was prepared in the same manner as in (1).
F was amplified and prepared. This vector DNA is transferred to a standard reagent.
Titium method [DNA Cloning, 2nd
Vol. 53, IRL Press, 198
5 years] with S. cerevisiae
visiae) Introduced to INVSC2 strain (Invitrogen)
Uracil-free minimal synthetic medium [50 μg / ml L
-Histidine, 2% casamino acid (manufactured by Difco), 0.
67% Bacto yeast nitrogen base (amino acid
Nothing. 2% glucose, 2% agar (Difco)
And the transformant was selected at 30 ° C.
When selected, 200 colonies per μg of DNA
was gotten. (2) The transformant prepared in the above step (1) is
Confirmation of producing ferricins Twelve strains were selected from the transformants obtained in the above (1) and (L
Named FSC1 to 12 strains. ) Sugar source from 2% glucose
Uracil-free minimal synthetic medium changed to 2% raffinose
Incubate with 5 ml at 30 ° C for 40 hours,
% Galactose was added, and the cells were further cultured for 8 hours. culture
The solution is centrifuged to collect the cells, and spheroplast buffer (1
2.5 μg / ml zymolyase, 1 M sorbitol,
10 mM 2-mercaptoethanol, 10 mM EDT
A) Suspend in 1 ml and incubate at 30 ° C for 1 hour
Plast, collect by centrifugation, and add PBS buffer
[150 mM sodium chloride, 16 mM hydrogen phosphate
Thorium, 4 mM sodium dihydrogen phosphate (pH 7.
3)] Resuspend in 500 μl and add spheroplast
After sonication, centrifuge again to melt GST-lactoferricins.
A cell-free extract expected to contain the synthetic protein was obtained. Next, in the same manner as in (2) of Example 2,
Anti-human lactoferrin polyclonal antibody IgG fraction
(Made by Kappel) and the ELISA method.
The GST-lactoferricins fusion protein was added to the cell-free extract.
Was confirmed to be included. (3) Producing lactoferricins in the step (2)
Culture of transformants confirmed to be The GST-lactoferricins fusion protein is produced in the above (2).
Transformants LFSC1-12 strains confirmed to produce
Of the LFSC1 strain, the sugar source was changed from 2% glucose to 2%.
Uracil-free minimal synthetic medium converted to 5% raffinose,
Preculture at 30 ° C., and add 1%
Add the volume and incubate at 30 ° C for 60
And then add 3% galactose to a final concentration of 3%.
And cultured for 12 hours. (4) Lactoferfate from the culture solution obtained in the step (3)
Purification of ricins The culture obtained in the above step (3) is centrifuged to collect cells,
Ultrasonic disruption of cells turbid in 0 ml of PBS buffer
And centrifuged at 12,000 xg for 20 minutes to remove cell-free extract
Got. This cell-free extract is used as an affinity gel for GST.
Glutathione Sepharose 4B column, rum
After washing with PBS buffer, dissolve
Buffer [5 mM reduced glutathione-50 mM
Squirrel (pH 8.0)]
The fusion protein was eluted, and the protein was eluted in the same manner as in item (4) of Example 2.
0.5% factor Xa based on the weight of the material
Cut the GST-lactoferricins fusion protein overnight.
And the resulting lactoferricins were treated with ODS-120T
Reversed-phase high-performance liquid chromatography using Tosoh Corporation as a carrier.
Fractionation and purification by electrophoresis, and electrophoretically uniform fractions (protein
About 4 mg as a white mass) was obtained. Example 7 (Recombinant vector using animal cell line vector
Construction) PGEXLF prepared in Example 1 was replaced with restriction enzymes BalI and
And EcoRI (all restriction enzymes used are manufactured by Toyobo Co., Ltd.)
From the methionine residue at position 81 of GST
And lactoferricins (the amino acid sequence of SEQ ID NO: 5)
Anti-bacterial peptide).
The ends of the 565 base pair DNA sequence are DNA blunted.
Blunting kit (Takara Shuzo) and DNA
Using a ligation kit (Takara Shuzo), NotI
NAKER (Takara Shuzo) in vitro
Cut with elementary NotI (Toyobo Co., Ltd.) to give a NotI site
DNA sequence, RSV-LTR promoter and
Containing the lac operator sequence in the
Plasmid pRSV containing the neomycin resistance gene
NotI site of Not (Stratagene) restriction enzyme
Cut with NotI (Toyobo Co., Ltd.)
Dephosphorylate the 5 'end using Vatase (Toyobo)
And the DNA ligation kit (Takara)
Using in vitro and recombinant vector
PRSVLF was constructed (see FIG. 4). This was transformed into E. coli JM109 strain (Toyobo Co., Ltd.)
To the same calcium chloride method as in item (2) of Example 1.
LB agar containing 50 μg / ml ampicillin
Transformants were selected using the medium. As a result, DNA
8 × 10 per 1 μg⁶Colonies were obtained. this
Among them, 12 clones (LFJMM1 to 12 strains and
Named. )), The same method as in item (2) of Example 1.
To prepare the plasmid DNA, and ligate it with the restriction enzyme NotI (E.
NotI digestion of the inserted fragment
Confirmed existence. Example 8 (using the recombinant vector created in Example 7)
Production of lactoferricins) (1) A form holding the recombinant vector prepared in Example 7
Confirm that transgenic plants can grow From the LFJMM1 strain obtained in Example 7, (1) of Example 2
Increase the recombinant vector pRSVLF by the same method
Width and prepared. This vector DNA was prepared by IPTG.
A plasmid containing a deregulated lacI repressor gene
With Rasmid p3'SS (Stratagene),
Kit containing Rasmid p3'SS and pRSVNot
Rack Switch Inducible Manmarian
・ Expression system [(LAC SWITCH Induc
ible Mammalian Expression System), Stratagene
According to the protocol (instruction manual)
Calcium phosphate method [Molecular cloning
(Molecular Cloning), Second Edition, Volume 3, Chapter 16, Chapter
Page 33, Cold Spring Harbor Labora
Tree Press (Cold Spring Harbor Laboratory Pres)
s), 1989] using hamster CHO (CH
O-K1) Transfected into cells (Dainippon Pharmaceutical Co., Ltd.)
Using DMEM containing 10% calf serum at 37 ° C.
_TwoAfter culturing for 18 hours in the incubator, the medium was renewed.
Replace with fresh one and incubate for 24 hours, 1 mM IPTG
Was added and the cells were further cultured for 12 hours. This culture plate
Cells were fixed with 3.7% formaldehyde [Cell Engineering
Science Experiment Protocol, page 204, Shujunsha, 199
1 year], rabbit anti-human lactoferrin polyclonal
Using the antibody IgG fraction (Kappel) as the primary antibody
Gimmun Blot Kit HRP (Bio-Rad)
Was used to perform immunochemical cell staining. As a result, 6% of all cells were GST
From the 81st methionine residue on and lactoferricins
It was confirmed that a fusion protein was produced. Also this
That the strain producing the fusion protein is viable
Was also confirmed. (2) A form holding the recombinant vector created in Example 7
Creating transmutants The recombinant vector pRSVLF prepared in the above (1) is
Using the calcium phosphate method, Stratagene
Introduce p3'SS prepared based on the protocol
Introduced into CHO cells stably expressing lac repressor
And 10% calf serum in DMEM at 37.degree.
_TwoIncubate for 18 hours in an incubator and add 10% medium
Calf serum and 400 μg / ml geneticin sulfate
Salt (G418, an antibiotic with the same effect as neomycin)
Substance) and exchanged for DMEM containing
Cultivated for an additional 2 weeks while exchanging
Colonies were obtained. (3) The transformant prepared in the step (2) is
Confirmation of producing ferricins Select 50 strains from the transformants obtained in the above (2)
(Named as LFCHO1-50 strain) Transfer to 96-well plate
And aliquot the culture in each well into a separate 96-well plate.
And transferred to each corresponding hole to create a replica plate.
Next, 1 mM IPTG was added, and the cells were further cultured for 12 hours.
The replica plate thus grown was treated similarly to (1) above.
Heron anti-human lactoferrin polyclonal antibody IgG
Using immunoassay (Kappel),
6 strains (LFCHO 7 strains, 13 strains, 19 strains, 24 strains
Shares, 39 shares and 48 shares) of GST
Between the first methionine residue and the lactoferricins
It was confirmed that the fusion protein was produced. (4) Producing lactoferricins in the step (3)
Culture of transformants confirmed to be In (3) above, the methionine residue after the 81st position of GST and
Confirmed to produce fusion protein with lactoferricins
Of the 6 transformed transformants, 7 strains of LFCHO were
% Calf serum and 400 μg / ml G418
Using 100 ml of DMEM at 37 ° C._Twoink
Preculture in an incubator.
At 37 ° C. in a rotating bottle,_TwoI
After culturing for 3 days in an incubator, 1 mM IPT
G was added and the cells were further cultured for 12 hours. (5) Lactoferfate from the culture solution obtained in step (4)
Purification of ricins The culture solution obtained in the step (4) is centrifuged to collect cells,
Sonicate cells suspended in 50 ml of PBS buffer
Crush and centrifuge at 10,000 xg for 20 minutes for cell-free extraction
A liquid was obtained. This cell-free extract is separated with Sephadex G-150 (Fal
Gel filtration column (25 mm, manufactured by Macia)
(Diameter, 20 cm length)
From the methionine residue at position 81 of GST
The fraction containing the fusion protein with elicins was identified. Next, the protein was prepared in the same manner as in item (4) of Example 2.
Add 0.5% Factor Xa to white matter weight and add at room temperature
The fusion protein was cleaved overnight and the resulting lactoferric
Using ODS-120T (manufactured by Tosoh Corporation) as a carrier
Purification and fractionation by two-phase high-performance liquid chromatography
Electrophoretically uniform fraction (about 3 mg as protein) was obtained
Was. [0041] As described in detail above, the present invention
Anti-microbial peptides (lactoferricins)
A recombinant vector into which a DNA sequence to be inserted is
A transformant carrying the recombinant vector, and
Provided is a method for producing lactoferricins using transformants
Is done. This is superior to natural lactoferrin.
Lactoferricins with high antibacterial properties
It is possible to easily supply a large amount. [0042] [Sequence list] SEQ ID NO: 1 Array length: 11 Sequence type: amino acid Topology: linear Sequence type: Peptide Sequence characteristics: Flag this peptide and this peptide
Pepdo included as a comment. In the following sequence, X01 is L-A
Lanine residue, L-arginine residue, L-asparagine residue,
L-aspartic acid residue, L-cysteine residue, L-glutami
Residue, L-glutamic acid residue, L-glycine residue, L-His
Tidine residue, L-isoleucine residue, L-leucine residue, L-
Lysine residue, L-methionine residue, L-phenylalanine residue
Group, L-proline residue, L-serine residue, L-threonine residue
Group, L-tryptophan residue, L-tyrosine residue or L-ba
Represents a phosphorus residue, and X02 represents an L-arginine residue or L-lysine
Indicates a residue. Array SEQ ID NO: 2 Array length: 11 Sequence type: amino acid Topology: linear Sequence type: Peptide Origin: Homo (human) Array SEQ ID NO: 3 Array length: 11 Sequence type: amino acid Topology: linear Sequence type: Peptide Origin: Bos (bovine) Array SEQ ID NO: 4 Array length: 11 Sequence type: amino acid Topology: linear Sequence type: Peptide Origin: Mus (mouse) Array SEQ ID NO: 5 Array length: 25 Sequence type: amino acid Topology: linear Sequence type: Peptide Origin: Homo (human) Array SEQ ID NO: 6 Array length: 54 Sequence type: amino acid Topology: linear Sequence type: Peptide Origin: Homo (human) Array SEQ ID NO: 7 Array length: 4 Sequence type: amino acid Topology: linear Sequence type: Peptide Array SEQ ID NO: 8 Array length: 90 Sequence type: nucleic acid Number of chains: double strand Topology: linear Sequence type: other nucleic acid synthetic DNA Array [Note 1] ↓ or △: Cut for oligonucleotide preparation
Cutting position [Note 2] Restriction enzyme name: for ligation to plasmid vector
Name of the restriction enzyme that acts on the restriction enzyme cleavage site [Note 3] Lowercase letters: for ligation to plasmid vector
Restriction enzyme site (base sequence) SEQ ID NO: 9 Sequence length: 35 (E-1), 63 (E-2), 54 (F-1), 40 (F
-2) Sequence type: nucleic acid Number of chains: single strand Topology: linear Sequence type: other nucleic acid synthetic DNA Array E-1 5'-GATCC ATCGAAGGTC GTACTAAATG CTTCCAGTGG-3 ' E-2 5'-CAGCGTAACA TGCGTAAAGT ACGTGGTCCG CCGGTTTCTT
 GCATCAAACG TGACTCCTAAGTC-3 ' F-1 3'-G TAGCTTCCAG CATGATTTAC GAAGGTCACC GTCGCATT
GT ACGCATTTCA TGC-5 ' F-2 3'-ACCAGGC GGCCAAAGAA CGTAGTTTGC ACTGAGGATTCAG
-Five' SEQ ID NO: 10 Array length: 180 Sequence type: nucleic acid Number of chains: double strand Topology: linear Sequence type: other nucleic acid synthetic DNA Array[Note 1] ↓ or △: Cut for oligonucleotide preparation
Cutting position [Note 2] Restriction enzyme name: for ligation to plasmid vector
Name of the restriction enzyme that acts on the restriction enzyme cleavage site [Note 3] Lowercase letters: for ligation to plasmid vector
Restriction enzyme site (base sequence) SEQ ID NO: 11 Sequence length: 43 (L-1), 50 (L-2), 50 (L-3), 40 (L
-4), 63 (M-1), 50 (M-2), 50 (M-3), 24 (M-4) Sequence type: nucleic acid Number of chains: single strand Topology: linear Sequence type: other nucleic acid synthetic DNA Array L-1 5'-CCA TCATCGAAGG TCGTGGCCGT CGTCGTCGTT CTGTTC
AGTG-3 ' L-2 5'-GTGCGCAGTA TCCCAGCCGG AAGCTACCAA ATGCTTCCAG
 TGGCAGCGTA-3 ' L-3 5'-ACATGCGTAA AGTACGTGGC CCGCCGGTTT CTTGCATCAA
 ACGTGACTCC-3 ' L-4 5'-CCGATCCAGT GTATCCAGGC AATCGCGGAA AACCGTTAAG
-3 ' M-1 3'-GGT AGTAGCTTCC AGCACCGGCA GCAGCAGCAA GACAAG
TCAC CACGCGTCATAGGGTCGGCC-5 ' M-2 3'-TTCGATGGTT TACGAAGGTC ACCGTCGCAT TGTACGCATT
 TCATGCACCG-5 ' M-3 3'-GGCGGCCAAA GAACGTAGTT TGCACTGAGG GGCTAGGTCA
 CATAGGACCG-5 ' M-4 3'-TTAGCGCCTT TTGGCAATTC CTAG-5 ' SEQ ID NO: 12 Array length: 90 Sequence type: nucleic acid Number of chains: double strand Topology: linear Sequence type: other nucleic acid synthetic DNA Array [Note 1] ↓ or △: Cut for oligonucleotide preparation
Cutting position [Note 2] Restriction enzyme name: for ligation to plasmid vector
Name of the restriction enzyme that acts on the restriction enzyme cleavage site [Note 3] Lowercase letters: for ligation to plasmid vector
Restriction enzyme site (base sequence) SEQ ID NO: 13 Sequence length: 35 (S-1), 61 (S-2), 54 (T-1), 42 (T
-2) Sequence type: nucleic acid Number of chains: single strand Topology: linear Sequence type: other nucleic acid synthetic DNA Array S-1 5'-GATCC ATCGAAGGTA GAACTAAGTG TTTCCAATGG-3 ' S-2 5'-CAAAGAAACA TGAGAAAGGT TAGAGGTCCA CCAGTTTCTT
 GTATCAAGAGAGACTCCTAA G-3 ' T-1 3'-G TAGCTTCCAT CTTGATTCAC AAAGGTTACC GTTTCTTT
GT ACTCTTTCCT ATC-5 ' T-2 3'-TCCAGGT GGTCAAAGAA CATAGTTCTC TCTGAGGATT CA
GCT-5 '

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a flow sheet for the construction of plasmid pGEXLF. FIG. 2 shows a flow sheet for the construction of plasmid pGEXLNL. FIG. 3 shows a flow sheet for the construction of plasmid pKOMLF. FIG. 4 shows a flow sheet for the construction of plasmid pRSVLF.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI C12R 1:91) (C12P 21/02 C12R 1:91) (72) Inventor Tomoko Kojima 5-1-83 Higashihara, Zama City, Kanagawa Prefecture Morinaga Milk Products Co., Ltd. Food Research Institute (56) References JP-A-5-238948 (JP, A) JP-A-5-92994 (JP, A) Tokuhyo Hei 4-505101 (JP, A) International publication 93 / 022348 (WO, A1) Hirotake Horikoshi et al., Genetic Engineering for Engineering, Kodansha Co., Ltd., March 10, 1995, p. 163-164, (6.2.3 Codon usage) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 15/00-15/90 C07K 14/00-14/825 JSTPlus (JOIS) BIOSIS / WPI (DIALOG)

Claims (1)

(57) [Claims] [Claim 1] The following steps: (1) A DNA sequence consisting of the nucleotide sequence of SEQ ID NO: 8 is inserted and ligated into a BamHI-SmaI cleavage site of an Escherichia coli vector pGEX 2T to perform recombination. Constructing the vector pGEXLF, (2) combining the recombinant vector pGEXLF with the restriction enzymes BalI and
DN consisting of a 565 base sequence obtained by digestion with EcoRI
A step of preparing a DNA sequence in which NotI sites are added to both ends of the A fragment;
(4) inserting the recombinant vector and inserting DN of the vector;
A step of introducing a plasmid p3'SS containing a lacI repressor sequence that controls the expression of the A sequence into animal cells to prepare a transformant; (5) culturing the transformant; and (6) culturing the IPTG. A step of culturing the transformant by releasing its expression control by the addition; (7) a fusion protein [GST +
A peptide consisting of the amino acid sequence of SEQ ID NO: 7 + an antimicrobial peptide consisting of the amino acid sequence of SEQ ID NO: 5), and (8) cutting the collected fusion protein with Factor Xa to obtain the amino acid sequence of SEQ ID NO: 5. Recovering the antimicrobial peptide.