JPH09295998A - Protein and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new protein having a cell adhesion activity, capable of adhering epithelial cells to a culture container to proliferate the cells, and useful as a culture substrate, etc., by extracting the cell surface protein of bifidobacterium with an alkali metal salt or a surfactant. SOLUTION: This new protein is originated from a bifidobacterium and has a cell adhesion activity. The protein can efficiently adhere cells, especially epithelial cells, to a culture container to proliferate the cells, and is useful as a substrate for the culture of the cells. The new protein is obtained by extracting the cell surface protein of a bifidobacterium such as Bifidobacterium longum, Bifidobacterium adlescentis, Bifidobacterium breve or Bifidobacterium infanitis with an alkali metal salt or a surfactant.

Description

Detailed Description of the Invention

[0001]

[0001] The present invention relates to a novel protein and a method for producing the same. More specifically, the present invention relates to a novel protein derived from Bifidobacterium and having epithelial cell adhesion activity, and a method for producing the protein, which comprises extracting a cell surface protein from the Bifidobacterium. INDUSTRIAL APPLICABILITY The protein of the present invention is very useful as a substrate for culture or the like for allowing epithelial cells to efficiently adhere to and grow in a culture vessel.

[0002]

2. Description of the Related Art At present, cultured cells are widely used for biological and medical research. When performing cell culture, especially when a new cell line is established or when cryopreserved cells are newly cultured, adhesion of the cells to the culture vessel is important. In particular, epithelial cells have an adhesion-dependent property (anchorage-
dependent) cells, the cell adhesion is low when a normal culture vessel is used, cell growth is delayed,
It is known that culture is not performed efficiently. Therefore, conventionally, in order to improve the adhesion of cells, particularly epithelial cells, to a culture vessel, it is generally practiced to coat the surface of the culture vessel with a cell adhesive protein such as collagen or gelatin derived from animal tissues. And coated containers are commercially available. Collagen is a cell-adhesive protein contained in animal connective tissue and having a role of adhering epithelial cells to connective tissue, and is divided into several types such as type I to type V.

[0003]

However, at present, collagen does not have a large-scale preparation method using genetic engineering techniques or the like, and must be separated and purified from animal tissues which are relatively difficult to obtain. Preparation is burdensome both in terms of labor and cost. In addition, commercially available collagen is very expensive and is not commonly used for cell culture on a daily basis. As other cell adhesion proteins, vitronectin, laminin, fibronectin and the like are known,
All are derived from animal tissues, are more expensive than collagen, and have not been commonly used for cell culture.

[0004] Accordingly, the present inventors aimed at solving the above-mentioned problems of the prior art with the object of providing a novel protein which has cell adhesiveness and can be easily and inexpensively produced. We continued our research. As a result, the inventors have found that a novel protein having cell adhesion activity is present in the cell surface protein of Bifidobacterium and a method for easily and inexpensively producing the protein has been completed, and thus the present invention has been completed.

[0005]

[Means for Solving the Problems] That is, the present invention is a protein derived from Bifidobacterium and having cell adhesion activity. The present invention is also the above protein, wherein the cell adhesive activity is an adhesive activity to epithelial cells. The present invention also provides that the Bifidobacteria are Bifidobacteria longum.
ium longum), Bifidobacterium adlescentis, Bifidobacterium breve, or Bifidobacteria infantis (Bifidobacter)
ium infantis). The present invention also relates to a case where Bifidobacterium longum is Bifidobacterium longum SBT2928 strain (FERM-P10.
657), Bifidobacterium addressacetis JCM1
275 strain, Bifidobacterium breve strain Bifidobacterium breve SBT2803 (FERM-
(P15532), Bifidobacterium infantis SBT
The above protein is the 2852 strain (FERM-P15533). The present invention is also a polyclonal antibody against the protein. The present invention is also the method for producing the above protein, which comprises extracting the cell surface protein from Bifidobacterium using an alkali metal salt or a surfactant.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention is described in detail below.
Bifidobacteria that can be used in the present invention,
Although not particularly limited, it is more preferable to select from bacterial species and strains that colonize the intestine of the animal from which the cultured cells are derived.
The Bifidobacterium that can be used for culturing human cells is not particularly limited, but examples of preferable ones include Bifidobacterium longum (Bifidobacterium longum) and Bifidobacterium adressentis (Bifidobacte).
rium adlescentis), Bifidobacterium breve
(Bifidobacterium breve), Bifidobacterium infantis, and the like. Among these, Bifidobacterium longum, particularly Bifidobacterium longum SBT2928 strain is preferable. The Bifidobacterium longum SBT2928 strain was designated as FERM-P10657 on April 13, 1989 by the Institute for Microbial Engineering, Ministry of International Trade and Industry (now the Institute of Biotechnology, Ministry of International Trade and Industry). Has been deposited. Also, Bifidobacterium adressesentis, particularly Bifidobacterium adressentis JCM1275 is preferable. In addition, Bifidobacterium breve, especially Bifidobacterium breve SBT2803 strain is preferable, and the Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology
Deposited on March 27, 1996 as FERM-P15532. In addition, Bifidobacterium infantis, particularly Bifidobacterium infantis SBT2852 strain is preferable, and is designated as FERM-P15533 by the Institute of Biotechnology, Ministry of International Trade and Industry on March 27, 1996. Has been deposited with.

[0007] The cells to which the protein of the present invention adheres are not particularly limited, but are highly useful for epithelial cells whose adhesion is particularly important in culture, and are suitable for most epithelial cells. Available. Further, since Bifidobacteria inhabit the intestines of animals, the protein of the present invention is effective for culturing intestinal epithelial cells. The protein having cell adhesion activity of the present invention includes not only its active fraction, but also those containing the active fraction, for example, those obtained by separating only the surface protein of the microbial cell from the bacterium of Bifidobacterium. . That is, the protein having cell adhesion activity of the present invention is a cell surface protein extracted from cells of Bifidobacterium,
And a protein obtained by purifying it.

[0008] The protein of the present invention can be produced, for example, by the following method. First, bifidobacteria are cultured overnight in a medium, and the bacterial cells are collected by centrifugation.
Preferred strains or strains of Bifidobacterium are as described above. As the medium, BL medium, BLA medium, GPV
A medium or the like can be used, and the culture is preferably performed at 37 ° C. for about 16 hours. Centrifugation is 5000 rpm
Then, it is preferable to perform it for about 15 minutes. Next, after the collected cells are suspended in ion-exchanged water, the cells are repeatedly recovered by centrifugation to prepare washed cells. In this case, the centrifugation is preferably performed at 5000 rpm for about 15 minutes, and it is preferable to repeat this three to four times.

Next, a surface protein is extracted from the washed cells thus obtained. For the extraction of the surface layer protein, a known method such as a method using an alkali metal salt or a method in which the cells are crushed and then a membrane fraction is obtained to use a surfactant can be used. Among them, in the case of the method using an alkali metal salt, an alkali metal salt solution having a high concentration of 1 M or more is prepared in advance, added to the washed cells, and extracted by vigorously stirring while cooling with ice. Is preferred. The alkali metal salt is not particularly limited, and examples thereof include lithium chloride, sodium chloride, and potassium chloride. Then, this is centrifuged at about 7,000 rpm for about 20 minutes to obtain a crude extract of Bifidobacteria surface layer protein in the supernatant. Next, the obtained crude extract is dialyzed against ion-exchanged water to precipitate a surface protein as a precipitate and to remove low molecular impurities.
For dialysis, it is preferable to use a dialysis membrane having a molecular weight fraction of 6,000 to 8,000. Next, the precipitated surface protein is collected by centrifugation, washed several times with ion-exchanged water, and then freeze-dried to obtain a dried sample of the protein of the present invention.

[0010] Further, in the case of a method using a surfactant by obtaining a membrane fraction after disrupting the cells, for example, first, N-acetyl is added to the washed cells prepared as described above. Add a muramidase solution (50 μg / ml) and add
By allowing to stand for 0 minutes, the cell wall is lysed and turned into protoplasts. Next, the cells are collected by centrifugation at 3000 rpm for 20 minutes, and then the cells are ruptured by adding distilled water. The disruption of the cells can also be performed by physical treatment such as ultrasonic treatment or French press. The disrupted bacterial solution was centrifuged at 5000 rpm for 10 minutes,
After removing the unbroken cells that precipitate, the membrane fraction is precipitated by centrifugation at 15,000 rpm for 30 minutes. Surface proteins are extracted from the precipitated membrane fraction using a surfactant. The surfactant used at this time is not particularly limited. However, considering that it is later removed by dialysis, 0.5 to 2% of CH is used.
AP or octylglucoside is desirable. Next, the obtained crude extract is dialyzed against ion-exchanged water to precipitate a surface protein as a precipitate and to remove low molecular impurities. For dialysis, it is preferable to use a dialysis membrane having a molecular weight fraction of 6,000 to 8,000. Next, the precipitated surface layer protein is collected by centrifugation, washed centrifugally with ion-exchanged water several times, and then freeze-dried,
A dry preparation of the protein of the present invention can be obtained.

[0011] Further, the thus-extracted bacterial cell surface protein of the present invention is further purified by a conventional purification method such as SDS-PAGE to obtain an active fraction of the protein of the present invention. it can.

The protein of the present invention thus produced is dissolved in distilled water and coated on a culture vessel such as a commercially available polystyrene dish or plate to easily obtain a substrate for cell culture. You can still,
The concentration at which the protein of the present invention is coated on a culture vessel is a coating solution (usually a 5M lithium chloride solution).
It is desirable that the amount of the protein of the present invention is 0.1 to 10 mg per ml.

The protein of the present invention can be identified, for example, by the following method. First, epithelial cells are cultured in a cell culture medium until they reach confluence, the cells are detached by trypsin-EDTA treatment, and then centrifugally washed using phosphate-buffered saline (PBS). Suspend in PBS. On the other hand, the protein of the present invention was subjected to SDS-polyacrylamide gel electrophoresis,
After separating the proteins by molecular weight, the separated proteins are transferred to a nitrocellulose membrane using a blotting apparatus, and blocked at 4 ° C. overnight in PBS containing 1% bovine serum albumin (BSA). Epithelial cells suspended in PBS are overlaid on the membrane after blocking,
, The epithelial cells adhere to the protein having cell adhesion activity. Subsequently, after the membrane was washed with PBS, the adhered cells were fixed with a 3% paraformaldehyde solution, immersed in a 40% amide black solution, and then decolorized with methanol. Thereby, it can be identified.

The proof that the protein of the present invention thus identified acts as a cell adhesion factor is as follows.
This can be done using an antibody. The antibody can be prepared for the purified cell adhesion protein by subjecting the protein of the present invention to SDS-polyacrylamide gel electrophoresis, staining with Coomassie Brilliant Blue solution, and then cutting out the gel. Antibodies can be produced by a known method, but polyclonal antibodies are preferable to monoclonal antibodies. A suspension of intestinal cells and the antibody obtained as described above are added to a cell culture vessel coated with the protein of the present invention. In addition, a cell culture vessel to which no antibody is added is prepared as a control. If the cell adhesion of the cell incubator to which the antibody is added decreases, it can be considered that the antibody first bound to the cell adhesion protein in the surface protein of the present invention, and the cell adhesion was lost. It is demonstrated that the protein plays a role of cell adhesion in the culture vessel.

The protein of the present invention can be purified by known column chromatography such as ion exchange and gel filtration, but can be more easily purified by using an antibody against the protein of the present invention. in this case,
Antibodies may be polyclonal or monoclonal. First, an antibody column was prepared by immobilizing the obtained antibody on a carrier for protein immobilization such as cyanogen bromide-activated Sepharose 4B, and the above-mentioned surface protein dissolved in PBS was applied to this column. Load. After washing the column, the protein bound to the antibody is eluted with a suitable eluate, whereby the target purified protein can be obtained. The purified protein thus obtained can also be used as a substrate for cell culture by the same operation as the case of the surface protein, and a high cell adhesion effect can be obtained with a smaller amount of protein than the surface protein. Is possible. However, surface cell proteins are sufficient for general cell culture, which is economically advantageous.

When the thus produced protein of the present invention is inoculated with intestinal epithelial cells, the cells can be more efficiently adhered and proliferated as compared with conventional culture substrates.
The culture period can be shortened. In particular, primary culture cells have a significantly higher engraftment rate and are useful for establishing cell lines.

[0017]

The present invention will be described in more detail with reference to the following examples, which are for the purpose of illustration only.
The present invention is not limited to these.

Example 1 (Production of Protein of the Present Invention) Precultured Bifidobacterium longum SBT2928 strain was inoculated into 5 L of BLA medium at 3% and cultured at 37 ° C. for 24 hours. The cultured cells were collected by centrifugation at 7000 rpm for 20 minutes, and the cells were washed by centrifugation using ion-exchanged water. To the obtained washed cells, 3 times a 5 M lithium chloride solution was added,
The surface layer protein was extracted by vigorous stirring for 30 minutes while cooling with ice, and the supernatant obtained by centrifugation was taken to obtain a surface layer protein extract. This extract was passed through a dialysis membrane (molecular weight fraction 6,00
After dialysis against ion-exchanged water (0 or less), the precipitated surface protein was recovered by centrifugation and freeze-dried to obtain a freeze-dried preparation.

Test Example 1 (Confirmation of Cell Adhesion Activity) Obtained from the freeze-dried preparation of the dialyzed precipitate fraction obtained in Example 1 and the protein solubilized in the supernatant (dialysis-soluble fraction). Lyophilized preparations (comparative products) were each dissolved in a 5M lithium chloride solution to give 0.001-
A solution having each concentration up to 10 mg / ml was prepared and 100 μL was overlaid on a 96-well microplate. Each protein was coated by allowing it to stand overnight at 4 ° C., and then washed twice with PBS. On the other hand, the small intestine-derived cells HCT-8
After culturing in a PMI1640 medium until reaching a confluent, cells were detached by trypsin-EDTA treatment, and then centrifugal washing with PBS at 1000 rpm for 5 minutes was repeated 3 times to suspend in PBS. This cell suspension was layered on the above plate and allowed to stand at 37 ° C. for 1 hour,
The plate was washed, observed under a microscope, and the number of attached cells was measured by hexosaminidase activity. The results are shown in FIG. As is clear from FIG. 1, stronger cell adhesion activity was observed in the dialysis-precipitated fraction than in the dialysis-soluble fraction.

Test Example 2 (Identification of Active Fraction and Measurement of Molecular Weight) 2.5 g of the dried protein of the surface layer protein of Bifidobacterium longum SBT2928 obtained in Example 1 was dissolved in 200 L of sample buffer, SDS-polyacrylamide gel electrophoresis was performed using 7 μL of the mixture to separate proteins by molecular weight. The separated protein was transferred to a nitrocellulose membrane using a blotting apparatus (TEFCO) and blocked overnight in phosphate buffered saline (PBS) containing 1% bovine serum albumin (BSA) at 4 ° C. . On the other hand, cells HCT-8 derived from the small intestine were transferred to RPMI164
After culturing in 0 medium until reaching confluent and detaching cells by trypsin-EDTA treatment, centrifugal washing with PBS at 1000 rpm for 5 minutes was repeated 3 times to obtain PB.
Suspended in S. The membrane after blocking was immersed in this cell suspension and gently shaken at 37 ° C. for 1 hour. Subsequently, this membrane was washed with PBS, cells adhered with a 3% paraformaldehyde solution were fixed, immersed in a 40% amide black solution, and then decolorized with methanol,
The protein to which the cells adhered was stained. The same procedure was used to test other strains of Bifidobacterium, Bifidobacterium adrensetis, Bifidobacterium breve, and Bifidobacterium infantis. . The results are shown in FIG. As shown in FIG. 2, it was revealed that the cell-adhesive protein is present in all these Bifidobacterium strains.

Test Example 3 (Determination of N-terminal amino acid sequence) The N-terminal amino acid sequence of the protein having a molecular weight of 28 kD derived from the Bifidobacterium longum SBT2928 strain obtained in Example 2 was analyzed. That is, the surface layer protein extracted with 5M lithium chloride in Example 1 was used as a preparative SD using 10% gel.
S-PAGE (TEFCO-CELL TC-16; T
Made by EFCO, and 491 PREP CELL; BIO
-Made by RAD), and the active fraction is 28 kD
The protein was recovered by elution. The recovered purified protein was again analyzed by SDS-PAGE (10% gel, C-808;
After being supplied to TEFCO, CAPS buffer (pH
11) under the condition of 50V, 60 minutes, PVDF membrane (0
3056; manufactured by TEFCO).
The protein (equivalent to 4 μg) on the obtained membrane was stained with Coomassie brilliant blue dissolved in 40% methanol, then decolorized with 40% methanol, and the stained band was cut out and air-dried. The N-terminal amino acid of the excised band was determined by a protein sequencer (476A; manufactured by Perkin Elmer) according to its protocol. The results are shown in SEQ ID NO: 1 in the sequence listing.

Test Example 4 (Cell Adhesion Test) According to the method of Test Example 2, a cell adhesion test of the protein of the present invention to Hudu-80 derived from duodenum, Caco-2 derived from large intestine and KATOIII derived from stomach was performed. . The results are shown in FIG. As is clear from FIG. 3, the protein of the present invention was stained as a cell-adhesive protein regardless of which cell was used. That is, the protein of the present invention has adhesiveness to many epithelial cells,
It was confirmed to be effective for culturing.

Example 2 (Preparation of Polyclonal Antibody) 2.5 g of a dried preparation of the protein of the present invention was dissolved in 200 L of sample buffer,
SDS-polyacrylamide gel electrophoresis was performed using 7 L of them. After staining with Coomassie Brilliant Blue solution, the protein of the present invention was excised and purified by electroelution. Balb / c mice were immunized with the purified protein of the present invention. Immunization was performed four times every other week, the first immunization was performed by mixing with Freund's complete adjuvant (FCA), and the subsequent immunization was performed by mixing with Freund's incomplete adjuvant (FIA). After the final immunization,
MethA cells that cause ascites-type cancer were injected into the abdominal cavity of mice. One week later, the ascites accumulated in the abdominal cavity was collected to obtain a polyclonal antibody against the protein of the present invention.

Test Example 5 (Cell Adhesion Test of Protein of the Present Invention by Polyclonal Antibody) Using the polyclonal antibody against the protein of the present invention obtained in Example 2, cell adhesion test of the protein of the present invention was carried out. . That is, using the microplate coated with the protein of the present invention, HCT was conducted according to the method of Test Example 1.
An adhesion test of -8 cells was performed. At that time, the obtained polyclonal antibody was added to the cell suspension at a dilution ratio of 3 to 50 times and overlaid on the plate. FIG. 4 shows the results. For comparison, the same test was performed in the case of adding an antibody (ascites fluid) of a non-immunized normal mouse,
Results are shown in FIG. Furthermore, the same test was performed in the case where a polyclonal antibody against all the Bifidobacterium longum SBT2928 strains was added, and the results are shown in FIG. As is clear from FIGS. 4 to 6, when the antibody to the protein of the present invention was added, compared with the case where the antibody of a normal mouse was added and the case where the polyclonal antibody against all strains of Bifidobacterium longum was added. , Cell adhesion was greatly reduced. From this, it was confirmed that the protein of the present invention was a protein having cell adhesion activity in a culture vessel.

[0025]

As described above, according to the present invention,
Provided is a novel protein having cell adhesion activity derived from Bifidobacterium, and a method for producing the protein, which comprises extracting a cell surface protein from Bifidobacterium. INDUSTRIAL APPLICABILITY The novel protein of the present invention is very useful as a substrate for culture and the like for efficiently adhering cells, especially epithelial cells, to a culture vessel and growing the cells.

[Brief description of drawings]

FIG. 1 shows the cell adhesiveness of a precipitate fraction (invention) and a soluble fraction (comparison) after dialysis of Bifidobacterium longum surface protein.

[Explanation of symbols]

 ○: dialysis precipitation fraction ●: dialysis soluble fraction

FIG. 2 shows the molecular weights of cell adhesion proteins obtained from each strain of Bifidobacterium by SDS-PAGE.

[Explanation of symbols]

 Lane 1: Molecular weight marker Lane 2: Cell surface protein Lane 3: Cell adhesion protein

FIG. 3 shows the cell adhesion of the protein of the present invention to various epithelial cells.

[Explanation of symbols]

 Lane 1: Molecular weight marker Lane 2: Cell surface protein Lane 3: Cell adhesion protein

FIG. 4 shows the cell adhesion inhibitory effect of Bifidobacterium longum by a polyclonal antibody against the protein of the present invention.

FIG. 5 shows the cell adhesion inhibitory effect of Bifidobacterium longum by the antibody of normal mice.

FIG. 6 shows the results of Bifidobacterium
8 shows the cell adhesion inhibitory effect of longum.

[Sequence list]

SEQ ID NO: 1 Sequence length: 19 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: N-terminal fragment Origin Biological name: Bifidobacte longum
rium longum) Strain name: SBT2928 sequence Ala Ser Thr Glu Asn Thr Pro Val Ala Val Xaa Lys Asn Lys Ala Xaa 1 5 10 15 Phe Trp Asp

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C12N 1/20 C12N 1/20 A (C12P 21/00 C12R 1:01) (C12N 1/20 C12R 1:01)

Claims (6)

[Claims] 1. A protein derived from Bifidobacterium and having cell adhesion activity. 2. The protein according to claim 1, wherein the cell adhesion activity is an adhesion activity to epithelial cells. 3. The bifidobacteria are Bifidobacterium longum and Bifidobacterium adlesentis.
scentis), Bifidob
Acterium breve) or Bifidobacterium infantis.
Or the protein according to 2. 4. The Bifidobacterium longum is a Bifidobacterium longum SBT2928 strain (FER
M-P10657), Bifidobacterium adressetis is Bifidobacterium adressetis JCM1275 strain, Bifidobacterium breve is Bifidobacterium breve SBT2803 strain (FERM-P15532), Bifidobacterium.・
The protein according to claim 3, wherein the infantis is Bifidobacterium infantis SBT2852 strain (FERM-P15533). 5. A polyclonal antibody against the protein according to any one of claims 1 to 4. 6. The method for producing a protein according to claim 1, wherein the cell surface protein is extracted from Bifidobacterium using an alkali metal salt or a surfactant.