JP5553329B2 - Tengu silk sericin medium - Google Patents

Description

  The present invention relates to a medium containing silk sericin derived from tengu.

  Conventionally, not only the use of rabbits produced by silk insects, but also the use of silk threads etc. has been studied for wild silkworms. For example, it has been attempted to use silk proteins obtained from these by means of extraction or the like in foods and drinks for health promotion or as cosmetic ingredients. In addition, studies on medical applications are underway.

  Here, a rabbit is a kind of insect belonging to the family Bombycidae and is not domesticated because it is a domesticated insect. On the other hand, barbarian is a synonym for rabbits, Bombyc mandarina of Bombycidae, Antheraea yamamai of Saturniidae, Anteraea pernyi, Eriogyna pyretorum, It is a collective term for wild insects that produce silk thread, such as Pilosamia Cynthia ricini, Saia cynthia, chestnut worm (Caligura japonica), Chuther moth (Antheraea mylitta), and Antheraea assama. The silk thread spouted by these insects is a hard protein mainly composed of fibroin and sericin (Non-patent Document 1).

  However, in the Chinese character notation, “蚕” is used in common, but as mentioned above, Bombycidae's Rabbit and Saturniidae's Antheraea yamamai are clearly taxonomically. They are distinguished and cannot be crossed. In addition, Tengu grows by eating kunugi, willow, konara, oak, etc. other than mulberry. It is known that it is completely different (Non-Patent Document 1).

  In recent years, there has been an increasing interest in the acquisition and use of silk proteins. For example, it has been reported that silk fibroin obtained by elution from silk spun from rabbits has a cell growth promoting effect ( Patent Document 1).

  On the other hand, sericin, a silk protein, is a protein that surrounds silk fibroin, and it is known that its acquisition and use is not as easy as fibroin, but in recent years it has been based on the research results of fibroin. Sericin is also a subject of research.

  For example, as a conventional method for extracting sericin from silk derived from B.mori, silk fibers are cut short, treated with water at 140 ° C. or less as a swelling process, then treated with steam, acidic aqueous solution, alkaline Various extraction methods such as a method of recovering sericin by a process of treating with an aqueous solution or water containing an enzyme (Patent Document 2) are known. From the difference in crystallinity between the extraction method and the sericin component, molecular species and The molecular weight is not uniform. Therefore, the studies on physical properties and functionality of sericin reported so far are not sericin separated and purified, but mostly mixtures. Furthermore, it has also been reported that the action effect of sericin having a molecular weight of about 400,000 obtained by elution from silk spun from B. mori has a cell growth promoting action (Patent Document 3).

However, the silk thread of B.mori and A.yamamai is completely different in amino acid composition and higher order structure as described above. Furthermore, the silk thread of Tengu is compared with B.mori. Because of its low sericin content, high levels of inorganic salts, especially Ca (COO) ₂ and active substances such as pigments, sericin from A.yamamai It is difficult to obtain pure sericin by adopting the same extraction method as that of the derived sericin, and it is impossible to analogize the action of the extracted sericin as the same type.

  Therefore, the research on sericin derived from A.yamamai is limited to amino acid composition analysis, inorganic component analysis, and physical structure analysis. Reference 4 partially mentions silk protein derived from A. yamamai, but in fact, it mainly examines fibroin derived from B. mori. As for the specific means for obtaining sericin derived from A.yamamai, the action of the obtained sericin and the cells to be acted on, including Patent Document 4, there is no report at all at present. .

JP 2004-123683 A Japanese Patent Laid-Open No. 2004-238312 Japanese Patent Laid-Open No. 2002-128691 JP 2004-300142 A

Komatsu Keiichi, Hokujo Tadashi, Continuation of silk, 352-377, published by Shinshu University, Faculty of Textiles, March 1980

  The present invention has been made in view of the circumstances as described above, and has as its object to provide a new cell culture medium and culture method using a sericin derived from sericin that has been extracted, isolated and purified from a sword layer. Yes.

In order to solve the above problems, the present invention provides the following cell culture medium and cell culture method.
<1> A medium for culturing insect cells, which contains sericin derived from tengu.
<2> The medium according to <1>, wherein the concentration of the sericin derived from the tempura in the medium is 0.05 to 1%.
<3> A culture medium for lymphocyte culture, characterized by containing sericin derived from a tengu.
<4> The medium according to <3>, wherein the lymphocyte is a mammalian lymphocyte.
<5> The medium according to <3> or <4>, wherein the concentration of sericin derived from the tempura in the medium is 0.1 to 1%.
<6> The medium according to any one of <1> to <5>, wherein the tengu-derived sericin is obtained through at least a scouring treatment step and a water dialysis step of the tengu layer.
<7> Tengu-derived sericin is obtained by freeze-drying after at least a scouring treatment step and a water dialysis step of the tengu layer, and the medium according to <1> to <5> .
<8> The medium according to <6> or <7>, wherein the scouring treatment step of the tengu layer is a treatment of immersing the tengu layer in a Na ₂ CO ₃ solution.
<9> The medium according to any one of <1> to <8>, wherein the tengu-derived sericin has a molecular weight of 35 kDa to 100 kDa.
<10> The medium according to any one of <1> to <9>, wherein the tengu-derived sericin is 35 kDa to 45 kDa.
<11> A method for culturing insect cells, which comprises culturing insect cells in a medium containing sericin derived from tengu.
<12> The culture method according to <11>, wherein the concentration of the sericin derived from the sea urchin in the medium is 0.05 to 1%.
<13> A method for culturing lymphocytes, comprising culturing lymphocytes in a medium containing tengu-derived sericin.
<14> The culture method according to <13>, wherein the lymphocytes to be cultured are derived from mammals.
<15> The culture method according to <13> or <14>, wherein the concentration of the sericin derived from the sea urchin in the medium is 0.1 to 1%.
<16> Tengu-derived sericin is obtained through at least a scouring treatment step and a water dialysis step of the tengu layer, and the culture method according to any one of <11> to <15>.
<17> Tengu-derived sericin is a culture according to any one of <11> to <15>, which has been subjected to a freeze-drying treatment after at least a scouring treatment step and a water dialysis step of the tengu layer Method.
<18> The culture method according to <16> or <17>, wherein the scouring treatment step is a treatment of immersing the tengu layer in a Na ₂ CO ₃ solution.
<19> Tengu-derived sericin has a molecular weight of 35 kDa to 100 kDa, The culture method according to any one of <11> to <18>.
<20> Tengu-derived sericin has a molecular weight of 35 kDa to 45 kDa, and any one of <11> to <18>.

  According to the present invention, a medium for insect cell culture and a medium for lymphocyte culture using sericin derived from Tendon are provided, and these media have an effect of promoting the growth of insect cells and lymphocytes. It can be widely used for the treatment and research of immune diseases involving lymphocytes and is highly useful.

It is a flowchart which illustrates the method of manufacturing the sericin solution and the solubilized powder which can be used for the culture medium of this invention. FIG. 5 shows the results of (A) Blue Native-PAGE electrophoresis and (B) Tricine SDS-PAGE electrophoresis using a soluble powder of sericin derived from Tendon. It is the result of HPLC which used the soluble powder of the sericin origin sericin as a sample. It is a result of (A) Blue Native-PAGE electrophoresis and (B) Tricine SDS-PAGE electrophoresis Tricine SDS-PAGE electrophoresis showing that purification of sericin-derived 41 kDa protein was confirmed. It is the result of HPLC which shows that the refinement | purification of the sericin 41kDa protein from a tengu was confirmed. It is a figure which shows the comparison of the result of having culture | cultivated Schneider S2 cell in the culture medium containing the sericin derived from a rabbit, and the culture medium of this invention containing the sericin 41kDa protein derived from a tengu. In addition, the symbol * and the symbol ** on the graph showing the results in the medium containing sericin 41 kDa protein indicate the significance level, * indicates p <0.01, and ** indicates p <0.001. It is a figure which shows the comparison of the result of having culture | cultivated the mouse | mouth spleen lymphocyte in the culture medium containing the sericin derived from a rabbit, and the culture medium of this invention containing the sericin 41kDa protein derived from a tengu. And the symbol * on the graph showing the results in the sericin-derived sericin 41 kDa protein-containing medium indicates a significant level, and p <0.02. It is a figure which shows the comparison of the result of having culture | cultivated the rat hepatoma cell (dRLh84) in the culture medium containing the rabbit sericin, and the culture medium of this invention containing the sericin 41kDa protein.

  The tengu used in the present invention may be any of those inhabiting or bred in these mountain fields. First, a ginger layer is obtained by incising a ginger discharged from these tengu. This tengu layer serves as a starting material for producing the medium of the present invention.

And the tengu-derived sericin added to the culture medium of this invention is manufactured through the following processes (A) and (B).
(A) Separation of the silkworm silk and the extract by the scouring treatment of the silkworm layer (B) Acquisition of the silkworm sericin solution by water dialysis of the extract To obtain the silkworm-derived sericin added to the medium of the present invention, First, the obtained tengu layer is scoured (step A). In this scouring treatment, the silk thread of the cocoon layer is separated as a solid component, and an aqueous liquid containing silk sericin as an extract is obtained.

  More preferably, in the scouring treatment in this step (A), the obtained tengu layer is preliminarily used in a temperature range of about 40 to 80 ° C. with pure water of about 20 to 200 times (weight ratio). It is considered that the dye substance is extracted with a methanol aqueous solution after washing with warm water as required. Thereby, the operation after the step (A) can be simplified with less burden in terms of impurity removal.

Further, in the scouring, it is preferably considered that the scouring is performed in a temperature range of about 70 to 105 ° C. with a weak alkali, for example, a 0.1 to ₂ % aqueous solution of Na ₂ CO ₃ . The processing time may be about 20 minutes to 3 hours. After this scouring treatment, for example, the cocoon layer silk thread and the extract are separated by filtration using a filter or the like.

  Thereafter, water dialysis is performed (step B). More preferably, as the pretreatment of Step B, concentration is performed at a temperature of less than 100 ° C., and the mixture is allowed to stand (stood) for several days and then filtered. As a result, it is possible to prevent deposits from adhering to the inside of the dialysis membrane during dialysis and insufficient dialysis.

  In the dialysis step in the step (B), a cellulose tube or the like can be used in a temperature range of about 5 to 25 ° C. Thereafter, a filtration treatment is also considered.

  A sericin solution derived from a bonito can be obtained through at least the above steps (A) and (B). And the soluble powder of sericin derived from a tengu can also be produced by freeze-drying the sericin solution derived from this tengu.

  The sericin-derived sericin solution usually contains a protein having a molecular weight of 35 kDa to 100 kD, although it depends on the above conditions for acquisition. Furthermore, since the protein has higher reactivity as the molecular weight is smaller, it is more preferably 35 kDa to 45 kDa. This protein can be purified by using a known method such as liquid chromatography. .

  And the culture medium containing the sericin derived from the above method, especially the said 35 kDa-45 kDa protein, can be preferably used as an insect cell culture medium. The insect cell culture medium of the present invention is based on the novel finding that sericin derived from Tendon has a remarkable insect cell growth effect.

  Specifically, the insect cell culture medium of the present invention contains at least the 35 kDa to 45 kDa protein, and may contain protein extracts, inorganic salts, saccharides, amino acids, vitamins, and other additives.

  Specifically, protein extracts include lactalbumin hydrolysate, yeast extract (Yeastlate) and tryptose phosphate broth (Fetptose Phosphate Broth), fetuin, cytochrome C, inosine, bovine plasma albumin V, etc. May be included. These protein extracts can all be commercially available.

Moreover, what can generally be added to the culture medium for animal cells should just be added as an inorganic salt, saccharides, an amino acid, and a vitamin. For example, NaH ₂ PO ₄ , NaHCO ₃ , KCl, CaCl ₂ , CuCl ₂ , CoCl, FeSO ₄ , MgCl ₂ , MgSO ₄ , MnCl ₂ , NaCl, NaH ₂ PO ₄ , (NH ₄ ) ₆ (Mo ₇ O ₂₄ · 4H ₂ O), ZnCl _{2 and the} like. Examples of the saccharide include glucose, fructose, sucrose, malic acid, α-ketoglutaric acid, succinic acid, fumaric acid, maltose and the like. As amino acids, α-alanine, β-alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, hydroxyproline, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, Such as valine. Examples of vitamins include biotin, calcium D-pantothenate, choline chloride, folic acid, i-inositol, nicotinic acid, pyridoxine, riboflavin, thiamine, vitamin B ₁₂ and paraaminobenzoic acid.

  These inorganic salts, saccharides, amino acids, and vitamins may contain all the above-mentioned substances, may lack some substances, or may contain other substances. These may use a commercially available inorganic salt composition, saccharide composition, amino acid composition and vitamin composition for medium addition, or a medium mainly composed of known inorganic salts, saccharides, amino acids and vitamins. These protein extracts may be added and used. In this case, the known medium includes known insect cell culture mediums such as Grace medium and Shneider's insect medium. Furthermore, antibiotics such as penicillin and streptomycin, glutathione and the like may be added to the medium. That is, as long as it is a culture medium for insect cells containing sericin derived from tengu, it is included in the medium of the present invention.

  And the culture medium of this invention can be used for culture | cultivation of all the insect cells, and exhibits a remarkable cell growth effect.

  Furthermore, a medium containing sericin derived from the above-mentioned method, particularly the 35 kDa to 45 kDa protein, can be preferably used as a lymphocyte culture medium. The culture medium for lymphocyte culture of the present invention is based on the novel finding that sericin derived from Tenji has a remarkable lymphocyte proliferation effect.

  As the "lymphocyte cell culture medium" in the present invention, similarly to the insect cell culture medium, the protein extract contains at least the 35 kDa to 45 kDa protein, and is also suitable for lymphocyte cell culture, It may contain inorganic salts, sugars, amino acids, vitamins and other additives.

  Specific examples include a standard medium containing growth factors and nutrients used to support cell proliferation and maintenance, and a medium in which various additives such as animal serum are added to the standard medium. it can. As the standard medium to be used, a medium such as Iskov medium, RPM1 medium, Dulbecco MEM medium or the like, which is usually used for animal cell culture, can be used, but factors other than serum known to be effective for cell growth and maintenance, For example, serum albumin, transferrin, lipid and fatty acid sources, cholesterol, pyruvate, glucocortisoid, growth factors (eg epidermal growth factor, fibroblast growth factor, platelet derived growth factor and insulin), and extracellular matrix cells (eg Collagen, fibronectin and laminin) may be added.

  The lymphocytes cultured in the lymphocyte culture medium of the present invention are one or more of lymphocytes such as animal-derived B cells, T cells, and NK cells. Mammals can be mentioned. Examples of mammals include humans, monkeys, cows, pigs, sheep, horses, rats and the like. As lymphocytes, for example, those obtained from known bone marrow, spleen cells, lymph nodes, peripheral blood and the like of the above animals can be used as appropriate.

  According to the lymphocyte culture medium of the present invention, lymphocytes can be efficiently cultured, and thus are useful in a wide range of fields such as treatment and research of immune diseases involving lymphocytes.

  And, the concentration of sericin derived from the tengu contained in the above insect cell culture medium and lymphocyte cell culture medium can be determined as appropriate according to the cultured cells, but considering denaturation due to the influence of the concentration, For example, it can be 0.05 to 1% in an insect cell culture medium and 0.1 to 1% in a lymphocyte cell culture medium.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to these Examples at all.

<Example 1> Extraction of Tendon-derived Sericin Solution The extraction process of Tendon-derived sericin solution will be described with reference to FIG.

  First, the cocoon layer obtained by incising the ginger of the tengu is washed with 100 times 65 ° C pure water for 3 minutes, and then solid-liquid separated with a 17G glass filter to quickly drain the water.

The pigment was extracted with a 70% methanol solution at 25 ° C. for 10 days. Further, the soot layer subjected to the above treatment was scoured for 2 hours at 98 ° C. with a 50% 0.5% Na ₂ CO ₃ solution. Tengu silk contains more Ca (COO) ₂ than silkworm silk, so in this scouring process, in addition to Na ₂ CO ₃ solution, weak acids such as citric acid and tartaric acid are relatively low molecular weight. The organic acid may be used. This is to effectively carry out the subsequent dialysis process. Soap and enzymes conventionally used to extract sericin from silkworm silk will solidify the dialysis membrane and cause clogging of the dialysis membrane. This is because they cannot.

Next, the extract after scouring is filtered using a glass filter, and the resulting filtrate is concentrated 30 times at 98 ° C for 30-60 minutes, and then left at 25 ° C for 2 days to precipitate impurities. Let This is because, in the solution immediately after scouring, precipitates adhere to the inside of the dialysis membrane during dialysis, resulting in insufficient dialysis, which is also due to the influence of Ca (COO) _{2 on} tengu silk. Conceivable.
Then, filter again with filter paper, fill the cellulose tube for dialysis (SPECTRUM Laboratories), perform dialysis with tap water for 3 days and pure water for 1 day at 20 ° C, then filter paper again. The sericin solution derived from Tendon was obtained.

  In the present invention, a sericin solution was freeze-dried at −20 ° C. using a lyophilizer (Tokyo Science Instrument Co., Ltd.) to obtain a soluble powder of sericin-derived sericin for use in a bioassay.

<Example 2> Tricine SDS-PAGE electrophoresis and liquid chromatographic analysis of sericin derived from Tendon (1) SDS-PAGE electrophoresis Tricine SDS-PAGE electrophoresis using the soluble powder of sericin derived from Tendon as a sample Electrophoresis was performed. More specifically, the soluble powder of sericin derived from Tendon was separated using a vertical slab gel in which a concentrated gel was layered on a 10% (T), 3% (C) separation gel. The cathode chamber is 0.1M Tris, 0.1M Tricine, 0.1% (W / V) SDS, the anode chamber is 0.2M Tris-HCl buffer (pH 8.9), and the sample for electrophoresis (20 μl) is the same amount. A sample buffer for Tricine SDS-PAGE was added and heated for 3 minutes. Proctin Marker Kit (Healthcare Bioscience) was used as the molecular weight marker. The electrophoresis was energized at a constant voltage of 30 V until entering the separation gel and then reaching the lower end of the separation gel at 80 V.

  The gel after electrophoresis was stained with Coomassie Blue R250. The protein concentration was determined using a biciconic acid (BCA) reagent.

(2) Blue Native-PAGE (BN-PAGE) Electrophoresis Blue Native-PAGE (BN-PAGE) electrophoresis was also performed using the soluble powder of sericin derived from Tenjiri obtained in Example 1 as a sample. Specifically, 4-16% gradient gel was used for BN-PAGE (Invitrogen). The electrophoresis method is basically the same as that of SDS-PAGE, but is characterized in that no reducing agent or surfactant is used in the sample buffer and the electrophoresis buffer, and the sample is not heat-treated. An electrophoresis buffer (Invitrogen) was placed in the electrophoresis tank, and an electrophoresis sample (20 μl) was prepared by adding an equal amount of sample buffer. As a molecular weight marker, Native Marker Protein Standerds (MW Range 1,236,000 to 20,000 Invitrogen) was used. The electrophoresis was performed at a constant voltage of 150 V, and the gel after the electrophoresis was decolorized according to Invitrogen BN-PAGE Manual. Specifically, the gel after electrophoresis was put into 100 ml of a fixing solution (40% ethanol, 10% acetic acid), warmed in a microwave for 45 seconds, shaken with a shaker at room temperature / 5 minutes, and the fixing solution was discarded. Subsequently, 100 ml of decolorizing solution (8% acetic acid) was added, heated in a microwave for 45 seconds, and shaken with a shaker until the background became clear.
(3) Results The results of Tricine SDS-PAGE electrophoresis are shown in FIG. The left lane of FIG. 2 (B) is a molecular weight marker, and the right lane is a band of sericin derived from Tendon. As shown in FIG. 2 (B), it was found that the sericin derived from the tengu obtained by the method of <Example 1> contains a protein having a molecular weight of 41 kDa and a polypeptide having a size of about 30 kDa or less. This polypeptide is thought to be due to a surfactant or a degradation product of the 41 kDa protein produced by SDS treatment.

  The result of BN-PAGE is shown in FIG. The left lane is a molecular weight marker, and the right lane is a band of sericin derived from Tendon. As shown in FIG. 2 (A), a band having a size of about 41 kDa as a whole molecule was also detected in BN-PAGE. In addition, BN-PAGE can measure the size of the whole molecule while maintaining the higher-order structure and complex structure of the protein. It was suggested that even when the H—H bond and the S—S bond forming the C are cleaved, the 41 kDa protein is not further cleaved and is maintained at a constant level.

In SDS-PAGE and BN-PAGE in this example, the molecular weight of sericin derived from Tendon was 41 kDa, but the molecular weight may vary depending on the extraction conditions.
(4) Liquid Chromatography Analysis Furthermore, the soluble powder of sericin derived from Tendon was adjusted with distilled water, then centrifuged at 10,000 rpm for 5 minutes, and the gel filtration Tsk-Gel G3000pw connected to the HPLC system connected to the column for gel filtration chromatography. Analysis was performed using a column (7.5 × 300 mm). The column was equilibrated with 0.2 M phosphate buffer (pH 6.8), separated at a flow rate of 0.5 ml / min, detected by ultraviolet absorption at 280 nm, and recorded with a recorder (C-R6A, Shimadzu Corporation).

  As a result, as shown in FIG. 3, four peaks were detected.

<Example 3> Purification of sericin 41 kDa protein from Tendon by liquid chromatography In order to purify the 41 kDa protein confirmed in Example 2, each peak detected by HPLC was collected and analyzed by Tricine SDS-PAGE and BN-PAGE. Analyzed.

  FIG. 4A shows the results of BN-PAGE, and FIG. 4B shows the results of tricine SDS-PAGE. Furthermore, the result of HPLC is shown in FIG. As shown in FIGS. 4 (A) and 4 (B), a clear band was detected in the vicinity of 41 kDa, and as shown in FIG. 5, the fraction containing the sericin 41 kDa protein derived from Tendon was eluted at around 17 minutes. .

  That is, the polypeptide having a size of about 30 kDa or less confirmed in Example 2 was removed, and the sericin-derived 41 kDa protein from Tendon was purified.

<Example 4> Insect cell proliferation effect by sericin 41 kDa protein derived from Tendon (1) Cell culture Using Schneider S2 cells (hereinafter referred to as S2 cells) derived from Drosophila embryos, the action of Tensei-derived sericin 41 kDa protein on insect cells was verified. did.

The medium used was a Schneider insect cell medium containing 10% FBS, antibiotic-antifungal mixed solution (100-fold concentrated) penicillin, streptomycin, amphotericin B.
(2) Measurement of cell proliferation activity After collecting S2 cells grown in a culture flask, the number of cells was measured using a Bürkerturk erythrocyte base, and the seeding cell density of S2 cells was adjusted to 1 × 10 ⁵ cells / ml. In a 96-well plate, 100 μl / well was added, and pre-culture was performed at 27 ° C. under dark conditions for 24 hours. Then, add 11 μl / well of a sericin-derived 41 kDa protein-containing sample prepared to a final concentration of 0.1% and 0.05% (W / V), and add the same amount of PBS buffer as a control. And cultured for 48 hours.

  On the other hand, as a positive control, commercially available silkworm-derived pure sericin (Wako Chemical) was prepared and added so that the final concentration was 0.1% and 0.05% (W / V), and as a control, The same amount of PBS buffer was added and cultured for 48 hours.

Further, 11 μl / well of WST-1 solution (Wako Chemical) was added and cultured for 4 hours, and the absorbance at 450 nm was measured with a microplate reader. The cell growth effect was evaluated by calculating the cell growth rate (%) = (A / A control) × 100, where the absorbance of the control group to which PBS buffer was added was A control and the absorbance of the sample addition group was A.
(3) Results As shown in FIG. 6, the cell proliferation activity of 15% and 48% was observed in the 0.1% and 0.05% sericin-derived sericin 41kDa protein-added group as compared to the non-added control group, respectively. On the other hand, cell proliferation activity was not observed in commercially available silkworm-derived sericin.

  From this, it has been clarified that the sericin 41 kDa protein derived from Tendon has an insect cell culture promoting effect.

Example 5 Mouse Spleen Lymphocyte Proliferation Effect by Tendon-derived Sericin 41 kDa Protein (1) Experimental Animal and Medium Composition As an experimental animal, an ICR mouse (4 weeks old, male) was used. As the cell culture medium, RPMI1640 medium (Nissui Pharmaceutical Co., Ltd.) containing 10% FBS, penicillin and streptomycin was used.
(2) Preparation of lymphocyte cell The spleen was extracted from the mouse, and the lymphocyte was collected in a dish containing PBS. Hemolysis buffer (0.14M ammonium chloride, 17 mM Tris-HCl buffer) and PBS washes were performed twice to sufficiently remove erythrocytes, and then incubated in a 5% CO ₂ incubator for 2 hours. Thereafter, the cells were stained with 0.4% trypan blue, and the number of cells was counted using a Bürkerturk erythrocyte base.
(3) Activity measurement Lymphocytes were prepared to a seeding cell density of 5 × 10 ⁶ cells / ml and added to a 96-well plate at 100 μl / well. After that, add 11μl / well of the sericin 41kDa protein-containing sample prepared to a final concentration of 0.1% and 0.05% (W / V), and add the same amount of PBS buffer as a control. Then, the cells were cultured for 48 hours at 37 ° C. in the presence of 5% CO ₂ .

On the other hand, as a positive control, commercially available silkworm-derived pure sericin (Wako Chemical) was prepared and added so that the final concentration was 0.1% and 0.05% (W / V), and as a control, The same amount of PBS buffer was added, and cultured in the presence of 5% CO ₂ at 37 ° C. for 48 hours.

Further, 11 μl / well of WST-1 solution (Wako Chemical) was added and cultured for 4 hours, and the absorbance at 450 nm was measured with a microplate reader. The cell growth effect was evaluated by calculating the cell growth rate (%) = (A / A control) × 100, where the absorbance of the control group to which PBS buffer was added was A control and the absorbance of the sample addition group was A.
(4) Results
As a result, as shown in FIG. 7, a cell proliferation activity of 16% was observed in the group added with 0.1% Tendon-derived sericin 41 kDa protein compared to the control group without addition. On the other hand, commercially available silkworm-derived sericin did not show cell proliferation activity, but rather cell proliferation tended to be suppressed as the concentration increased.

  From this, it became clear that the sericin 41 kDa protein derived from Tendon has a lymphocyte culture promoting effect.

Example 6 Rat Hepatoma Cell (dRLh84) Cell Proliferation Effect by Tendon-derived Sericin 41 kDa Protein (1) Cell Culture Rat hepatoma cell (dRLh84) is 10% NBS, 4 mM glutamine, 50 U / ml penicillin, 100 ng A DMEM medium containing / ml kanamycin and cultured at 37 ° C. for 24 hours in the presence of 5% CO ₂ was used.
(2) Measurement of cell proliferation activity Rat hepatoma cells (dRLh84) were prepared to a cell density of 5 × 10 ⁴ cells / ml, added to a 96-well plate at 100 μl / well, and cultured for 24 hours. Thereafter, 11 μl / well of a sericin-derived 41 kDa protein-containing sample prepared to a final concentration of 0.1% (W / V) was added. Similarly, as a positive control, the final concentration was 0.1% (W / V). A commercially available silkworm-derived pure sericin (Wako Chemical) was added. In the control group, the same amount of PBS buffer solution was added, and cultured in the presence of 5% CO ₂ at 37 ° C. for 48 hours. Further, according to the MTT method (Oka et al., 1992), 11 μl / well of MTT solution (Wako Chemical) was added and cultured for 4 hours, DMSO (dimethyl Sulfoxide, Wako Chemical) was added, and the generated formazan was dissolved. Absorbance at −620 nm was measured with a microplate reader. The cell proliferation effect was calculated and evaluated in the same manner as in Example 4.
(3) Results As shown in FIG. 8, the results were not significantly different between the group containing the sericin 41 kDa protein added from the Tendon, the commercially available silkworm-derived sericin added group, and the non-added control group.

  From this, it was suggested that the cell proliferation effect of the sericin 41 kDa protein derived from Tendon was not observed in arbitrary cells.

Claims (10)

At least the following steps:
(1) A scouring treatment step of immersing the tengu layer in a 70 to 105 ° C. and 0.1 to 2.0% Na ₂ CO ₃ solution to obtain an extract,
(2) a water dialysis step for obtaining a tengu silk sericin solution by hydrodialyzing the extract;
(3) A step of freeze-drying the tengu silk sericin solution,
A medium for insect cell culture, characterized by containing sericin derived from Tendon, which has a molecular weight of 41 kDa, obtained by passing through .   2. The medium according to claim 1, wherein the concentration of sericin derived from the sea urchin in the medium is 0.05 to 1%. At least the following steps:
(1) A scouring treatment step of immersing the tengu layer in a 70 to 105 ° C. and 0.1 to 2.0% Na ₂ CO ₃ solution to obtain an extract,
(2) a water dialysis step for obtaining a tengu silk sericin solution by hydrodialyzing the extract;
(3) A step of freeze-drying the tengu silk sericin solution,
A medium for lymphocyte culture, characterized by containing a sericin derived from Tendon, which has a molecular weight of 41 kDa .   The medium according to claim 3, wherein the lymphocytes are mammalian lymphocytes.   The culture medium according to claim 3 or 4, wherein the concentration of sericin derived from the sea urchin in the culture medium is 0.1 to 1%. At least the following steps:
(1) A scouring treatment step of immersing the tengu layer in a 70 to 105 ° C. and 0.1 to 2.0% Na ₂ CO ₃ solution to obtain an extract,
(2) a water dialysis step for obtaining a tengu silk sericin solution by hydrodialyzing the extract;
(3) A step of freeze-drying the tengu silk sericin solution,
A method for cultivating insect cells, comprising culturing insect cells in a medium containing sericin derived from Tendon, having a molecular weight of 41 kDa obtained by passing through   7. The culture method according to claim 6, wherein the concentration of sericin derived from the sea urchin in the medium is 0.05 to 1%. At least the following steps:
(1) A scouring treatment step of immersing the tengu layer in a 70 to 105 ° C. and 0.1 to 2.0% Na ₂ CO ₃ solution to obtain an extract,
(2) a water dialysis step for obtaining a tengu silk sericin solution by hydrodialyzing the extract;
(3) A step of freeze-drying the tengu silk sericin solution,
The method of culturing lymphocytes characterized that you culturing lymphocytes in a medium molecular weight obtained contains wild silkworm derived sericin 41kDa by undergoing. 9. The culture method according to claim 8, wherein the lymphocytes to be cultured are derived from a mammal. 10. The culture method according to claim 8 or 9, wherein the concentration of sericin derived from the sea urchin in the medium is 0.1 to 1%.