JP4132151B2 - How to deactivate prions - Google Patents

Description

〔表１〕から明らかなとおり、LEO処理により中和抗体価は低下せず、グロブリンの活性に影響のないことが確かめられた。
【００１０】
実施例２
１．実験材料の調製
健康な食肉牛それぞれ２０頭から得られた混合牛血清を３ロット用意し、以下の操作によりサンプルＣ，Ｄ，Ｅを調製した。
各サンプルの調製
牛血清１Lに硫酸アンモニウム351gを少しずつ加えて溶解させ、30分放置した後、生じた沈澱を濾過により除去した。得られた上清液にさらに硫安103gを少しずつ加えて溶解させ、30分放置の後、生じた沈澱を濾過により集めた。得られた固体を約100mlの蒸留水に溶解し、氷冷撹拌下 LEO を0.7ml滴下し、滴下後25℃で約４０時間放置した。これを透析膜（ビスキング社製）に入れて、生理食塩液で透析した。透析内液を取り出し、蛋白量が100mg／mlになるように生理食塩液で調整した。メンブランフィルター（0.22μm、ミリポア社製）で除菌濾過し、動物細胞培養用組成物を得た。この組成物を蛋白量にして3mg／mlになるようにダルベッコ改変イーグル培地（DMEM）／ハム１２培地（F12）（DMEM／F12）（1：1）の基礎培地300mlに加え、さらにインシュリン3mg、トランスフェリン6mg、エタノールアミン36.6μg、亜セレン酸ナトリウム1.4μgを添加し、メンブランフィルター（0.22μm、ミリポア社製）で除菌濾過し、動物細胞培養用培地サンプルを得た。３ロットそれぞれに以上の操作を施し、サンプルＣ、Ｄ及びＥとした。
【００１１】
２．測定方法
サンプルＣ、Ｄ、Ｅ及び牛胎児血清（FBS）（LEO 未処理）を10％v/vとなるように DMEM／F12（1：1）の基礎培地で調整したものを対照培地として用いた。細胞は、チャイニーズ ハムスターの腎細胞である CHO-K1 細胞（大日本製薬社製）と、ハムスターの腎細胞である BHK-21 細胞（大日本製薬社製）を使用した。サンプルＣ、Ｄ、Ｅ及び対照培地を各々２４穴マルチディッシュに１ml／ウエルずつ分注した後、各細胞浮遊液（細胞数2.4〜2.6×10⁴／ml）を0.1mlずつ分注し、5％CO₂インキュベーターで37℃で７日間培養した。培養後、各ウエルの培地を廃棄後0.25％トリプシン溶液で細胞を剥離し、コールターカウンターで細胞数を測定した。細胞増殖促進効果は、対照培地の細胞増殖率（培養後の細胞数÷培養開始時の細胞数）を100としたときの各サンプルの細胞増殖率で示した。その結果を〔表２〕に示した。
【表２】

上記〔表２〕から明らかなとおり、LEO処理した動物細胞培養用組成物は、10v/v％ FBS を使用した対照培地に比べ遜色のない細胞増殖促進効果を示した。
【００１２】
実施例３
１w/v％スクレイピー感染マウス脳乳剤に、 LEO を２v/v％になるように添加し、25℃で90時間放置した後に透析した LEO 処理群と、 LEO を添加せずに25℃で90時間放置した後に透析した LEO 無処理群、及び10w/v％スクレイピー感染マウス脳乳剤を10⁶倍まで10倍段階で希釈した各感染価測定群のそれぞれを IcR 系生後４週齢のマウスに投与して、スクレイピーの発症、斃死を観察した。なお、脳乳剤のマウスへの投与は0.02mlを脳内に接種した。
【表３】

感染対照群の結果から、接種後339日経過の時点で、スクレイピー感染マウス10W/V％脳乳剤の10⁵倍希釈液接種マウスが100％斃死し、10⁶倍希釈液接種マウスに斃死が確認されていないことから、スクレイピー感染マウス10W/V％脳乳剤の50％致死率（LD₅₀）を、10^5.5倍希釈液とし、これを５.５（log₁₀）感染単位とした。
【００１３】
感染対照群の潜伏日数と感染単位から試験群の感染単位を求める標準曲線を作成し、試験群の潜伏日数から感染単位を測定したところ、次のような結果が得られた。
処理群の感染単位（log₁₀）＝−０.２
未処理群の感染単位（log₁₀）＝４.９
これらから、LEO 処理による感染単位の減衰値（log₁₀）は 4.9-（-0.2）=5.1（log₁₀）＝10^5.1となる。即ち、LEO処理することにより、スクレイピー感染マウス10W/V% 脳乳剤を10^5.1＝1.26×10⁵倍に希釈した液と同等の感染単位にまでプリオンの感染力を減衰せしめたことになる。
【００１４】
【発明の効果】
本発明において、従来法による細菌、ウイルスの失活法に強い抵抗性を示すプリオンを、蛋白の生理活性、特質や物性等を保持させる緩和な条件下において効果的に不活化ないしはその感染力を有意に減衰させることができる。
【図面の簡単な説明】
【図１】は感染単位を求める標準曲線を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method of inactivating or attenuating the infectivity of prions mixed in, for example, a protein having physiological activity, characteristics, or the like, or a substance containing the same, or a protein for food, for example.
[0002]
[Prior art]
When proteins having physiological activity or properties or proteins for food materials are prepared from tissues or body fluids of animals including humans, there is a risk of contamination by pathogenic particles mixed in those tissues and body fluids. A typical example of the source is HIV-virus, ie AIDS virus. However, the nature of this AIDS virus has been gradually elucidated, and inactivation methods have been established, and it has become possible to almost certainly prevent new infections caused by protein products derived from human or animal tissues or body fluids such as blood products. .
In recent years, however, a new pathogenic factor called prion has been highlighted and has shaken the world. Although there are still many unclear points regarding prions, they are said to be infectious proteins that do not have DNA or RNA because the existence of nucleic acids cannot be proved.
Known human diseases caused by prion pathogens include kuru, Creutzfeldt-Jakob disease, and Gerstmann-Strreisler syndrome. Other than humans, scrapie in sheep and goats, and spongiform encephalopathy in cattle (madness) Cow disease) is known. There is evidence that iatrogenic Creutzfeldt-Jakob disease was transmitted to patients through corneal and dural transplants, administration of growth hormone extracted from the human pituitary gland, and contaminated EEG electrodes.
[0003]
This transmissible pathogen has been shown to be present in large amounts in the brain and spinal cord of Kuru, and in patients with Creutzfeldt-Jakob disease and Gerstman-Strreisler syndrome, similar transmissible pathogens are found in the brain, spleen, and liver. , Detected in lymph nodes, lungs, spinal cord, kidney, cornea, lens, cerebrospinal fluid, blood.
However, since prions do not produce an immune response such as antibody formation, there is no vaccine and diagnosis of morbidity is extremely difficult.
Although various attempts have been made to inactivate prions or attenuate infectivity, the pathogens are radiation, boiling, dry heat, chemicals (formalin, beta-propiolactone, alcohol, iodine, acetone. The conventional inactivation method or infectivity reduction method against pathogenic microorganisms including potassium permanganate, hydrogen peroxide, ethylene oxide gas, etc. is extremely resistant.
It is known that prions are inactivated by treatment with high concentration mineral acid or alkali solution at high temperature, treatment with hypochlorous acid-alkali solution, or high pressure steam (134 ° C., 1 hour). The treatment under such severe conditions denatures the protein, for example, loses the physiological activity and properties of the protein, or irreversibly deteriorates the physical properties.
[0004]
[Problems to be solved by the invention]
Under such circumstances, the development of a method for inactivating or attenuating the infectivity of prions mixed in proteins or their contents while maintaining the physiological activity, characteristics, and physical properties of the proteins has been awaited.
[0005]
[Means for Solving the Problems]
In order to solve the above-mentioned problems, the present inventors have examined many methods including methods conventionally used to inactivate this kind of pathogenic particles and attenuate infectivity. It was discovered that ethylene oxide actually inactivates prions in liquid form or significantly attenuates infectivity, and that proteins retain their physiological activity, properties and physical properties. Was completed. That is, the present invention provides (1) a method for inactivating prions or attenuating their infectivity, characterized by treating prion mixed proteins or their contents with liquid ethylene oxide,
(2) treatment with ethylene oxide to -10 to 60 ° C., intends 0.5 to 168 hours row (1) The method according,
( 3 ) The method according to (1), wherein the liquid ethylene oxide is an aqueous solution,
( 4 ) The method according to (1), wherein the concentration of ethylene oxide in the protein to be treated or the content thereof is 0.05 v / v% or more,
It is.
[0006]
DETAILED DESCRIPTION OF THE INVENTION
The object of the liquid ethylene oxide treatment in the present invention is a protein containing prions or a substance containing the same. These include hormones such as insulin, glucagon, thyroid stimulating hormone, chorionic gonadotropin, calcitonin, growth hormone, eg fibrinolytic factors such as plasminogen, plasminogen activator, urokinase, eg blood coagulation VII, Blood coagulation factors such as factor VIII, IX, XI, factor XII, fibrinogen, thrombin, such as antithrombin III, α ₁ -antithrombin, protein C, protein S, such as interleukin 1-15, lymphokine, Bioactive proteins such as tumor necrosis factor, perforin, cytokines such as α, β, and γ-interferon and various antibodies, biologically excised tissues containing them, or their ground products, proteins for food materials such as gelatin and collagen, or foods containing these Material Is included. The ethylene oxide used in the present invention is used in the form of a solution. The solvent used for forming the solution is usually water, but may be a mixture of water and a lower alcohol such as ethanol or other hydrophilic organic solvent in an appropriate amount, for example, a ratio of 10% by weight or less. The concentration of ethylene oxide in the object to be treated is 0.05 v / v% or more, usually 0.1 to 10 v / v%, preferably 0.2 to 3 v / v%, more preferably 0.3 to 2 v / v. %, Most preferably 0.7 to 1.5 v / v%.
[0007]
The treatment temperature of the prion-mixed protein or its inclusion with this liquid ethylene oxide is usually -10 to 60 ° C, preferably 0 to 60 ° C, more preferably 10 to 40 ° C, most preferably 15 to 30 ° C. The time is usually 0.5 to 168 hours, preferably 1 to 120 hours, more preferably 10 to 108 hours, and most preferably 24 to 96 hours. Under the prion inactivation or infectivity reducing process conditions used in the present invention, the infectivity of prions can be significantly attenuated while maintaining the physiological activity, properties, physical properties, etc. of the protein. In order to inactivate the mixed prion or to remove ethylene oxide used for attenuating the infectivity from the treated product, a method known per se, for example, dialysis, membrane filtration or gel filtration chromatography can be used. The effect of the present invention is to inject each of the scrapie-infected mouse brain emulsion treated with liquid ethylene oxide and the untreated one into the mouse brain, rearing and observing for a certain period, and grasping the onset, moribund status, etc. You can check with
[0008]
【Example】
The present invention will be further described below with reference to examples, but the present invention is not limited thereto.
Example 1
1. Preparation of Experimental Materials Samples A and B were prepared as follows using mixed bovine serum obtained from 20 healthy beef cattle.
Sample A
To 1 L of bovine serum, 7 ml of liquid ethylene oxide (LEO) was added dropwise with stirring under ice cooling, and the mixture was allowed to stand at 25 ° C. for about 40 hours. To this was added 351 g of ammonium sulfate little by little and dissolved, and after standing for 30 minutes, the resulting precipitate was collected by filtration. The obtained solid was dissolved in about 100 ml of distilled water, put into a dialysis membrane (manufactured by Visking), and dialyzed with a phosphate buffer solution (PBS (−)). The dialyzed solution was taken out and adjusted with PBS (-) so that the protein amount was 120 mg / ml. Bacterial filtration was performed with a membrane filter (0.22 μm, manufactured by Millipore) to obtain a LEO-treated globulin fraction. This was designated as sample A.
Sample B
To 1 L of bovine serum, 351 g of ammonium sulfate was added little by little to dissolve, and after standing for 30 minutes, the resulting precipitate was collected by filtration. The obtained solid was dissolved in about 100 ml of distilled water, put into a dialysis membrane (manufactured by Visking), and dialyzed with PBS (-). The dialyzed solution was taken out and adjusted with PBS (−) so that the protein amount was 120 mg / ml. Bacterial filtration was performed with a membrane filter (0.22 μm, manufactured by Millipore) to obtain an LEO-untreated globulin fraction. This was designated as Sample B.
[0009]
2. Method for measuring neutralizing antibody titer The anti-rotavirus neutralizing antibody titer is defined as the reciprocal of the dilution factor of the sample that was measured by the fluorescent-focus neutralization test (Fluorescent-focus neutralization test) and inhibited the growth of rotavirus by 60% or more [Table 1].
[Table 1]

As is clear from [Table 1], it was confirmed that the neutralizing antibody titer was not decreased by the LEO treatment and the globulin activity was not affected.
[0010]
Example 2
1. Preparation of Experimental Materials Three lots of mixed bovine serum obtained from 20 healthy beef cattle were prepared, and samples C, D, and E were prepared by the following operations.
Preparation of each sample 351 g of ammonium sulfate was added little by little to 1 L of calf serum and allowed to stand for 30 minutes, and the resulting precipitate was removed by filtration. Further, 103 g of ammonium sulfate was added little by little to the obtained supernatant and dissolved, and after standing for 30 minutes, the resulting precipitate was collected by filtration. The obtained solid was dissolved in about 100 ml of distilled water, 0.7 ml of LEO was added dropwise with stirring under ice cooling, and the mixture was allowed to stand at 25 ° C. for about 40 hours. This was put into a dialysis membrane (manufactured by Visking) and dialyzed with physiological saline. The dialyzed solution was taken out and adjusted with physiological saline so that the amount of protein was 100 mg / ml. Bactericidal filtration was performed with a membrane filter (0.22 μm, manufactured by Millipore) to obtain a composition for animal cell culture. This composition was added to 300 ml of basal medium of Dulbecco's modified Eagle medium (DMEM) / Ham 12 medium (F12) (DMEM / F12) (1: 1) so that the amount of protein was 3 mg / ml, and further 3 mg of insulin, 6 mg of transferrin, 36.6 μg of ethanolamine, and 1.4 μg of sodium selenite were added, and sterilization filtration was performed with a membrane filter (0.22 μm, manufactured by Millipore) to obtain a medium sample for animal cell culture. The above operation was applied to each of the three lots to obtain samples C, D, and E.
[0011]
2. Measurement method Samples C, D, E and fetal bovine serum (FBS) (LEO untreated) adjusted to 10% v / v with DMEM / F12 (1: 1) basal medium are used as control medium It was. CHO-K1 cells (Dainippon Pharmaceutical Co., Ltd.), which are Chinese hamster kidney cells, and BHK-21 cells (Dainippon Pharmaceutical Co., Ltd.), hamster kidney cells, were used as the cells. Samples C, D, E and control medium were each dispensed at 1 ml / well in a 24-well multi-dish, and then 0.1 ml of each cell suspension (cell count 2.4-2.6 × 10 ⁴ / ml) was dispensed. The cells were cultured at 37 ° C. for 7 days in a% CO ₂ incubator. After culturing, the medium in each well was discarded, the cells were detached with a 0.25% trypsin solution, and the number of cells was measured with a Coulter counter. The cell growth promoting effect was shown by the cell growth rate of each sample when the cell growth rate of the control medium (number of cells after culture / number of cells at the start of culture) was 100. The results are shown in [Table 2].
[Table 2]

As is clear from the above [Table 2], the LEO-treated animal cell culture composition showed a cell growth promoting effect comparable to the control medium using 10 v / v% FBS.
[0012]
Example 3
LEO was added to the brain emulsion of 1 w / v% scrapie-infected mouse to 2v / v%, left to stand at 25 ° C for 90 hours and dialyzed, and LEO treatment group without adding LEO for 90 hours at 25 ° C administered was left unattended and dialyzed LEO untreated group after, and each of infectivity measured group that has been diluted with 10-fold serial to 10 ⁶ fold 10w / v% scrapie-infected mouse brain homogenate to IcR system 4-week old mice We observed the onset and dying of scrapie. In addition, for administration of brain emulsion to mice, 0.02 ml was inoculated into the brain.
[Table 3]

From the results of infection control group, at the time of lapse 339 days after inoculation, 10 ⁵ fold dilutions inoculated mice scrapie infected mice 10 W / V% brain homogenate is mortality 100% mortality is checked 10 ⁶ fold dilutions inoculated mice Since this was not done, the 50% lethality (LD ₅₀ ) of 10 W / V% brain emulsion of scrapie-infected mice was used as a 10 ^5.5- fold dilution, and this was used as 5.5 (log ₁₀ ) infectious units.
[0013]
A standard curve for determining the infection unit of the test group was prepared from the incubation days of the infection control group and the infection unit, and the infection unit was measured from the incubation days of the test group, and the following results were obtained.
Infectious unit of treatment group (log ₁₀ ) = -0.2
Infectious unit of untreated group (log ₁₀ ) = 4.9
From these, the attenuation value (log ₁₀ ) of the infection unit by LEO treatment is 4.9-(-0.2) = 5.1 (log ₁₀ ) = 10 ^5.1 . In other words, the LEO treatment attenuated the infectivity of prions to the same infection unit as a solution obtained by diluting 10 W / V% brain emulsion of scrapie-infected mice to 10 ^5.1 = 1.26 × 10 ⁵ times.
[0014]
【The invention's effect】
In the present invention, prions that are highly resistant to conventional methods for inactivating bacteria and viruses are effectively inactivated or infectious under mild conditions that retain the physiological activity, properties, and physical properties of proteins. Can be significantly attenuated.
[Brief description of the drawings]
FIG. 1 shows a standard curve for determining infectious units.

Claims (4)

A method of inactivating prions or attenuating their infectivity, characterized by treating prion mixed proteins or their contents with liquid ethylene oxide. The process according to claim 1, wherein the treatment with ethylene oxide is carried out at -10 to 60 ° C for 0.5 to 168 hours. The process according to claim 1, wherein the ethylene oxide is an aqueous solution. 2. The method according to claim 1, wherein the concentration of ethylene oxide in the protein to be treated or its content is 0.05 v / v% or more.

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