JPH0778079B2 - Protein with anti-cancer activity - Google Patents
Protein with anti-cancer activityInfo
- Publication number
- JPH0778079B2 JPH0778079B2 JP61215623A JP21562386A JPH0778079B2 JP H0778079 B2 JPH0778079 B2 JP H0778079B2 JP 61215623 A JP61215623 A JP 61215623A JP 21562386 A JP21562386 A JP 21562386A JP H0778079 B2 JPH0778079 B2 JP H0778079B2
- Authority
- JP
- Japan
- Prior art keywords
- protein
- body fluid
- present
- activity
- cancer cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 102000004169 proteins and genes Human genes 0.000 title claims description 20
- 108090000623 proteins and genes Proteins 0.000 title claims description 20
- 230000001093 anti-cancer Effects 0.000 title description 2
- 241000238631 Hexapoda Species 0.000 claims description 12
- 230000000259 anti-tumor effect Effects 0.000 claims description 8
- 230000001418 larval effect Effects 0.000 claims description 5
- 239000012588 trypsin Substances 0.000 claims description 4
- 108090000631 Trypsin Proteins 0.000 claims description 3
- 102000004142 Trypsin Human genes 0.000 claims description 3
- FADYJNXDPBKVCA-UHFFFAOYSA-N L-Phenylalanyl-L-lysin Natural products NCCCCC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FADYJNXDPBKVCA-UHFFFAOYSA-N 0.000 claims description 2
- GHKXHCMRAUYLBS-CIUDSAMLSA-N Lys-Ser-Asn Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O GHKXHCMRAUYLBS-CIUDSAMLSA-N 0.000 claims description 2
- MEQLGHAMAUPOSJ-DCAQKATOSA-N Lys-Ser-Val Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O MEQLGHAMAUPOSJ-DCAQKATOSA-N 0.000 claims description 2
- YCJCEMKOZOYBEF-OEAJRASXSA-N Lys-Thr-Phe Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O YCJCEMKOZOYBEF-OEAJRASXSA-N 0.000 claims description 2
- WBAXJMCUFIXCNI-WDSKDSINSA-N Ser-Pro Chemical compound OC[C@H](N)C(=O)N1CCC[C@H]1C(O)=O WBAXJMCUFIXCNI-WDSKDSINSA-N 0.000 claims description 2
- JAIZPWVHPQRYOU-ZJDVBMNYSA-N Val-Thr-Thr Chemical compound C[C@H]([C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)O)NC(=O)[C@H](C(C)C)N)O JAIZPWVHPQRYOU-ZJDVBMNYSA-N 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 2
- 239000012530 fluid Substances 0.000 claims description 2
- 108010026333 seryl-proline Proteins 0.000 claims description 2
- 108010073969 valyllysine Proteins 0.000 claims description 2
- 210000001124 body fluid Anatomy 0.000 description 19
- 239000010839 body fluid Substances 0.000 description 19
- 210000004027 cell Anatomy 0.000 description 17
- 238000000034 method Methods 0.000 description 15
- 206010028980 Neoplasm Diseases 0.000 description 12
- 201000011510 cancer Diseases 0.000 description 12
- 230000000694 effects Effects 0.000 description 10
- 239000000126 substance Substances 0.000 description 8
- 238000000746 purification Methods 0.000 description 7
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 5
- 230000001472 cytotoxic effect Effects 0.000 description 5
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 5
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 239000013543 active substance Substances 0.000 description 4
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 4
- 238000004007 reversed phase HPLC Methods 0.000 description 4
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 3
- 101000972324 Cynodon dactylon Leaf protein Proteins 0.000 description 3
- 108010053229 Lysyl endopeptidase Proteins 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 3
- 235000011130 ammonium sulphate Nutrition 0.000 description 3
- 230000003013 cytotoxicity Effects 0.000 description 3
- 231100000135 cytotoxicity Toxicity 0.000 description 3
- 230000008260 defense mechanism Effects 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 241000254173 Coleoptera Species 0.000 description 2
- MQUQNUAYKLCRME-INIZCTEOSA-N N-tosyl-L-phenylalanyl chloromethyl ketone Chemical compound C1=CC(C)=CC=C1S(=O)(=O)N[C@H](C(=O)CCl)CC1=CC=CC=C1 MQUQNUAYKLCRME-INIZCTEOSA-N 0.000 description 2
- 239000012190 activator Substances 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 210000000601 blood cell Anatomy 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 231100000433 cytotoxic Toxicity 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000002523 gelfiltration Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 231100000636 lethal dose Toxicity 0.000 description 2
- 230000001665 lethal effect Effects 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 241000490557 Allomyrina Species 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 241000255789 Bombyx mori Species 0.000 description 1
- 241001481710 Cerambycidae Species 0.000 description 1
- 241000255749 Coccinellidae Species 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- 241000254158 Lampyridae Species 0.000 description 1
- 102000004856 Lectins Human genes 0.000 description 1
- 108090001090 Lectins Proteins 0.000 description 1
- 241000131077 Lucanidae Species 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 241000130993 Scarabaeus <genus> Species 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 229940100198 alkylating agent Drugs 0.000 description 1
- 239000002168 alkylating agent Substances 0.000 description 1
- 238000003453 ammonium sulfate precipitation method Methods 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 230000000340 anti-metabolite Effects 0.000 description 1
- 229940100197 antimetabolite Drugs 0.000 description 1
- 239000002256 antimetabolite Substances 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000002532 enzyme inhibitor Substances 0.000 description 1
- 229940125532 enzyme inhibitor Drugs 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000002523 lectin Substances 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000001932 seasonal effect Effects 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 238000000539 two dimensional gel electrophoresis Methods 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Description
【発明の詳細な説明】 〔産業上の利用分野〕 本発明は制癌活性を有する蛋白に関するものである。TECHNICAL FIELD The present invention relates to a protein having an antitumor activity.
従来の癌化学療法剤はいわゆるアルキル化剤及び代謝拮
抗剤に見られるように核酸蛋白合成系を阻害することに
より癌細胞を致死せしめるものである。しかしながらこ
れらの薬剤においては癌細胞と正常細胞の識別という点
で問題があり、必然的に重大な副作用を引き起こすとい
う欠点を有している。また、動物細胞より生産される制
癌物質を単離しようとする研究が展開されているが、こ
のような物質の制癌活性は十分と言えるものではなく、
また副作用の問題は依然として未解決の問題として残つ
ている。Conventional cancer chemotherapeutic agents kill the cancer cells by inhibiting the nucleic acid protein synthesis system as seen in so-called alkylating agents and antimetabolites. However, these drugs have a problem in that they distinguish cancer cells from normal cells, and have the drawback of inevitably causing serious side effects. In addition, studies have been conducted to isolate antitumor substances produced from animal cells, but the antitumor activity of such substances is not sufficient,
Also, the problem of side effects remains an unsolved problem.
動物生体防御機構については、従来、哺乳動物を主たる
対象として免疫系を中心に研究されてきたが、本発明者
らは昆虫類の生体防御機構を解明する為の研究過程で、
多食亜目に属する昆虫の幼虫体液に癌細胞に対して強い
細胞傷害性を示す物質が含まれていることを見出した
が、更に検討を重ねた結果、本発明を完成するに至つ
た。すなわち、本発明の要旨は多食亜目に属する昆虫の
幼虫体液から得られ、分子量が約42,000であり、等電点
がpI8.9〜9.0であり、トリプシンに耐性であり、かつ、
下記のＮ末端アミノ酸配列を有することを特徴とする、
制癌活性を有する蛋白に存する。Regarding the biological defense mechanism of animals, conventionally, the mammal has been mainly studied mainly on the immune system, but the present inventors, in the research process for elucidating the biological defense mechanism of insects,
It was found that the larval body fluid of insects belonging to the polyphagous suborder contained a substance having strong cytotoxicity against cancer cells, but as a result of further studies, the present invention was completed. That is, the gist of the present invention is obtained from the larval fluid of insects belonging to the polyphagous suborder, the molecular weight is about 42,000, the isoelectric point is pI8.9-9.0, is resistant to trypsin, and,
Characterized by having the following N-terminal amino acid sequence:
It exists in a protein having anti-cancer activity.
Val Thr Thr Lys Ser Val Lys Thr Phe Lys Ser Asn Pr
o Ser Pro 哺乳動物の免疫系とは異なつた生体防御機構が昆虫類に
も存在することがすでに知られており、例えば細菌感染
に対する防御物質を生産している昆虫としてハエ及びカ
イコ等が知られている（Gregoire,C.,Physiology of In
secta,1974年,5巻,309頁，アカデミツクプレス社）。Val Thr Thr Lys Ser Val Lys Thr Phe Lys Ser Asn Pr
o Ser Pro It is already known that insects also have a biological defense mechanism different from the mammalian immune system. For example, flies and silkworms are known as insects that produce protective substances against bacterial infections. (Gregoire, C., Physiology of In
secta, 1974, Vol. 5, p. 309, Academic Press Co.).
しかしながら、本発明による物質は抗菌作用は示さず、
癌細胞に対する直接細胞傷害性を有するという点で従来
公知の昆虫由来生理活性物質とは明らかに異なる。また
本発明による物質は昆虫の正常細胞自身が生産するもの
であるから、癌細胞に対する選択的毒性を示すことによ
り動物体内で常時出現しうる癌細胞あるいはその前駆細
胞を排除しうるような生体防御物質の１つとして理解さ
れるべきである。以下、本発明を詳細に説明する。However, the substances according to the invention show no antibacterial action,
It is clearly different from conventionally known insect-derived physiologically active substances in that it has direct cytotoxicity to cancer cells. In addition, since the substance according to the present invention is produced by normal insect cells themselves, it shows a selective toxicity against cancer cells, and thus a biological defense that can eliminate cancer cells or progenitor cells thereof that can constantly appear in the animal body. It should be understood as one of the substances. Hereinafter, the present invention will be described in detail.
本発明の蛋白の単離方法及びその特性を以下に記載す
る。The method for isolating the protein of the present invention and its characteristics are described below.
まず、本発明における多食亜目に属する昆虫の例として
は、カブトムシ、ホタル、テントウムシ、コガネムシ、
クワガタムシ及びカミキリムシ等が挙げられる。First, as examples of insects belonging to the polyphagous order of the present invention, beetles, fireflies, ladybirds, scarabs,
Examples include stag beetles and long-horned beetles.
これらの昆虫のうちいずれかを用いるかは、幼虫の繁殖
の容易さ、得られる体液の量及び／又は制癌活性の強さ
等により規定されるがいずれの昆虫を使用してもよい。
又、一般に昆虫幼虫は脱皮を繰り返して成長するが、こ
のいずれの時期のものでも使用することができる。幼虫
から体液を得る方法としては例えば背脈管に注射針を穿
刺して殺生することなく体液を得る方法が挙げられ、こ
の幼虫を再飼育して頻回体液を得ることも可能である。
又、腹部切断して体液を得たり、あるいは幼虫全体をホ
モジネートして得ることも可能であるが、以後の精製工
程等を考慮すると、ホモジネートによる方法より体液の
みを純粋に取りだす方法の方が望ましい。Which of these insects is used is determined by the ease of larval reproduction, the amount of body fluid obtained, and / or the strength of the antitumor activity, and any insect may be used.
In general, insect larvae grow by repeating molting, but any larva can be used. As a method for obtaining body fluid from the larva, for example, a method for obtaining body fluid without sterilization by puncturing the dorsal vein with an injection needle, and this larva can be re-bred to obtain frequent body fluid.
It is also possible to obtain a body fluid by cutting the abdomen or obtain a homogenate of the whole larva. However, in consideration of the subsequent purification step and the like, a method of purely extracting body fluid is preferable to a method using homogenate. .
取得した体液は、半透明の粘稠液体で、通常遠心分離に
より血球を除去して用いる。又、以後の精製工程に悪影
響をきたす恐れがあるので適切な透析膜を用いて透析し
て粘度を低下させたり、あるいは不要の加水分解酵素及
び／又は酸化酵素を失活させるために加熱処理を行なう
かあるいは適切な酵素阻害剤を添加しておくことが好ま
しい。この場合の加熱処理は、本発明に係る制癌活性蛋
白が失活されない条件下、すなわちpH5〜９の範囲で60
℃、30分間の加熱を行うことが好ましく、更にはpH7付
近で加熱することが好ましい。The obtained body fluid is a translucent viscous liquid, which is usually used after removing blood cells by centrifugation. Further, since it may have an adverse effect on the subsequent purification process, it is dialyzed with an appropriate dialysis membrane to reduce the viscosity, or heat treatment is performed to inactivate unnecessary hydrolase and / or oxidase. It is preferable to carry out or to add an appropriate enzyme inhibitor. The heat treatment in this case is carried out under the condition that the antitumor protein according to the present invention is not inactivated, that is, in the range of pH 5-9.
It is preferable to heat at 30 ° C. for 30 minutes, and it is more preferable to heat at around pH 7.
次に、精製過程について説明する。Next, the purification process will be described.
本発明に係る制癌活性蛋白は硫安塩析法によつて体液か
ら部分精製することが可能である。この時の硫安濃度は
40％飽和以上であれば、活性蛋白は90％以上塩析され
る。また硫安のみならず硫酸ナトリウム又はエタノー
ル、アセトンなどの有機溶媒等の蛋白塩析効果を有する
ものはいずれも使用することが可能である。The antitumor active protein according to the present invention can be partially purified from the body fluid by the ammonium sulfate precipitation method. Ammonium sulfate concentration at this time is
If it is 40% or more, 90% or more of the active protein is salted out. Further, not only ammonium sulfate but also any one having a protein salting-out effect such as sodium sulfate or an organic solvent such as ethanol or acetone can be used.
体液中にはガラクトース特異的レクチンなどが大量に含
まれていることが知られており（梅津ら,Biochem.Inter
national,10巻,549頁,1985年）、最初にこれらの不要物
を除去しておくことが好ましい。その方法としては例え
ばDEAE−“セフアデツクス（Sephadex）”等のイオン交
換体で体液を処理する方法が挙げられ、非吸着部分を以
後の精製に用いればよい。It is known that body fluid contains a large amount of galactose-specific lectins (Umezu et al., Biochem.
National, Vol. 10, p. 549, 1985), it is preferable to first remove these unwanted substances. Examples of the method include a method of treating the body fluid with an ion exchanger such as DEAE- "Sephadex", and the non-adsorbed portion may be used for the subsequent purification.
体液から本発明に係る制癌活性蛋白を更に精製するに
は、上記処理の後、更にイオン交換体で処理し、非吸着
画分を逆相液体クロマトグラフイーに付し、Ｌ−929細
胞を用いた細胞傷害活性アツセイを行なう。To further purify the antitumor active protein of the present invention from body fluid, after the above treatment, it is further treated with an ion exchanger, the non-adsorbed fraction is subjected to reverse phase liquid chromatography, and L-929 cells are Perform the cytotoxic activity assay used.
また、本発明による制癌活性蛋白に対する抗体を用いる
アフイニテイークロマト手法なども精製手法として使用
し得る。Further, an affinity chromatography method using an antibody against the antitumor protein according to the present invention can be used as a purification method.
以上に述べたような一般的な蛋白精製手法を組み合わせ
ることにより本発明による制癌活性蛋白は単離精製でき
る。そのような組み合わせの１例として次のような精製
過程を取り得る。The antitumor protein according to the present invention can be isolated and purified by combining the above-mentioned general protein purification techniques. As an example of such a combination, the following purification process can be taken.
このような工程により得られた本発明に係る蛋白は次の
ような特性を有する。 The protein of the present invention obtained by such a process has the following characteristics.
１） 分子量が約42,000である。1) The molecular weight is about 42,000.
２） 等電点がpI8.9〜9.0である。2) The isoelectric point is pI8.9-9.0.
３） トリブシンに耐性である。3) Resistant to tribucine.
４） 40％飽和硫安により塩析される。4) Salt out with 40% saturated ammonium sulfate.
５） ガラクトース残基を有する不溶性担体に吸着され
ない。5) Not adsorbed on an insoluble carrier having a galactose residue.
６） pH5〜９で60℃、30分間加熱しても失活されな
い。6) It is not inactivated even if it is heated at pH 5-9 at 60 ° C for 30 minutes.
７） セルロース膜で透析されない。7) Not dialyzed against cellulose membrane.
８） エタノール、アセトンなどの有機溶媒に不溶であ
る。8) Insoluble in organic solvents such as ethanol and acetone.
９） 癌細胞に対して強い細胞傷害活性を有し、抗菌作
用は有しない。9) It has a strong cytotoxic activity against cancer cells and has no antibacterial action.
以下、実施例により本発明を更に具体的に説明するが、
本発明はその要旨を越えない限り以下の実施例によつて
限定されるものではない。Hereinafter, the present invention will be described in more detail with reference to Examples.
The present invention is not limited to the following examples unless it exceeds the gist.
本発明における制癌活性の活性評価は次の方法で行なつ
た。The activity evaluation of the antitumor activity in the present invention was performed by the following method.
マウスＪ−774癌細胞あるいはＬ−929細胞のRPMI−1640
倍地中浮遊液を96穴マイクロプレート中に入れ、104/we
llになるようにした。これに被検体を加えて37℃３日間
CO2培養器で培養し、検鏡により細胞が完全に死滅して
いる被検体の希釈倍率あるいは濃度を最少致死濃度（MI
C）とした。RPMI-1640 of mouse J-774 cancer cells or L-929 cells
Place the double suspension in a 96-well microplate, 10 4 / we
ll. Add the sample to this and incubate at 37 ℃ for 3 days
Culturing in a CO 2 incubator and completely dying the cells under a microscope is used to determine the dilution ratio or concentration of the analyte to the minimum lethal concentration (MI
C).
実施例１ ＜単離＞ （１） アロミリナ デイコトーマ（Allomyrina dicho
toma L.）（カブト虫）の幼虫（体重約30g）の背脈管部
に注射針を穿刺することにより半透明粘稠液体を得た
（１匹当たり約2ml）。この液体を5000回転で30分間遠
心して血球を除去後、60℃、15分間加熱し、さらにセル
ロース透析膜を用いて一昼夜水道水に対して透析した後
以後に実験に用いた。（以後これを体液と称す）。Example 1 <Isolation> (1) Aromyrina dekotomas (Allomyrina dicho)
A semitransparent viscous liquid was obtained (about 2 ml per animal) by puncturing the dorsal vascular portion of a larva of Toma L.) (beetle bug) (body weight: about 30 g) with an injection needle. This liquid was centrifuged at 5000 rpm for 30 minutes to remove blood cells, heated at 60 ° C. for 15 minutes, dialyzed against tap water for one day using a cellulose dialysis membrane, and then used in the experiment. (Hereafter referred to as body fluid).
夏季および冬季に得た幼虫体液のＪ−774癌細胞に対す
る最少致死濃度（MIC）はいずれも１×105希釈倍率で、
材料源の季節変動、成育過程の変動で活性物質の濃度に
差は認められなかつた。The minimum lethal concentration (MIC) for J-774 cancer cells in larval body fluid obtained in summer and winter was 1 × 10 5 dilution ratio,
No difference was found in the concentration of the active substance due to the seasonal variation of the material source and the variation of the growth process.
（２） 体液を下記表１に示されるpHで60℃30分間加熱
した後の体液のＪ−774癌細胞に対する致死活性を測定
した。(2) The lethal activity of the body fluid against J-774 cancer cells was measured after the body fluid was heated at the pH shown in Table 1 below at 60 ° C for 30 minutes.
活性物質はpH5〜９の範囲で60℃30分間加熱しても失活
することはなかつた。また100℃30分間加熱したときpH2
〜11の範囲で活性は完全に消失した。The active substance was not inactivated by heating at 60 ° C for 30 minutes in the pH range of 5-9. When heated at 100 ° C for 30 minutes, pH2
The activity completely disappeared in the range of -11.
（３） 体液を下記表２に示される濃度になるように硫
安を加え、その沈殿物を遠心分離により集め、さらに蒸
留水に対して透析し、元の容積に戻した後Ｌ−929癌細
胞致死活性を測定した。(3) Ammonium sulfate was added to the body fluid to a concentration shown in Table 2 below, the precipitate was collected by centrifugation, dialyzed against distilled water, and returned to the original volume, and then L-929 cancer cells Lethal activity was measured.
活性物質は50％飽和硫安以上の濃度で塩析された。The active substance was salted out at a concentration above 50% saturated ammonium sulfate.
（４） 上記の体液をイオン交換（DEAE−“セフアデツ
クス"A50）にて処理（0.02M Tris−HCl,pH7.4）し、そ
の非吸着画分を逆相系液体クロマトグラフイー（RP−HP
LC,C8カラム）により分離精製すると、図１に示すよう
な回析パターンが得られた。図１のそれぞれのピークを
有する画分を分取りし、Ｌ−929を用いたアツセイを行
なうと細胞傷害活性のピークはFr.2（17.787mm）に現れ
た。 (4) The above body fluid was treated with ion exchange (DEAE- “Sefadex” A50) (0.02M Tris-HCl, pH7.4), and the non-adsorbed fraction was analyzed by reverse phase liquid chromatography (RP-HP).
After separation and purification by LC, C8 column), a diffraction pattern as shown in FIG. 1 was obtained. Fractions having the respective peaks shown in FIG. 1 were collected and assayed with L-929, and the peak of cytotoxic activity appeared at Fr.2 (17.787 mm).
＜物性＞ （１） 分子量 逆相液体クロマトグラフイー（RP−HPLC）で分離後、活
性ピークを有する画分を分取し、レムリ（Laemmli）の
系のSDSゲルろ過（SDS−PAGE）にかけたところ、分子量
42Kにバンドが表れた。<Physical properties> (1) Molecular weight After separation by reverse phase liquid chromatography (RP-HPLC), fractions having an activity peak were collected and subjected to SDS gel filtration (SDS-PAGE) of a Laemmli system. Where the molecular weight
A band appeared at 42K.
更にこのバンドを切り出し、ゲルを1mm角にスライス
し、0.1％ウシ血清アルブミン（BSA）／リン酸緩衝液
（PBS）中で４℃において、一夜振とう後、タンパクを
ゲルより抽出し、Ｌ−929によりアツセイしたところ、
明らかに活性が認められ、活性タンパクの分子量は42K
であることがわかつた。Further, this band was cut out, the gel was sliced into 1 mm squares, shaken in 0.1% bovine serum albumin (BSA) / phosphate buffer (PBS) at 4 ° C. overnight, and the protein was extracted from the gel. When I made an adjustment by 929,
Clearly active, molecular weight of active protein is 42K
I knew it was.
（２） 等電点 DEAE非吸着画分をオフアーレル（Ｏ´Farrell）の方法
により２次元電気泳動を行なつた。SDS−PAGEの結果よ
り分子量42Kのタンパクが活性タンパクであることが確
認されているので分子量42Kのタンパクのスポツトの等
電点を求めたところ、pI8.9〜9.0であることがわかつ
た。(2) Two-dimensional electrophoresis was performed on the non-adsorbed fraction of isoelectric point DEAE by the method of O'Farrell. It was confirmed from the results of SDS-PAGE that the protein having a molecular weight of 42K was an active protein. Therefore, the isoelectric point of the spot of the protein having a molecular weight of 42K was found to be pI8.9 to 9.0.
（３） 蛋白であることの確認 6M尿素及び0.1％SDS中でDEAE非吸着画分200に対しリシ
ル エンドペプチダーゼ（Lysil endo peptidase,LEP）
を１の割合で加え（モル比）、37℃において一夜消化し
た。(3) Confirmation of protein Lysyl endopeptidase (LEP) against DEAE non-adsorbed fraction 200 in 6M urea and 0.1% SDS
Was added at a ratio of 1 (molar ratio) and digested overnight at 37 ° C.
消化後、SDSゲルろ過（SDS−PAGE）及び液体クロマトグ
ラフイー（HPLC）により分析したところ、SDS−PAGEで
はバンドは全て低分子化の為、検出されなかつた。又、
HPLCでは図４及び５のような分解パターンが示された。After digestion, when analyzed by SDS gel filtration (SDS-PAGE) and liquid chromatography (HPLC), all bands were not detected in SDS-PAGE due to low molecular weight. or,
HPLC showed decomposition patterns as shown in FIGS. 4 and 5.
次にこの消化物を、Ｌ−929を用いたアツセイにかけた
ところ活性は無くなつていた。Then, when this digest was subjected to an assay using L-929, the activity was lost.
以上の様にこの細胞傷害活性因子はLEPで分解され、失
活することによりタンパク質であることが確認できた。As described above, it was confirmed that this cytotoxic activator was a protein when it was degraded by LEP and inactivated.
（４） トリプシン消化 DEAE非吸着画分をトシル フエニルアラニルクロロメチ
ルケトン（TPCK）−トリプシンと/:/で37℃において一
夜反応させ、SDS−PAGEで分析したところ、活性バンド
である42Kバンドはほとんど分解されなかつた。またＬ
−929でアツセイを行つたところ活性は保持されてい
た。(4) Trypsin-digested DEAE non-adsorbed fraction was reacted with tosylphenylalanyl chloromethyl ketone (TPCK) -trypsin at //: / overnight at 37 ℃ and analyzed by SDS-PAGE. Was almost never broken down. Also L
The activity was retained after conducting an exercise with -929.
以上より、この細胞傷害活性因子はトリプシンに耐性で
あることがわかつた。From the above, it was found that this cytotoxic activator was resistant to trypsin.
（５） アミノ酸配列 RP−HPLCで分取した活性画分を更に同じC8 RP−HPLCで
再びクロマトグラフイーを行なつた後気相蛋白シークエ
ンサー（Applied Biosystem Protein Sequencer470A）
によりＮ末端アミノ酸配列を分析したところ、図２のご
とくであることがわかつた。(5) Amino acid sequence The active fraction collected by RP-HPLC was further chromatographed again by the same C8 RP-HPLC, and then gas phase protein sequencer (Applied Biosystem Protein Sequencer470A)
When the N-terminal amino acid sequence was analyzed by, it was found to be as shown in FIG.
本発明に係る蛋白は癌細胞に対する直接細胞傷害性を示
す。The protein of the present invention exhibits direct cytotoxicity to cancer cells.
図１は、実施例１における体液のRP−HPLCによる溶出パ
ターンを示す図である。 図２は、本発明に係る蛋白のＮ末端アミノ酸配列を示す
図である。FIG. 1 is a diagram showing an elution pattern of body fluid in Example 1 by RP-HPLC. FIG. 2 is a diagram showing the N-terminal amino acid sequence of the protein according to the present invention.
フロントページの続き (72)発明者 滝沢 寿男 神奈川県横浜市緑区鴨志田町1000番地 三 菱化成工業株式会社総合研究所内Continuation of the front page (72) Inventor Hisao Takizawa 1000 Kamoshida-cho, Midori-ku, Yokohama-shi, Kanagawa Sanryo Kasei Co., Ltd.
Claims (1)
れ、分子量が約42,000であり、等電点がpI8.9〜9.0であ
り、トリプシンに耐性であり、かつ、下記のＮ末端アミ
ノ酸配列を有することを特徴とする、制癌活性を有する
蛋白。 Val Thr Thr Lys Ser Val Lys Thr Phe Lys Ser Asn Pr
o Ser Pro1. Obtained from larval fluid of an insect belonging to the subfamily Polyphagida, having a molecular weight of about 42,000, an isoelectric point of pI8.9 to 9.0, is resistant to trypsin, and has the following N-terminal. A protein having an antitumor activity, which has an amino acid sequence. Val Thr Thr Lys Ser Val Lys Thr Phe Lys Ser Asn Pr
o Ser Pro
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61215623A JPH0778079B2 (en) | 1986-09-12 | 1986-09-12 | Protein with anti-cancer activity |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61215623A JPH0778079B2 (en) | 1986-09-12 | 1986-09-12 | Protein with anti-cancer activity |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS6372700A JPS6372700A (en) | 1988-04-02 |
| JPH0778079B2 true JPH0778079B2 (en) | 1995-08-23 |
Family
ID=16675465
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP61215623A Expired - Fee Related JPH0778079B2 (en) | 1986-09-12 | 1986-09-12 | Protein with anti-cancer activity |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0778079B2 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP6512681B2 (en) * | 2014-09-03 | 2019-05-15 | 学校法人 東洋大学 | Polypeptide from a scale insect |
-
1986
- 1986-09-12 JP JP61215623A patent/JPH0778079B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JPS6372700A (en) | 1988-04-02 |
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