TW201755B - - Google Patents
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- TW201755B TW201755B TW080104225A TW80104225A TW201755B TW 201755 B TW201755 B TW 201755B TW 080104225 A TW080104225 A TW 080104225A TW 80104225 A TW80104225 A TW 80104225A TW 201755 B TW201755 B TW 201755B
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
- C07K14/4703—Inhibitors; Suppressors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Chemical & Material Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pharmacology & Pharmacy (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Description
經濟部中央標準局員工消費合作社印製 01755 Λ 6 _Β6 五、發明説明(1) 發明背景 發明範圍. 本發明是有關新穎的腫瘤細胞生長抑制劑。更特別地 ,本發明是關可得自3T3衍生細胞培養物上清液的新穎 的腫瘤細胞生長抑制劑,其可於腫瘤細胞生長上呈現抑制 活性。 相關技藝陳述 合成藥物如化學治療劑及免疫治療劑被廣泛地充作抗 腫瘤劑,但涉及特異性低且副作用嚴重等問題。另一方面 ,已於組織培養細胞中鑑定出各種腫瘤細胞生長抑制劑。 認為這些抑制劑可為具有高度特異性且副作用最小的抗腫 瘤劑。此類例子中已知有例如:干擾素,淋巴毒素及腫瘤 壞死因子(TNF)。近年有報告關於腫瘤胞毒因子,其 係得自衍自人類的纖維母細胞(日本專利案特許公開第 1 一 148197號),及腫瘤細胞生長抑制因子,其係 得自衍自人類的肺癌細胞(日本專利案特許公開第 1-187094 號)。 另一方面,某些細胞生長抑制劑也分離自纖維母細胞 細胞株3T3細胞,其係由得自瑞士胎鼠之細胞所發展成 的。即,Natraj等人曾報告一種穩定期3T3細胞之表面 層所得的生長因子[Proc. Natl. Acad. Sci. USA,7 5 -61 15-61 19 (1978) 〕。Hare 1 等人也曾 報告一種分子量為4 0 k P a的生長抑制劑係得自 (請先閲讀背面之注意事項再塡寫本頁) 裝- 本紙張尺度通用中國國家樣準(CNS)甲4規格(210x297公婕) -3 - 經濟部中央標準局员工消費合作社印製
i.017S5 A 6 __B6_ 五、發明説明(2) 3 T 3細胞之培養物上清液〔J. Cell. Physio) 119, 1 0 1 - 1 0 6 (1 9 8 4) ; ibid. , 123, 139— 143 (1985)〕。無論如何,已知這些生 長抑制劑於腫瘤細胞上不顯出任何顯著的抑制活性。 發明要點 因此,本發明的一個目的是提出一種對腫瘤細胞生長 具抑制活性的新穎的腫瘤細胞生長抑制劑。 依據本發明,提出一種腫瘤細胞生長抑制劑,其是一 種蛋白質可得自3T 3衍生細胞之培養物上清液,且具有 以下特性: (a )分子量 當以S D S聚丙烯醯胺凝膠電泳於還原及非還原條件 下偵測具3700土370道耳呑之分子量; (b )管柱特性 該抑制劑於pH約7. 4時並不實質地吸附於陰離子 交換樹脂管柱上,但於pH約5. 0時可實質地吸附在陽 離子交換樹脂管柱上, (c )生理活性: 抑制劑於至少人類前骨髄性白血病細胞及人類子宮頸 腫瘤衍生細胞上具有抑制活性。 附圖簡要說明 圖1圖示本發明腫瘤細胞生長抑制劑於CM — 3 SW (請先閱讀背面之注意事項再填寫本頁) 裝- 訂_ 本紙張尺度边用中國S家標準(CNS〉甲4規格(210x297公釐) 一 4 一 經濟部中央標準局員工消費合作社印製 L01755 Λ 6 ________Β_6 五、發明説明(3) 陽離子交換層析中之溶離略圖; 圖2示出本發明腫瘤細胞生長抑制劑於苯基5 PW-RP逆相HPLC中之溶離略圖; 圖3示出本發明腫瘤細胞生長抑制劑於苯基5 PW_ RP逆相HPLC中之溶離略圖; 圖4為本發明腫瘤細胞生長抑制劑接受S D S — P A G E所得結果之照片。 圖5示出本發明腫瘤細胞生長抑制劑(P — 1)的一 级結構。 圖6示出本發明腫瘤細胞生長抑制劑(P — 2)的一 级結構。 圖7示出本發明腫瘤細胞生長抑制劑(P — 1)於人 I類宮癌細胞HeLa,人類前骨髄性白血病細胞' HL — 6 ◦及人類肺癌細胞A - 549上之生長抑制作用 Ο 圖8示出本發明腫瘤細胞生長抑制劑(P — 2)於人 類子宮癌細胞HeLa,人類前骨髓性白血病細胞HL— 6 ◦及人類肺癌細胞A — 54 9上之生長抑制作用。 圖9示出本發明腫瘤細胞生長抑制劑(P-1)於人 類子宮癌細胞衍生的H e L a細胞上之生長抑制作用。 發明之詳細說明 本發明的腫瘤細胞生長抑制劑可如下獲得。 (請先閲讀背面之注意事項再填寫本頁) 裝- 訂- 本紙張尺度边用中困國家楳準(CNS)甲4規格(210x297公龙) -5 - 經濟部中央標準局貝工消費合作社印製 L01755 Λ 6 ___Β6_ 五、發明説明(4) 3 T 3衍生細朐株之製備 腫瘤細胞生長抑制劑可製備自如N I Η 3Τ3細胞 〔J. Virol., 4, 549 (1969)〕,其僳纖維母細 胞株3T3細胞中的一種,由得自瑞士胎鼠細胞所發展的 即,欲求的由3T3細胞衍生之細胞株,其製備可將如 N I Η 3 T 3細胞繼代培養於添加有血清的D F培養基上 ,其為由 Dulbecco's 經修飾 MEM[ Virology,8,3 9 6 (1 9 6 9)〕及Ham F-12 [ Proc. Nat1 . Acad . Sci. 53, 288 (1965)〕混合之培養基,之後培養於 含有激素如胰島素之DF培養基中,以分離或篩選增殖的 純条,進一步將純条培養於單獨的DF培養基中,並篩選 可於單獨的DF培養基中增殖之純条。 培赛物上淸液之魁備 衍生自3T3細胞衍生細胞株,最先培養於含血清之 DF培養基中。當細胞呈融合狀時,則移去培養基。於進 一步培養於無血清之DF培養基一定時期後,移去培養基 。之後,培養於無血清的DF培養基中,如歴約96至約 120小時,且毎96—120小時更新培養基,可收集 培養物上淸液。所收集之培養物上清液離心以製備培養物 上清液。 晡瘤細朐生長抑制酬:> 姊化 培養物上清液接受超過濾,以進行分子量分段分離且 (請先閱讀背面之注意事項再填寫本頁) 裝- 線. 本紙張尺度逍用中國國家標準(CNS)甲4規格(210x297公婕) -6 - L01755 Λ 6 Β6 經濟部中央標準局員工消費合作社印製 五、發明説明(5 ) 濃縮上清液。上清液必要且企求時再接受鹽析及透析。 接下來,含有欲求組份之適當缓衝溶液通過陰離子交 換樹脂管柱,如 Q-Sepharose 管柱(Pharmacia)及 DEAE-Se-pharose (Pharmacia),以部份純化.。_本發明的腫瘤細胞 生長抑制劑具有並非實質地吸附在#離子交換樹脂管柱上 之特性。因此,部份純化的抑制劑可由通過pH約7. 4 V - 之#_字交換樹脂管柱及收集未吸附流份而得。 本發明的抑制劑可實質地吸附在pH約5. 0之陽離 子交換樹脂管柱上。因此,通過pH約5. 0的陽離子交 換樹脂管柱',如S-Sepharose管柱(Pharmacia),. CM Seph-arose管柱(Pharmacia)及 T S K 凝膠(Μ - 3 S W (Toyo Soda),可得到更純的抑制劑。 利用如上述之陰離子及陽離子交換樹脂純化,也可變 化這些純化步驟次序而適度地進行,依必要性而定。 藉著適當地進行純化步驟,如利用羥基磷灰石管柱之 吸附性層析;及利用T S K凝膠CM— 3 SW或苯基 5 PW_RP逆相管柱之高性能液相層析,可獲得本發明 高度純化的腫瘤細胞生長抑制劑。 睡瘤生#抑制割之特件 本發明腫瘤生長抑制劑之特性説明於下。 (a )分子量 本抑制劑當以SDS聚丙烯醯胺凝膠電泳,於還原及 非還原條件下偵測,具有3, 700±370道耳呑之分 (請先閱讀背面之注意事項再塡寫本頁) 本紙張尺度逍用中《國家標準(CNS) f 4規格(210x297公;¢) -7 - 01755 A 6 B6 五、發明説明(6 ) 子量。此抑制劑是一種蛋白質具單股結構,因為於還原及 非還原條件下不見其分子量變化。 (b )管柱特性 所上文中所述,此抑制劑具有於pH約7. 4不賁質 吸附於陰離子交換樹脂管柱,而於pH約5. 0時可實質 地吸附於陽離子交換樹脂管柱上之特性。 (c )生理活性 抑制劑具有在至少人類前骨強性白血病細胞,如 HL—60,及人類子宮頸腫瘤衍生細胞,如HeLa細 胞上之抑制活性。因此,本發明的抑制劑可用於治療白血 病,或固態腫瘤如子宮腫瘤。 (d )胺基酸序列 依據自動式愛德曼降解作用法,以氣相蛋白質定序儀 定序胺基酸之結果顯示,抑制劑具有下列二種胺基酸序列 〔(P—1)及(P — 2)〕中任一序列。 (請先閱讀背面之注意事項再填寫本頁) 經濟部中央標準局員工消費合作社印製 本紙張尺度逍用中國Η家標準(CKS)甲4規格(210x297公;¢) -8 - L〇itS[> Λ 6 B6 五、發明説明(7 (P-1) 1 2 3 4 5 6 7 8 9 Val -Gin -lie -Thr -Lys -Crys _ -Ser · -Sei :-Asp 10 11 12 13 14 15 16 17 18 Met -Asp -Gly -Tyr -Cys -Leu 一 His - Gly -Gin 19 20 21 22 23 24 25 26 27 Cys — lie -Tyr -Leu -Val -Asp - Met - Arg -Glu 28 29 30 31 32 33 34 35 36 Lys -Phe -Cys -Arg -Cys —Glu ~ Val - Gly -Tyr 37 38 39 40 41 42 43 44 45 Thr 46 Leu -Gly -Lys -Arg -Cys —Glu ~ His - Phe -Phe (請先閱讀背面之注意事項再填寫本頁) 裝- 訂' 經濟部中央標準局貝工消費合作社印製 (P-2) 1 2 3 4 5 6 7 8 9 Val —Gin — lie -Thr -Lys -Crys . -Ser -Ser — Asp 10 11 12 13 14 15 16 17 18 Met -Asp - Gly -Tyr -Cys -Leu 一 His - Gly - Gin 19 20 21 22 23 24 25 26 27 Cys -He - Tyr -Leu -Val -Asp - Met - Arg - Glu 28 29 30 31 32 33 34 35 36 Lys -Phe - Cys -Arg -Cys —Glu ~ Val - Gly - Tyr' 37 38 39 40 41 42 43 44 Thr -Gly - Lys -Arg -Cys _ Glu — His — Phe 表紙張尺度边用中B國家標準(CNS)甲4規格(210x297公龙) 9 - 01^55 A 6 B6 經濟部中央標準局員工消費合作社印製 五、發明説明(8 ) 腫瘤細胞生長抑制劑像一種新穎的蛋白質,其可得自 由3T3紳胞衍生的細胞株之培養物上淸液。此抑制劑可 顯著地抑制人類前骨髓性白血病細胞及人類子宮頸腫瘤衍 生細胞之生長。因此,本發明的抑制劑可用於治療白血病 或固態腫瘤,如子宮癌。 文後,本發明參照實例更詳細説明。 實例 1 .由3 T 3細朐衍牛的細朐抶之靱備 於Ν ΙΉ 3 T 3細胞繼代培養於含有1 0%胚牛血清 之D F培養基(Dulbe CCO 1 S經修飾MEM : Ham F — 12=1 : 1)後,細胞培養於DF培養基中,並含 有5微克/毫升胰島素,5撤克/毫升鐵傳遞蛋白及2X 1 0_βΜ硒酸鹽。取得可於培養基中生長之純糸。 再者,也自純条中選擇可於單獨的DF培養基中增殖 之純糸。此純糸經繼代培養以獲得其細胞株。發展出的細 胞株命名為ΝΙ H 3T3—s f。培養進行於37它, 5 % C〇2氣相下。培養基3天以新鮮培養基做70% 更新。繼代培養之進行則是於培養細胞達次融合狀時稀釋 2倍。至於培養基,準備50%調適培養基及50%新鮮 培養基之混合物供使用。 2 .進備無細朐T H#3T3—s f細胞培養物上清 (請先閲讀背面之注意事項再填寫本頁) 裝< 訂· 線. 本紙張尺度逍用中國國家楳準(CNS)甲4規格(210x297公着) -10 - 經濟部中央標準局貝工消费合作社印製 l755 A 6 __B6_ 五、發明説明(9 ) N I H3T3 — S f細胞培養於有1 0%胚牛血清之 DF培養棊中。當細胞欲融合時移去培養基。待以PBS (―)洗一次後,細胞培養於DF培養基中48小時。移 去培養基,再繼績於新鮮的DF培養基中培養96— 120小時。培養基每96—120小時更新,並收集 1〇〇升培養基。所收集的培養基離心(2000rpm X10分鐘)以回收上清液,並貯於一 20Ϊ:下。 3 .純化 l)Q-Seph’arose 管柱層析 利用Pel icon匣条統(超過濾作用濾膜条,分出分子 量100 ◦者:FUJI FILTER),可將回收的 1 0 0升培養物上清液濃縮約5 0倍。濃縮物再接受 90%硫酸銨飽和作用之鹽析,之後以8000rpm離 心60分鐘。沈澱物溶於20mM Tr i s — HC5缓 衝溶液(pH7. 4),溶液再透析至缓衝溶液。透析物 加至 Q-Sepharose 管柱(Pharmacia) (Φ 5 公分 X 5 公分) ,其先前已用相同的缓衝溶液平衡,以收集未吸附及吸附 的流份。 溶離條件如下: 流速: 8毫升/分 分段分離:2毫升/管 溶離劑:20mM T r i s— HC$缓衝溶液( P Η 7 . 4 ) (請先閲讀背面之注意事項再填寫本頁) 裝- 訂* 本紙張尺度逍用中國國家標準(CNS)甲4規格(210x297公龙) -11 - A 6 B6 經濟部中央標準局貝工消費合作社印製 五、發明説明(10) 2 ) S-Pepharose管柱層析 未吸附附流份之pH諝至5. 0後,令其通過S-Sep- harose管柱(Phermacia) (Φ5公分X6公分),其先前 已用20mM醋酸鹽缓衝溶液(PH5. 0)平衡。活性 部份被吸附。之後,以20mM Tris — HCJ2緩衝 溶液(P Η 7 . 4 )進行溶離以獲得活性流份。 溶離條件如下。 流速··‘ 0. 85毫升/分 分段分離:4毫升/管 溶離劑:20mM Tr i s - HC5缓衝溶液( P Η 7 . 4 ) 3)羥基磷灰石管柱層析 自S-Sepharose管柱中溶離之活性流份,其PH值以 醋酸調至6. 0。活性流份填料於羥基磷灰石管柱(Pent- ax 0 7. 5公分Xl〇公分,Asahi Kogaku),其先目U 已用20mM醋酸鹽缓衝溶液(pH6. ◦)平衡,以收 集未吸附流份。 (請先閲讀背面之注意事項再填寫本頁) 裝< 訂- -線, 本紙張尺度边用中國國家楳準(CNS)甲4規格(210x297公潑) 12 - A 6 Β 6 五、發明説明(π) 溶離條.件如下。 流速: 1毫升/分 分段分離:1毫升/管 溶離劑:2〇mM 醋酸鹽缓衝溶液(ρΗ6· 0) 4) TSK凝膠CM— 3SW管柱靥析HPLC 活性流份之pH以醋酸調至5.◦。活性流份填料至 TSK凝膠CM — 3SW管柱(07. 5公分X7. 5公 分,Toso),其先前已用20m Μ鍵?. T 3)醋酸鹽缓 丨己一 衝溶液(ρΗ5. ◦),含5%乙睛予以平衡。 (請先閱讀背面之注意事項再填寫本頁) 經濟部中央標準局員工消費合作社印製 溶離條件如下: 流速: 1毫升/分 分段分離:1毫升/管 溶離劑:A) 20mM醋酸鹽缓衝溶液(ρΗ5 )/ 5 % C Η 3 C Ν B) 20mM醋酸鹽缓衝溶液(ΡΗ5 )/ 5 % C Η 3 C Ν / 0 . 2 Μ Ν a C ί A — Β之直線梯度(120分鐘) 0 〇 活性出現於2個流份,且分別以86mM (Ρ—1) 及lOOmM (Ρ — 2)之NaCi濃度溶離(參見圖1 本紙張尺度逍用中a國家標準(CNS)甲4規格(210x297公茇) 一 13 - 01755 A 6 B6 經濟部中央標準局貝工消費合作社印製 五、發明説明(12) )〇 5)苯基5PW—RP逆相管柱層析HPLC 由CM— 3SW HPLC所得的活性流份,填料至 苯基v 5PW— RP管柱(Φ4. 6毫米X7. 5公分, Tosf),其先前已用含5% CH3 CN之磷酸鹽緩 衝溶液(PH7. 4)平衡。經含20% CH3CN之 5mM磷酸鹽緩衝溶液(pH7. 4)溶離20分鐘後, 進一步的溶離則是利用溶離程序為80分鐘,由磷酸鹽缓 衝溶液(P‘h7.4)/20% CHaCN至磷酸鹽缓 衝溶液(pH7.4)/40% CH3CN之直線梯度 。流速為1毫升/分,且分段分離為2毫升/管。P-1 及P—2分別於59—60分鐘及60—61分鐘位置處 被溶離。 4 . SDS-PAGE 以逆相Η P L C所得的2値活性流份再接受S D S — PAGE,係將樣品缓衝溶液(0. 0625Μ Tr i s— HC5 缓衝溶液,pH6. 8, 2% SDS 3ΜΜ糖)加至其一部份中,再於l〇〇t:下加熱 3分鐘。以經修改的Laemmli法進行電泳〔Nature, 227, 680 (1970)〕,其中採用含有0.1% SDS的20%聚丙烯醯胺凝膠(厚1毫米)。電泳後, 蛋白質帶以銀染色偵測(銀染色套件,<ako)。至於分子 (請先閲讀背面之注意事項再填寫本頁) 裝. 訂 線- 本紙張尺度逍用中國S家標準(CNS)甲4規格(210x297公龙) -14 - A 6 B 6 經濟部中央標準局貝工消費合作社印製 五、發明説明(13) 量標誌則採用:肌蛋白(17201),肌蛋白I + I ( T 14632),肌蛋白(8235),肌蛋白E ( 6383),肌蛋白I (2556)及肌蛋白1 一 14 ( 1696)。結果,P—1及P—2二種均可於分子量 3, 70〇±370道耳呑位置處呈單一帶被偵測。於還 原及非邇原條件下,分子量均未見化。SDS—PAGE 之結果示於圖4。 5 .胺某酸序列之決宙 關於二値純産物,其胺基酸序列以自動式愛德曼降解 法,利用氣相丨蛋白質定序儀()Model 470A, Applied Biosystems , Co. , Ltd.,) 予以決定 。可顯示, 蛋白質具有如 上所述的胺基酸序列(參見圖5及6)。 6.生物活袢 1)於人類子宮腫瘤細胞HeLa,人類前骨髄性白血病 細胞H L — 6 0及人類肺腫瘤細胞A - 54 9之生長 抑制活性 每一細胞以5X103/100微升/孔洞,接種於 9 6孔洞盤上(Falcon),之後培養於371,5% C〇2氣相下24小時。培養基換上含有每毫升5毫微克 或50毫微克,由苯基5PW— RP逆相HPLC所得的 純化産物〔(P—1)或(P - 2)〕之培養基,細胞再 培養6天。每3天更新培養基。6天後,以錐藍貿染色計 (請先閲讀背面之注意事項再填寫本頁) 裝- 訂- 線. 本紙張尺度逍用中國《家標準(CNS)甲4規格(210x297公釐) { A 6 B 6
經濟部中央標準局貝工消t合作社印製 五、發明説明(]4) 數活細胞數目。HL — 60細胞培養於RDF — 2% FBS培養基,且HeLa及A — 549細胞培養於 D F - 2 96 FBS培養基。於個別細胞上之生長抑制作 用,示於圖7及8。由圖7及8可明顯看出,本發明的抑 制劑可顯著地抑制He La細胞,A — 549細胞及 H L — 6 0細胞之生長。 2)人類子宮癌細胞衍生的He La細胞外形變化 HeLa細胞以5X1 04細胞/260微升含 1 0 % F B S — D F /孔洞接種於4 8孔洞盤中(Corning Ltd.), 之後於 37 °C, 5% C〇2之氣相下培養 24小時。每一培養基以含有每毫升10毫微克苯基 5PW—RP逆相HPLC得到的純産物(P — 1)之新 鮮培養基更換,細胞再繼缠培養4 8小時。於位差顯微鏡 下觀察培養細胞之外形變化。結果示於圖9。圖9A及 9B分別示出對照用細胞及經(P - 1)處理細胞之外形 變化。由圖9可明白看出,本發明的抑制劑(P_l)可 顯著地抑制H e L a細胞之生長。 (請先閱讀背面之注意事項再填寫本頁) 裝· 訂- 線. 本紙張尺度逍用中a a家標準(CNS)甲4規格(210X297公¢) -16 _
Claims (1)
- r- -务-告衣-^ .I 六、申請專利类r圊_ J 1 . 一種腫瘤細胞生長抑制劑,其係一種蛋白質,可 得自3 T 3衍生細胞株培養物上淸液,且具有以下特性: (a )分子置 當以SDS聚丙烯醯胺凝膠電泳,於還原及非還原條 件下偵測,具3700±37 ◦道耳呑之分子量; (b )管柱特性 此抑制劑於pH約7. 4時並不會實質地吸附於陰離 子交換樹脂管柱上,但於p Η約5 . 0時可實質地吸附在 陽離子交換樹脂管柱上, (c )生理活性: 抑制劑具有於至少人類前骨髓性白血病細胞及人類子 宮頸腫瘤衍生細胞上之抑制活性。 2.根據申請專利範圍第1項之腫瘤細胞生長抑制劑 ,其具有以下二個胺基酸序列〔(Ρ-1)及(Ρ — 2) 〕中任一値序列: (請先聞讀背面之注意事項再填寫本頁) 經濟部中央揉準局貝工消费合作社印製 -17 - 、本紙張尺度適用中國國家標準(CNS)甲4規格(210x297公釐) 六'申請專利範ffi AT B7 C7 D7 經濟部中央標準局員工消费合作社印製 V (P-1): 1 2 3 4 5 6 7 8 9 Val —Gin — lie - Thr -Lys -Crys - Ser - Ser - Asp " 10 11 12 13 14 15 16 17 18 Met 一 Asp - Gly - Tyr -Cys -Leu - His - Gly - Gin — 19 20 21 22 23 24 -25 26 27 Cys -lie - Tyr - Leu -Val -Asp - Met - Arg - Glu - 28 29 30 31 32 33 34 35 36 Lys —Phe — Cys - Arg -Cys -Glu - Val - Gly - Tyr - 37 38 39 40 41 42 43 44 45 Thr -Gly - Lys - Arg -Cys -Glu - His - Phe - Phe - 46 Leu (P-2): 1 2 3 4 5 6 7 8 9 Val -Gin - lie - Thr -Lys -Crys -Ser - Ser -Asp - 10 11 12 13 14 15 16 17 18 Met -Asp " Gly - Tyr -Cys -Leu - His - Gly - Gin - 19 20 21 22 23 24 25 26 27 Cys -lie - Tyr 一 Leu -Val -Asp - Met - Arg - Glu - 28 29 30 31 32 33 34 35 36 Lys -Phe - Cys - Arg —Cys _ Glu - Val - Gly ~ Tyr 一 37 38 39 40 41 42 43 44 Thr -Gly - Lys — Arg -Cys —Glu — His 一 Phe (請先閱讀背面之注意事項再填窝本頁) k. •綠, K紙張尺度適用中國國家標準(CNS) 規格(210X297公釐) -18
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US5155217A (en) * | 1987-05-29 | 1992-10-13 | The Trustees Of Columbia University In The City Of New York | Ongogene encoding polypeptide having growth factor activity |
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