TW557299B - Novel antiproliferative protein from bacillus thuringiensis var. thuringiensis - Google Patents
Novel antiproliferative protein from bacillus thuringiensis var. thuringiensis Download PDFInfo
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- TW557299B TW557299B TW085114980A TW85114980A TW557299B TW 557299 B TW557299 B TW 557299B TW 085114980 A TW085114980 A TW 085114980A TW 85114980 A TW85114980 A TW 85114980A TW 557299 B TW557299 B TW 557299B
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- protein
- bacillus thuringiensis
- thuringiensis
- cells
- toxin
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
- C07K14/325—Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins
-
- 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
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- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Crystallography & Structural Chemistry (AREA)
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557299 經濟部中央標準局員工消費合作社印製 A7 ___B7___五、發明説明（3 ) ϋ-9 37細胞之抗增生活性。使用此種分析時，致腫瘤毒素 藉差別溴化鈉梯度超離心，蛋白質分解性消化，繼以DEAE 阿非凝膠（affigel)藍親和層析而予分離〇該致腫瘤毒素 活性結合於DEAE阿非凝膠藍樹脂，且可用0.05M NaCl流洗 。其在變性條件下具有大約20 kDa之分子量。 已純化蛋白質之生物活性對各種蛋白質分解性酵素包 括胰蛋白_、胰凝乳蛋白_及鏈徽蛋白_敏感，對酸性條 件（pH低於4 )敏感，對三氟乙酸（0·1 %)敏感，且對 乙腈（90%)敏感。然而，致腫瘤毒素活性耐高溫（100 Ό，3 0分鐘）及還原條件（1 mM二硫蘇糖醇）〇此種蛋白 質之胺基末端胺基酸序列由NM-Pro-Ser-Thr-Val-Val-Asn-Val-Ser-Asn-Leu-Lys-Pro-Gly-Asp-Thr-Ile-GIu-Lys-Glu-Phe-構成〇此序列在與其他蛋白質之已公開序列 相較具獨特性Q —種以此序列爲基礎之合成舦曾用以在兔 體內製備多株抗體，而此等抗體甚至在1:10,〇〇〇之抗血清 稀釋率時將致腫瘤毒素之生物活性完全中和。用此等抗體 進行之西方印跡分析亦顯示在20 kDa處之致腫瘤毒素帶〇 除胸t滲入法外，台盼藍排斥及MTT法亦顯示高度純 化致腫瘤毒素對ϋ- 9 3 7細胞長期生長之完全抑制作用〇後 者顯示對抗廣泛之不同種類腫瘤細胞系包括骨髓性（U-9 3 7 、ΤΗΡ-1、HL40)、淋巴性（Raji、Jurkat)、成紅細胞性 (K-562 )、乳癌（CL0、MCF — 7)、卵巢癌（0VCA429、 0VCA4 32 、0VCA433)、腎（A-293)及肝癌（Hep3B、HepG2)等之抗增 生效應〇在類似條件下，人類成神經膠質細胞瘤（U-251) 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297处楚) _ (S 一 (請先閲讀背面之注意事項再填寫本頁) 557299 A7 B7 五、發明説明（5 ) 爲達成並能詳細了解本發明之上述特色、優點與目的 以及其他將成爲明顯之事物，可參考對以上所扼述發明之 一些於各附圖中加以例示之各具體形式所作更明確之說明 。此等圖式構成本說明書之一部分。然請注意，各附圖乃 例示本發明之各項較佳具體形式，故而不應視爲對其範疇 設限〇 圖1顯示致腫瘤毒素之十二硫酸鈉聚丙烯醯胺凝膠分 析。如下述將1a g之致腫瘤毒素溶於15 %丙烯醯胺上〇 圖2顯示致腫瘤毒素之胺基末端胺基酸序列分析。 圖3顯示藉由西方印跡分析對致腫瘤毒素之偵測，且 亦顯示藉由對抗合成致腫瘤毒素肽之抗體對致腫瘤毒素生 物活性之中和〇 圖4顯示致腫瘤毒素以其胺基酸序列爲準與一合成肽 之效應比較〇 圖5顯示各種蛋白_對致腫瘤毒素生物活性之效應。 圖6顯示pH (圖6A)及有機溶劑（圖6B)對致腫瘤毒 素生物活性之效應〇 經濟部中央標準局員工消費合作社印製 (請先閲讀背面之注意事項再填寫本頁) 圖7顯示對致腫瘤毒素之生物分析〇於37 °C在各96井 平皿內以致腫瘤毒素培育5 X 103個細胞達72小時，然後如 下述藉m處理胸苷滲入法測定細胞存活埤。所有測定均作 成三份〇 圖8顯示致腫瘤毒素對不同骨髓細胞系之劑量相依效 應。 圖9 A顯示致腫瘤毒素對不同淋巴細胞系之劑量相依效 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297令签） 557299 A7 B7 五、發明説明（8 ) 移性）之性質及其群體、特定免疫毒素之特性（亦即其治 療指標）、病患、病歷以及其他因素〇蛋白質投施量典型 上在約0 , 1至10 mg/kg病患體重之範圍內0該計畫將繼續 使功效成最佳化，同時平衡治療之各項負面效應〇請參閱 Remington’s Pharmaceutical Science，17版（1990)賓州 伊士頓市 Mark Publishing Co.;以及Goodman 及 Gilman :The Pharmacological Basis of Therapeutics 第8 版 (1990) Perga mon Press ;凡此均以指述方式納入本文。 對於胃腸外投施，蛋白質最常配製成單位劑量形式（ 溶液、懸浮液、乳液）而與一製藥上可接受之胃腸外載體 聯用。此等載體較佳爲無毒性及非治療性〇此等載體之實 例爲水、鹽水、林格氏溶液、葡荀糖溶液、以及5 %人類 血清蛋白〇亦可使用非水性載體譬如不揮發油類及油酸乙 酯0脂質體可用作載體◦載體可含有小量之添加物譬如提 升等滲性及化學穩定性之物質，例如緩衝劑及防腐劑〇免 疫毒素典型上係配製成濃度約爲0 · 1 mg / m 1至1 0 mg / m 1之載 體。 經濟部中央標準局員工消費合作社印製 (請先閲讀背面之注意事項再填寫本頁) 分子生物學界內平均科學家之普通技術水準近年來已 實質增筒◦業界普通技術入士在已知本案說明書之敎示後 ，將能迅即就此新穎細胞毒性蛋白質之基因進行殖株〇 本發明之標的亦爲一種處理贅生細胞之方法，包含對 所述細胞投施一治療有效劑量之本發明組合物〇該贅生細 胞較佳爲由骨髓細胞、B淋巴細胞、T淋巴細胞、成紅細 胞性細胞、乳房腫瘤細胞、卵巢腫瘤細胞及肝癌細胞所組 木紙張尺度適用中國國家標準（CNS ) A4規格（21〇χ297合義丄 557299 經濟部中央標準局貝工消費合作社印製 A7 B7五、發明説明（9 ) 成集團中選出〇 一般而言，贅生細胞可在人體或動物體內 〇其特別企圖爲該新穎組合物將阻滯一贅生情況之復現， 並延長所述贅生細胞之宿主之生存時間0 本發明蛋白質亦可用於活體外方法。舉例言之，該方 法可用於殺死骨髓內之腫瘤細胞〇在此方法中，骨髓先予 摘離患有贅生疾病之個體◦嗣後，用一細胞殺死有效劑量 之本發明蛋白質處理，以消除殘餘之腫瘤細胞〇經處理之 骨髓細胞可再投施於病患，以便利在接受强化學治療及/ 或放射治療以消除所有內生性贅生血毒性細胞之後重建免 疫系統◦ 以下實例係就例示本發明各種不同具體形式之目的而 予列示，故無意以任何方式對本發明設限〇 實例1 物質 RPMi-l(340 係得自 Whittaker MA Bioproducts 公司（ Walkerville，MD) 〇胎牛血清（FBS)及艮他黴素由GIBC0 公司（Grand island, NY)獲得〇其他化學品及生物化學品 係獲自 Sigma Chemical 公司（St Louis ’ M0)。ϋ - 937 (組 織細胞性淋巴瘤，CKL 1 5 9 3 )、前髓細胞性白血病HL-60 (CCL 240)、急性骨髓致生白血病KG-la (CCL 246·1)、 乳腺癌 MCF-7 (ΗΤΒ 22)、表皮癌 HepG-2 (CCL 23)、乳癌 BT - 20 (ϋϊβ 19)、伯奇氏淋巴瘤 Raji (CCL 86)及 Jurkat (急性T細胞白血病，TIB 152) 等細胞系均得自美國種 細胞培養物收集中心（Kochi 1 le，MD) 〇各細胞以例常方 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 29J會美) " (請先閱讀背面之注意事項再填寫本頁) 557299 A7 B7 五、發明説明（12 ) )以2 5,0 0 0 rpm差動超離心（Beckman離心機L5-50E,轉 子SW_2T) 9U分鐘將結晶與孢子分離。將含有結晶之各帶 匯集，用去離子水沖洗三次，凍乾並儲存於-20 ( Ang及
Nickerson， 1978) 〇 實例6 結晶之溶化 此步驟如Prasad及Shethna，1974所述者予以實施。 簡言之，Ιϋϋ mg結晶蛋白質於室溫懸浮於20ml之1M NaOH 在0·1Μ甘胺酸中之溶液內5分鐘，然後離心（ 20,000 r pm 於4 eC 30分鐘）〇上澄液（稱作鹼溶化結晶）對pH 7 .2磷 酸鹽緩衝鹽水透析◦將試樣凍乾並儲存於-20 〇 實例7 塍蛋白_對鹼溶化結晶之活化作用 經濟部中央標準局員工消費合作社印製 (請先閲讀背面之注意事項再填寫本頁) 將Choma等（1 9 9 0 )之方法用於此一步驟。簡言之，在 pH 7 ·2之ϋ . 1M甘胺酸緩衝液內用胰蛋白廳（10%，w/w)於 37¾處理可溶於鹼之各分量（5 mg/ml)達30分鐘。試樣於 4 °C以2ϋ，ϋϋϋ rpm離心（Beckman離心機）30分鐘，然後 對pH 7.2磷酸鹽緩衝鹽水透析上澄液〇 實例8 glA E阿非凝膠藍親和層析 將一得自以上步驟之致腫瘤毒素分量（約2mg/ml)施 加於預平衡於pH 8之20 mM Tris內之DEAE阿非凝膠藍柱（ 1 X 6 . 5 em) 〇用平衡緩衝液沖洗該柱，然後用〇 . 05至1 ·0Μ
NaCl之昇高梯度予以流洗。各致腫瘤毒素分量藉生物分析 本紙張尺度適用中國國家標準（CNS ) A4規格（210'〆297公釐） -15- 557299 A7 B7__ 五、發明説明（13 ) 予以識別，並以〇r ad法測量蛋白質濃度〇 m m 9 DNA斷裂分析 用致腫瘤毒素（5〇/χ g/ml)處理各細胞（5X 106/ml)達 24小時或72小時，然後旋轉沉降，用PBS沖洗，而再懸浮 於PH 7.4之 10 mM tris-Cl 及pH 8之ImM EDTA內。然後， 用溶胞緩衝液·Π〇 mM tris-Cl pH 8、100 mM NaCl、25mM EDTA、及ϋ.5 %SL)Sj將細胞溶解，並添加RNA_(1U1之 l〇mg/ml )移除KNA 〇以50eC培育30分鐘後，將1 /z 1之蛋白 酶Κ (2ϋ mg/ml)添加於所有之試管，並於50°C繼續多培育 3〇分鐘。添加ϋ·4/χ1之荷載染料（ 0.02 5 %溴酚藍、0.25 二甲苯氰醇FF、及%甘油在水中），將各試樣在1 . 2 % 瓊脂糖凝膠上溶於ΤΑΕ緩衝液（0·(ΗΜ Tris乙酸、0.001Μ EDTA中）〇 實例1 〇 胺基酸序列分析 經濟部中央標準局員工消費合作社印製 (請先閲讀背面之注意事項再填寫本頁) 致腫瘤毒素之胺基酸序列藉B a y 1 〇 r醫學院（Η 〇 u s t ο η， TX)之蛋白質定序設備實施〇將蛋白質自SDS-PAGE凝膠轉 移於PVDl·'膜上，然後在4 7 3A型微定序器（Applied Biosys -t e m s公司，F 〇 s t e r C i t y，C A )上接受序列分析。序列同源 性硏究係在國家生物技術資訊中心（NCBI )用BLAST網路服 務執行0 實例11 肽合成 本紙張尺度適用中國國家標準（CNS ) A4規格（210X2!7^^ ) 557299 ΚΊ Β7 五、發明説明（14 ) 以致腫瘤毒素之胺基酸序列爲基礎’藉B a y 1 0 Γ醫學院 (Houston, ΤΧ)之蛋白質化學核心設備合成一 18個胺基酸 長度之狀。fmc)e複抗原取（MAP)樹脂予用於致腫瘤毒素狀 之合成。該合成肽之反相fiPLC予以純化並將胺基酸組成定 性〇 實例1 2 抗體生成 將如上合成之肽用以製作抗體（Beth5^ Laboratories 公司，Montgomery，TX)，方法爲用完全弗羅因德氏佐劑 中之ΙϋΟ/xg之抗原經皮下（多部位）使兔子免疫0在14曰 及2 1日上繼以各在不完全弗羅因德氏佐劑中之二次50 a g 肌內注射◦其後，血清在致腫瘤毒素生物分析中測試中和 滴度，並藉西方印跡分析測試免疫反應力。 實例1 3 西方印跡分析 經濟部中央標準局員工消費合作社印製 (請先閲讀背面之注意事項再填寫本頁) 各蛋白質試樣在SDS-聚丙烯醯胺凝膠（15% )上電泳。 電泳後，將各試樣轉移至含有Tris-HCl (25mM，pH 8·3) 、甘胺酸mM)、及甲醇（20 %，ν/ ν)之緩衝液內之硝 酸纖維素濾紙上。於室溫用一含有BSA (5%)及在PBS中 之 Tween 2ϋ (ϋ·1%，v/v)之阻斷緩衝液（PBS-Tween 緩 衝液）將該硝酸纖維素濾紙上之非專一性結合作用減小達 1小時。用PliS-Tween緩衝液沖洗三次後，於室溫用抗致 腫瘤毒素抗體（ι:ιυ，〇ϋ〇稀釋率）培育濾紙1小時〇再次 沖洗該濾紙，然後用山羊抗兔I gG-辣根過氧化物辭接合體 本紙張尺度適用中國國家標準（CNS ) A4規格（21〇Χ2^7&釐） 557299 A7 B7 五、發明説明（15 ) (1 :10，Οϋϋ稀釋率）於室溫培育1小時。其後，四次沖洗 濾紙’並用强化化學發光系統（A m e r s h a m )使各帶視覺化。 實例1 4 之分離及物化定性 藉由使用細胞毒素生物分析及包含梯度超離心 、蛋白質水解活化及DEAE阿非凝膠藍親和層析之純化協定 ，蛋白質即以ϋ · 05Μ NaCl自DEAE流洗出而予分離。此種蛋 白質在本文中稱作致腫瘤毒素〇在變性條件下以SDS-PAGE 分析（藉庫瑪西U· ϋ g m a s s i e )藍及藉銀染色二者）顯露一 於大約2ϋ kDa分子量之主帶（圖1) 〇致腫瘤毒素予電印 跡於一PVDF膜上，然後實施胺基酸序列分析。所得胺基末 端序列示於圖2 〇當兼用肽及核苷酸序列資料庫檢査時發 現，致腫瘤毒素之序列係屬新穎〇 經濟部中央標準局員工消費合作社印製 (請先閱讀背面之注意事項再填寫本頁) 以此序列爲基礎將肽合成，然後將此肽用以將兔子免 疫。所得對抗致腫瘤毒素敢之抗體與西方印跡分析上之致 腫瘤毒素蛋白質交又反應。此抗體甚至亦能以1至10, 〇〇〇 稀釋率之抗血清將致腫瘤毒素之生物活性中和（圖3 ) 〇 雖然遠低於全長蛋白質，合成之致腫瘤毒素肷具有一些對 抗U-9 3 7細胞之活性（圖4 ) 〇 致腫瘤毒素對各種蛋白_亦即胰蛋白靡、胰凝乳蛋白 酶、及鏈黴蛋白酸·（1ϋ%，w/w)之處理達24小時即廢除蛋 白質之活性（圖5 ) 〇此等結果指示全長蛋白質內存有生 物活性。除蛋白廳:外，致腫瘤毒素之活性亦經發現對酸性 條件敏感〇在致腫瘤毒素暴露於pH 2時，有一顯著量（雖 本紙張尺度適用申國國家標準（CNS ) A4規格（210X2^7邊巷） 557299 Α7 Β7 五、發明説明（u ) 非全部）之活性被破壞（圖6 A)。致腫瘤毒素活性亦經發 現對用三氟乙酸（ϋ，1%)或用乙腈（5〇%)處理係屬敏感（ 圖6 Β) 〇然而，該活性對用二硫蘇糖醇處理（：達2小 時）及暴露於Ιϋϋ t1溫度達30分鐘（資料未示出）具有抗 性〇 實例1 5 、 致腫瘤毒素之生物定座 經濟部中央標準局員工消費合作社印製 (請先閲讀背面之注意事項再填寫本頁) ϋ- 9 3 7細胞之72小時致腫瘤毒素處理抑制此等細胞之 生長，如胸甘滲入所確定（圖7 ) 〇在1 5 “ g / m 1之致腫瘤 毒素濃度上觀察到5 〇 %抑制作用〇致腫瘤毒素對若干其他 類型細胞系之效應亦予審查。致腫瘤毒素對其他骨髓細胞 (圖8 )、淋巴（B及T二者）細胞（圖9A)、成紅細胞 性細胞（圖9β)、乳房腫瘤細胞（圖1〇)、卵巢腫瘤細胞 (圖11)及肝癌細胞（圖12)亦具抑制性。胚腎細胞對致 腫瘤毒素較具抗性（圖1 3) 〇人類成神經膠質細胞瘤細胞 及鼠類纖維肉瘤經發現對致腫瘤毒素具完全抗性（圖14及 圖15) 〇在分析誘生5ϋ %細胞生長抑制作用所需之致腫瘤 毒素量時，其視腫瘤細胞類型而有顯著之變化（表1 ) 〇 表 丄 致腫瘤毒素對人類腫瘤細胞系生長之抑制 細胞系ί濃度〕 50%生長抑制（U g/ml) AAMUfi—iM.胞玉丄 組織細胞性淋巴瘤（U-937) 15 急性單細胞白血病（ήρ-1) 32 本紙張尺度適用中國國家標準（CNS ) Α4規格（) 557299 A7 B7 五、發明説明（i7 ) 經濟部中央標準局員工消費合作社印製 前髓細胞性白血病（HL_60) 52 人類肝細胞癌： 肝細胞癌（Hep 3B) 15 肝細胞癌（Hep G2) 78 人類T及β淋巴瘤細胞： 急性Τ細胞白血病（J u r k a t ) 25 伯奇氏B細胞淋巴瘤Ua ji ) 83 人類乳腺癌： 乳腺癌（MCF-7 ) 35 乳腺癌（CLO) 90 人類紅白血病： 急性骨髓致生白血病（K- 5 6 2 ) 80 人類卵巢腺癌： 卵巢腺癌（〇 V C A - 4 2 9 ) 40 卵巢腺癌（〇 V L； A - 4 3 2 ) 50 卵巢腺癌㈧VCA-433 ) 100 人類成神經膠質細胞瘤： 成神經膠質細胞瘤（U 4 5 1 ) >100 其他： 人類轉形胚腎（A - 2 9 3 ) >100 鼠類纖維肉瘤U - y 2 9 ) >100 正常細胞 人類二倍體包皮成纖維細胞 >100 人類周圍血液淋巴細胞 >100 本紙張尺度適用中國國家標準（CNS ) A4規格（210 X 297&巻） (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装 557299 A7 B7__ 五、發明説明（22 )
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Yamamoto, T· entomocidal toxins in thuringiensis kurstaki. Yokoyama, Y· ^
本紙張尺度適用中國國家標準（CNS ) A4規格（210X291^禁J (請先閱讀背面之注意事項再填寫本頁) -裝· 訂 557299 A7 B7 五、發明説明（27 ) (i v )通信地址：（略） (v )電腦可讀形式： (幻媒體類型：DS，HD 1·44 Mb/1.44 Mo (B) 電腦：IBM相容個人電腦 (C) 操作系統：PC-D0S/MS-D0S (D )軟體：W 〇 r d P e r f e c t 6 · 0 (v i )囚前申請資料：（略） (vi i i )代埋人資訊：（略） (i幻電信資訊：（略） (2 )序列辨識編號1之資訊： (i )序列特徵： U J長度：2 ϋ (ιυ類型：胺基酸 (C )股別： (L))外形：直線形 (i i )分r類型： U )說明：蛋白質 (i i i )假設：無 (i v)反義：無 (v i )原始來源：（略） (X i )序列說明：序列辨識編號1 :
Pro S e r T h r Val V a 1 Asa V a 1 Ser A s n Leu L y s Pro 1 5 10 G 1 y Asp T ii r 1 1 e C! 1 u L y s G i u P h e 15 20 本紙張尺度適用中國國家標準（CNS ) A4規格（210'〆2^^ ) (請先閲讀背面之注意事 裝-- :寫本頁) 經濟部中央標準局員工消費合作社印製
Claims (1)
- 557299 5. 如申請專利範圍第3項之藥學組合物，其中所述組 合物阻滯贅生情況之復現〇 6. 如申請專利範圍第3項之藥學組合物，其中所述處 理延長所述贅生細胞之宿主之生存時間〇 7. 如申請專利範圍第3項之藥學組合物，其中所述贅 生細胞係在活體外〇
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US45463695A | 1995-05-31 | 1995-05-31 | |
| PCT/US1996/008189 WO1996038477A1 (en) | 1995-05-31 | 1996-05-31 | Novel antiproliferative protein from bacillus thuringiensis var. thuringiensis |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| TW557299B true TW557299B (en) | 2003-10-11 |
Family
ID=23805447
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| TW085114980A TW557299B (en) | 1995-05-31 | 1996-12-04 | Novel antiproliferative protein from bacillus thuringiensis var. thuringiensis |
Country Status (12)
| Country | Link |
|---|---|
| US (1) | US5824636A (zh) |
| EP (1) | EP0835260B1 (zh) |
| JP (1) | JP3786962B2 (zh) |
| CN (1) | CN1144816C (zh) |
| AU (1) | AU6028196A (zh) |
| CA (1) | CA2234794C (zh) |
| DE (1) | DE69624570T2 (zh) |
| ES (1) | ES2181889T3 (zh) |
| RU (1) | RU2178798C2 (zh) |
| TW (1) | TW557299B (zh) |
| WO (1) | WO1996038477A1 (zh) |
| ZA (1) | ZA964359B (zh) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| MX2011000894A (es) * | 2010-12-17 | 2012-06-18 | Ct De Investigacion Cientifica Y De Educacion Superior De Ensenada B C | Proteinas cry insecticidas de bacilus thuringiensis con actividad anticancerígena. |
-
1996
- 1996-05-29 ZA ZA9604359A patent/ZA964359B/xx unknown
- 1996-05-31 WO PCT/US1996/008189 patent/WO1996038477A1/en active IP Right Grant
- 1996-05-31 JP JP53671196A patent/JP3786962B2/ja not_active Expired - Fee Related
- 1996-05-31 CN CNB961960361A patent/CN1144816C/zh not_active Expired - Fee Related
- 1996-05-31 DE DE69624570T patent/DE69624570T2/de not_active Expired - Fee Related
- 1996-05-31 CA CA002234794A patent/CA2234794C/en not_active Expired - Fee Related
- 1996-05-31 AU AU60281/96A patent/AU6028196A/en not_active Abandoned
- 1996-05-31 EP EP96917890A patent/EP0835260B1/en not_active Expired - Lifetime
- 1996-05-31 RU RU97121930/13A patent/RU2178798C2/ru not_active IP Right Cessation
- 1996-05-31 ES ES96917890T patent/ES2181889T3/es not_active Expired - Lifetime
- 1996-11-04 US US08/743,553 patent/US5824636A/en not_active Expired - Fee Related
- 1996-12-04 TW TW085114980A patent/TW557299B/zh active
Also Published As
| Publication number | Publication date |
|---|---|
| JP3786962B2 (ja) | 2006-06-21 |
| ES2181889T3 (es) | 2003-03-01 |
| WO1996038477A1 (en) | 1996-12-05 |
| EP0835260B1 (en) | 2002-10-30 |
| RU2178798C2 (ru) | 2002-01-27 |
| CN1192220A (zh) | 1998-09-02 |
| EP0835260A1 (en) | 1998-04-15 |
| DE69624570D1 (en) | 2002-12-05 |
| EP0835260A4 (en) | 1999-06-02 |
| US5824636A (en) | 1998-10-20 |
| JPH11507025A (ja) | 1999-06-22 |
| MX9709247A (es) | 1998-10-31 |
| AU6028196A (en) | 1996-12-18 |
| ZA964359B (en) | 1998-07-29 |
| CA2234794C (en) | 2008-05-13 |
| DE69624570T2 (de) | 2003-03-20 |
| CA2234794A1 (en) | 1996-12-05 |
| CN1144816C (zh) | 2004-04-07 |
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