585921 Α7 Β7 五、發明説明（1 ) 發明背景 發明領域 (請先閲讀背面之注意事項再填寫本頁) 本發明係關於一種免疫學和基因工程技術。定言之， 本發明係關於製造新穎反應試劑的免疫工程，該試劑鎖定 毒物在進口火蟻后之中腸裡微絨毛上的細胞表面分子。 相關技術的描述 進口火蟻爲一種德洲和美國南部其它洲的生態及財政 災難。進口火蟻在1 9 3 0年代意外地進口美國。這些害 蟲完全地擾亂及破壞生態系統並在農業（大量損壞機械） ，蓄牧業（損失新生家畜）和娛樂及觀光業（損失娛樂鳥 類和休閒公園和熱鬧場所最多感到不舒服）上產生不利的 經濟影響。 經濟部智慧財產局員工消費合作社印製 火蟻控制的特定問題在於提供應該控制或抑制進口昆 蟲物種而不破壞本土昆蟲物種。該問題係關於多種非本土 的動物物種，其已引進在美國各洲。進口物種通常具有比 本土物種更具競爭性，因爲，在許多情況裡，其已發展出 更強的再生能力並且在其新的環境中沒有天敵（1 )。因 此，明顯地，消除外來物種是很重要的。另一方面，本土 物種應該不被消除，因爲特別生態系統的適當平衡包括本 土物種的存在。 目前的方法包括各種火蟻控制的方法。化學毒物，例 如A M D R〇爲此項技藝所知並經常地使用。然而，此等 毒物可能污染環境，而且沒有區別地將本土物種與外來物 本紙張尺度通用中國國家標準（CNS ) Α4規格（210Χ297公釐） 585921 A7 B7 五、發明説明（2 ) 種一起消除。 (請先閱讀背面之注意事項再填寫本頁) 因此，習知技術的缺點爲進口火蟻根除產品，其對環 境沒有造成破壞，特別鎖定及只消除進口火蟻。本發明實 現此項技藝中這個長久以來的需要及需求。 發明摘要 本發明係關於一種安全、符合成本效益、對環境溫和 及生態健全，用來控制進口火蟻的生物工程產物，及其製 法。免疫及基因工程技術係用於產生單株抗體（mA b § )及展現抗體片段的病毒（噬菌體），所謂單鏈V -基因 片段（scFv)或V —基因重及輕鏈片段（Fab)， 其對進口火蟻后中腸微絨毛有高度特定結合力。特定的單 株抗體及展現抗體s c Fv及F a b的噬菌體係與毒素（ 膠洛尼（gelornn )或其它毒素）或前凋亡誘使劑結合，細 胞周期阻斷劑或細胞增殖抑制劑之c D N A編碼配位至鎖 定運送及解構進口火蟻，但不針對本土物種的F a b片’段 ，藉此恢復自然生態系統。 經濟部智慧財產局員工消費合作社印製 本發明之目的在於提供一種管理及控制害蟲的特殊方 法，該方法對環境溫和而且不傷害本土動物物種。 在本發明裡，其係提供一種特定輸送前凋亡，細胞周 期肄制劑至目標細胞的組成物。 本發明之另一目的係提供一種輸送毒素及細胞生長肄 制劑給目標細胞的姐成物。 在本發明又一目的裡，其係提供一種製造傳送毒物給 本纸浪尺度適用中國國家標準（CNS M4規格（210X 297公釐） ' ~ 585921 A7 B7 五、發明説明（3 ) (請先閱讀背面之注意事項再填寫本頁) 目標細胞但不是給非目標細胞之試劑的方法，其包括：以 該目標細胞使動物免疫，產生單株抗體；收成，使脾臟富 含養份；使用聚乙二醇將該脾臟細胞與骨髓髓癌熔化助質 雜交；藉由在HAT培養基上生長選擇融合瘤細胞，及使 用E L I S A技術篩選製造老鼠抗體所用及使用免疫組織 學技術篩選針對中腸微絨毛抗原之特定抗體的融合瘤上淸 液；藉由限制稀釋，篩選融合瘤上淸液，膨漲無性生殖系 及冷凍陽性無性生殖系來進行無性繁殖；及獲得安定、單 株抗體生產融合瘤，產生上淸液或腹水流質或製備純化的 單株抗體。 本發明的其它及進一步的覲點，特徵及優點將從下列 較佳具體實施例的說明名顯得知。這些具體實施例僅供說 明之用。 圖式的簡單說明 經濟部智慧財產局員工消費合作社印製 所附之圖式係包括於其中，所以上述之本發明的特徵 ，優點及目的將淸楚可知並可詳細地了解。這些圖式係形 成本專利說明書的一部分。然而，應注意的是，所附之圖 式係舉例說明本發明的較佳具體實施例而且不應該被認爲 用來限制本發明的範疇。 圖1係槪略地顯示本發明的方法，其包括首先進行免 疫；cDNA製備；產生噬菌體群，噬菌體選擇；確定 s c F v和 F a b ;及其測試。 本紙浪尺度適用中國國家標準（CNS ) A4規格（210X297公釐） -6 - 585921 A7 _B7___ 五、發明説明（4 ) 本發明的詳細說明 (請先閲讀背面之注意事項再填寫本頁) 以下具體實施例對於熟悉該項技藝人士而言，可能根 .據上述說明而對該具體實施例作部分變更或修改，卻不脫 離出本發明之精神範疇，因此，本發.明之專利範圍僅由附 錄之申請專利範圍加以說明之。 在此所用的v'單細包胞繁殖抗體〃或” m A b 〃係指 包括針對目標細胞之重及輕聚縮氨酸鏈的抗體而且產生及 選自無性生殖的抗體產生細胞。 在此所用的，抗體片段〃或％ F a b "係指以最小尺 寸之辨識單元爲基質的免疫球蛋白。來自免疫重及輕鏈的 V -基因片段對目標抗原展現高親和力。 在此所用的'' s c F v 〃片段係指以最小尺寸之辨識 單元爲基質的免疫球蛋白，一種對目標細胞有高親和力的 單鏈V —基因片段。 在此所用的毒素”係指在倂入目標細胞時殺死細胞，發 揮毒性作用的任何一種化學物。定言之， ''膠洛尼〃係指 眾所皆知的核糖酸小體鈍活蛋白質或其重組形式。 經濟部智慧財產局員工消費合作社印製 在此所用的'^噬菌體展現族群〃係指高達2 X 1 0 8 個免疫球蛋白F a b或s c F v片段之獨立無性生殖系的 族群。 在此所用的 ''前凋亡〃，”細胞周期阻斷劑〃，及' 細胞增殖抑制劑〃和 '細胞增殖劑〃係指來自控制細胞增 殖及細胞死亡，配位至單株抗體之基因的c D N A。 F a b片段或s c F v片段係供特定輸送至目標細胞之用585921 Α7 Β7 V. Description of the invention (1) Background of the invention (Please read the notes on the back before filling out this page) The present invention relates to an immunology and genetic engineering technology. In a word, the present invention relates to an immunoengineering for producing a novel reaction reagent, which locks the cell surface molecules of the poison on the microvilli in the midgut of the imported fire ant. Description of related technologies Imported fire ants are an ecological and financial disaster in Texas and other continents in the southern United States. Imported fire ants were accidentally imported into the United States in the 1930s. These pests completely disrupt and destroy ecosystems and create disadvantages in agriculture (lots of machinery damage), livestock (lost new livestock) and recreation and tourism (lost recreational birds and leisure parks and lively places at most) Economic impact. Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs. The specific problem of fire ant control is to provide that imported insect species should be controlled or suppressed without destroying native insect species. This question concerns a variety of non-native animal species, which have been introduced on all continents in the United States. Imported species are often more competitive than native species because, in many cases, they have developed greater regenerative capabilities and are free of natural enemies in their new environment (1). It is therefore clear that the elimination of alien species is important. On the other hand, indigenous species should not be eliminated, as the proper balance of particular ecosystems includes the presence of indigenous species. Current methods include various fire ant control methods. Chemical poisons such as AM DRO are known in the art and are often used. However, these toxicants may pollute the environment, and the native paper and foreign objects are not distinguished. The paper size is common Chinese National Standard (CNS) A4 specification (210 × 297 mm) 585921 A7 B7. 5. Description of invention (2). (Please read the precautions on the back before filling out this page) Therefore, the disadvantage of the conventional technology is that imported fire ant eradication products do not cause damage to the environment, and specifically lock and eliminate imported fire ants. The present invention fulfills this long-standing need and demand in the art. Summary of the Invention The present invention relates to a bioengineering product that is safe, cost-effective, mild to the environment, and ecologically sound, and is used to control imported fire ants, and a method for making the same. Immune and genetic engineering technology is used to produce monoclonal antibodies (mA b §) and viruses (phages) that display antibody fragments, so-called single-chain V-gene fragments (scFv) or V-gene heavy and light chain fragments (Fab), It has a high specific binding force to the midgut microvilli of imported fire ants. Specific monoclonal antibodies and phage systems displaying antibodies sc Fv and Fab combined with toxins (gelornn or other toxins) or pro-apoptotic inducers, cell cycle blockers or cell proliferation inhibitors c The DNA code is coordinated to lock the transport and deconstruction of imported fire ants, but does not target the fab segment of native species, thereby restoring the natural ecosystem. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economics The purpose of the present invention is to provide a special method for managing and controlling pests which is mild to the environment and does not harm native animal species. In the present invention, it is a composition that provides a specific pre-apoptotic, cell cycle 肄 preparation to target cells. Another object of the present invention is to provide a sister product that delivers a toxin and a cell growth agent to a target cell. In another object of the present invention, it is to provide a method for manufacturing and transmitting poisons to the paper. Applicable to Chinese national standards (CNS M4 specification (210X 297 mm) '~ 585921 A7 B7 V. Description of the invention (3) (Please read first Note on the back, please fill in this page again.) A method for target cells but not for non-target cells, including: immunizing animals with the target cells to produce monoclonal antibodies; harvesting, enriching the spleen with nutrients; using polyethylene Glycol hybridizes the spleen cells with the bone marrow medullary cancer fusion; selects fusion tumor cells by growing on HAT medium, and uses ELISA to screen mouse antibodies and uses immunohistology to screen for midgut microvilli antigens Antibodies for fusion tumors of specific antibodies; screening for fusion tumors by limiting dilution, expansion of asexual germline and cryo-positive asexual germline for asexual reproduction; and obtaining stable, single antibody production of fusion tumors To produce sera or ascites fluid or to prepare purified monoclonal antibodies. Other and further aspects, features and advantages of the present invention will be compared from the following The description names of the specific embodiments appear to be known. These specific embodiments are for illustrative purposes only. A brief description of the drawings The attached drawings printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs are included therein, so the above-mentioned The features, advantages, and objectives will be clearly known and understood in detail. These drawings form part of this patent specification. However, it should be noted that the accompanying drawings illustrate preferred embodiments of the invention and It should not be considered to limit the scope of the present invention. Figure 1 shows the method of the present invention, which includes immunization first; cDNA preparation; phage population generation, phage selection; determination of sc F v and F ab; and The test of this paper applies the Chinese National Standard (CNS) A4 specification (210X297 mm) -6-585921 A7 _B7___ 5. Description of the invention (4) Detailed description of the invention (please read the precautions on the back before filling this page) ) For those skilled in the art, the following specific embodiments may be partially changed or modified according to the above description, but not Departing from the spirit of the present invention, the patent scope of the present invention is only explained by the appended patent application scope. As used herein, the v'single cell cytoplasmic antibody or "m A b" refers to Antibodies to heavy and light poly-peptide chains of the target cells and produce and are selected from asexual reproduction of antibody-producing cells. As used herein, antibody fragment 〃 or% F ab " refers to the smallest size recognition unit as the substrate The V-gene fragment from the immunoheavy and light chains exhibits high affinity for the target antigen. The "sc F v 〃 fragment" as used herein refers to an immunoglobulin with the smallest size recognition unit as a matrix, a A single-stranded V-gene fragment with high affinity for target cells. As used herein, "toxin" refers to any chemical that kills cells when they are inserted into target cells and exerts a toxic effect. In the final analysis, "gelonitil" refers to the well-known inactivation of ribosomal bodies. Protein or its recombinant form. The "phage display ethnic group" printed here by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs refers to independent asexual reproduction of up to 2 X 108 immunoglobulin F ab or sc F v fragments. The "pro-apoptotic 〃", cell cycle blocker 〃, and 'cell proliferation inhibitors' and 'cell proliferation agents' used here refer to the control of cell proliferation and cell death, coordinated to a single C DNA of the antibody gene. F a b fragment or s c F v fragment is for specific delivery to target cells
本紙張尺度適用中.國國家標準（CNS ) A4規格（2Η)Χ 297公H 585921 A7 ___B7_ 五、發明説明（5 ) 〇 (請先閲讀背面之注意事項再填寫本頁) 在此所用的a噬菌體展現的F a b及噬菌體展現的 s c F v 〃係指展現在噬菌體及經由鍵結至目標細胞之抗 原選擇的Fab or scfv片段群。 根據本發明，其可以使用此項技藝中傳統的分子生物 學，微生物學，及重組D N A技術。此等技術係完全地解 釋於文獻中。請參考例如Maniatis， Fritsch & Sambrook, "Molecular Cloning: A Laboratory Manual (1982);丨丨 DNA Cloning: A Practical Approach,"第 I 和 11 冊（D.N. Glover 編輯 1985);’· Oligonucleotide Synthesis1· (M.J. Gait 編輯 1 984);"Nucleic Acid Hybridization1· (B.D. Haines & S.J· Higgins 編輯（1985));"Transcription& Translation" (B.D. Hames & S. J. Higgins 編輯（1984));n Animal Cell Culture" (R.I. Freshney，編輯（1986));11 Immobilized Cells 和 Enzymes1· (IRL Press, ( 1 986));B. Perbal, ，·Α Practical Guide To Molecular Cloning丨丨 1984) o 經濟部智慧財產局員工消費合作社印裝 因此，如果上述的名詞出現的話，則其將具有上述的 定義。 >載體〃係爲複製體，例如質體或噬菌體，其可連接 另一 D NA片段以複製所連接的片段。載體被認爲是藥理 上可接受的，如果其服用方式可爲服用者所接受。此等試 劑被認爲是以 ''治療有效的數量〃服用，如果服用量在藥 理上來說是有效的。如果試劑的存在造成服用者生理上的 改變，則稱該試劑爲在藥理上來說是有效的。例如在治療 一本紙張尺^用中國國家標準（cNS)A4規格（2ΐ〇χ297公釐） 77^ 585921 A7 B7 五、發明説明（6 ) 逆轉濾過性病毒感染時，減少感染機率或由於感染而致之 生理受損的化合物將被視爲有治療效果。 (請先閱讀背面之注意事項再填寫本頁) 細胞時已由外生或或異性D N A在此等細胞已置入細 胞內部時〜轉移〃。轉移D N A可以或不可以倂入（共價 連結）細胞基因。在原核生物，酵母和晡乳動物細胞，例 如轉移D N A，可以保持在游離基因元素，例如質體。有 關真核細胞，穩定轉移的細胞係爲其中轉移D N A已變成 倂入染色體，以致於經由染色體複製而遺傳至子細胞的細 胞。該穩定性係由真核細胞建立細胞株或包括一群含轉移 D N A之子細胞的無性生殖系的能力展現。”無性生殖系”係 爲一群衍生自單細胞或一般原體經由細胞有絲分裂的細胞 。 >細胞株〃係爲能穩定地活體外生長許多代之一級細胞 的無性生殖系。 轉錄及轉譯控制序列係爲D N A調節序列，例如啓動 子，促進子，聚腺苷訊號，中止子和相似物，其用於宿主 細胞中編碼序列的表現。 經濟部智慧財產局員工消費合作社印製 w D N A分子〃係指去氧核糖核苷酸（腺嘌呤，鳥糞 鹼，胸腺嘧啶或胞嘧啶）依單股形式或雙股螺旋的聚合形 式。該名詞係僅指分子的一級及二級結構，而且不限於任 何特別的三級形式。因此，該名詞包括所發現的雙股 D N A，尤其是在線性D N A分子（例如限制片段），病 毒，質體，及染色體裡。一般傳統的結構係只在5 /到 3 /方向沿著D N A之非轉錄股得到序冽（即該股具有與 m R N A同型的序列）。 本紙張尺度適用中.國國家標準（CNS ) A4規格（210X297公釐） 585921 A7 _ __ B7 五、發明説明（7 ) (請先閱讀背面之注意事項再填寫本頁) 本發明係關於一種火蟻根除產品，其對環境建全，及 特定鎖定目標及只消除進口火蟻。另外經過深思熟慮的是 ，本發明的方法可以用來鎖定任一種動物害蟲。 製造及篩選對特定抗原表位具高度特定性的單細包繁 殖抗體係爲一種已知且標準的文獻技術。熟習此項技藝者 所知的D N A技術係得以將小免疫球蛋白辨識單元（所謂 抗體片段，即展現與完整較大型母抗體相同抗原特定性之 重及輕免疫球蛋白鏈的N -終端可利用區域）的D N A編 碼置入病毒展現載體（噬菌體），在其表面上產生及展現 s c F v及抗體片段。然而，至今，該技術一直未應用到 解構特定目標害蟲身上。 經濟部智慧財產局8工消費合作社印製 噬菌體展現方法比傳統單株抗體多一項主要的優點， 其中可以產生F a b基因的大型及分歧群並顯示在丙毒的 表面上；藉此得以快速篩選及選擇具高度（緊緊結合）目 標特定性的F a b片段。重要的是，一但特定噬菌體展現 的F a b片段以選定針對抗原表位的特定性，則針對特定 F a b片段編碼的D N A可供基因工程進行之細胞死亡（ 凋亡）基因以及將細胞增殖分裂成F a b D N A的系胞 所利用。因此，產生輸送誘使凋亡作用或成塊細胞增殖之 基因的鎖定系統。殺死細胞或避免細胞增殖的作用基因係 造成目標組織解構。 本發明係利用免疫學和基因工程技術來產生展現抗體 Fab片段的單株抗體和病毒（噬菌體），該抗體Fab 片段係選來與進口火蟻后的中腸微絨毛細胞反應，但不與 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） -10- 585921 A7 ____B7 五、發明説明（8 ) (請先閲讀背面之注意事項再填寫本頁) 本土火蟻后的中腸細胞反應。這些展現F a b片段的單株 抗體及噬菌體係與毒素，例如膠洛尼，核糖體不活化蛋白 質（其沒有進入細胞之機製及無毒性，除非特定輸送到細 胞內部）結合，以將毒速輸送到進口火蟻后的消化部位。 爲了進一步瞭解本發明之特徵及技術內容，請詳細參 閱以下有關本發明之詳細說明、實施例暨圖式，然而下列 實施例僅供參考與說明用，而非用來對本發明做任何限制 〇 實施例1 以中腸微絨毛細胞使老鼠免疫 將B a 1 b / c老鼠以從進口火蟻后所孵的蛋而得的 中腸作免疫。使用抗原複合切碎的中腸來免疫，然後進行 雙選擇程序（請參考實施例4 )及免疫組織化學程序（請 參考實施例5 )，以選頓隊進口火蟻后之微絨毛細胞具特 性性的F a b片段。 經濟部智慧財產局員工消費合作社印製 使用取自免疫老鼠的脾臟細胞，利用標準細胞培養程 序來產生特定物種的單株抗體。單株抗體對進口火蟻之中 腸微溶毛抗原具有特定性。使用實施例5所述的免疫組織 化學程序來選則所得的融合瘤上淸液的特定性。 除此之外，產生了展現s c Fv及F a b片段，與中 腸微絨毛細胞的特定物種抗體噬菌體。發展展現噬菌體之 F a b片段的步驟式程序係顯示於圖1。 本纸張尺度適用中.國國家標準（CNS ) A4規格（210 X 297公釐） 585921 A7 __B7________ 五、發明説明（9 ) 實施例2 從免疫老鼠脾臟所得之全部R N A 製備，放大及純化 (請先閲讀背面之注意事項再填寫本頁)The standard of this paper is applicable. National National Standard (CNS) A4 specification (2Η) X 297 male H 585921 A7 ___B7_ 5. Description of the invention (5) 〇 (Please read the precautions on the back before filling this page) A used here The phage-displayed F ab and phage-displayed sc F v 群 refer to a group of Fab or scfv fragments displayed on the phage and selected by an antigen bound to a target cell. According to the present invention, it is possible to use conventional molecular biology, microbiology, and recombinant DNA technology in this technology. These techniques are fully explained in the literature. Please refer to, for example, Maniatis, Fritsch & Sambrook, " Molecular Cloning: A Laboratory Manual (1982); 丨 丨 DNA Cloning: A Practical Approach, " Volumes I and 11 (DN Glover edit 1985); '· Oligonucleotide Synthesis1 · (MJ Gait editor 1 984); " Nucleic Acid Hybridization1 · (BD Haines & SJ · Higgins editor (1985)); " Transcription & Translation " (BD Hames & SJ Higgins editor (1984)); n Animal Cell Culture " (RI Freshney, editor (1986)); 11 Immobilized Cells and Enzymes1 (IRL Press, (1 986)); B. Perbal,, · Α Practical Guide To Molecular Cloning 丨 丨 1984) o Intellectual Property Bureau, Ministry of Economic Affairs Employee Consumer Cooperative Copies So if the above term appears, it will have the above definition. > A vector is a replica, such as a plastid or a phage, which can be linked to another DNA fragment to replicate the linked fragment. The carrier is considered to be pharmacologically acceptable if it is administered in a manner acceptable to the user. These agents are considered to be taken in a `` therapeutically effective amount '' if the amount taken is pharmacologically effective. If the presence of the agent causes a physiological change in the user, the agent is said to be pharmacologically effective. For example, when treating a paper ruler using the Chinese National Standard (cNS) A4 specification (2 × χ297 mm) 77 ^ 585921 A7 B7 V. Description of the invention (6) Reverse filtering virus infection, reduce the chance of infection or because of infection Physiologically impaired compounds will be considered therapeutic. (Please read the precautions on the back before filling this page) When the cells have been exogenous or heterogeneous D N A When these cells have been placed inside the cell ~ Transfer 〃. Transfer D N A may or may not be incorporated into (covalently linked) cellular genes. In prokaryotes, yeast and lactating mammalian cells, such as transferred D N A, can remain in free genetic elements, such as plastids. In the case of eukaryotic cells, a stably transferred cell line is a cell in which the transferred DNA has been transformed into a chromosome so that it is inherited to daughter cells via chromosomal replication. This stability is demonstrated by the ability of eukaryotic cells to establish cell lines or clonal germ lines including a population of daughter cells containing metastatic DNA. A "asexual germ line" is a group of cells derived from a single cell or general protozoa via cell mitosis. > The cell line 〃 line is an asexual germ line capable of stably growing many first-generation cells in vitro. Transcription and translation control sequences are DNA regulatory sequences such as promoters, promoters, polyadenylation signals, terminator and analogs, which are used for the expression of coding sequences in host cells. Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs, w D N A molecular 〃 refers to the aggregation of deoxyribonucleotides (adenine, guanine, thymine or cytosine) in a single strand or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule and is not limited to any particular tertiary form. Therefore, the term includes the double-stranded DNA found, especially in linear DNA molecules (such as restriction fragments), viruses, plastids, and chromosomes. Generally, the traditional structure system only obtains the sequence sequence along the non-transcribed strand of D N A in the 5 / to 3 / direction (that is, the strand has a sequence of the same type as m R N A). The paper size is applicable. National National Standard (CNS) A4 specification (210X297 mm) 585921 A7 _ __ B7 V. Description of the invention (7) (Please read the precautions on the back before filling this page) This invention is about a fire Ant eradication products, which are environmentally friendly, have specific targets and eliminate only imported fire ants. It is also well thought out that the method of the present invention can be used to target any kind of animal pest. It is a known and standard literature technique to make and screen for a single-cell proliferation resistant system that is highly specific for a particular epitope. DNA technology known to those skilled in the art is able to use small immunoglobulin recognition units (so-called antibody fragments, that is, N-terminals of heavy and light immunoglobulin chains that exhibit the same antigen specificity as intact larger parent antibodies) Region) DNA code is placed in a virus display vector (phage) to generate and display scFv and antibody fragments on its surface. However, to date, the technique has not been applied to deconstruct specific target pests. The phage display method printed by the 8th Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs has one more major advantage over traditional single-body antibodies. Among them, large and divergent groups of Fab genes can be generated and displayed on the surface of propylenid. Screen and select Fab fragments with high (tightly binding) target specificity. Importantly, once the Fab fragment displayed by a specific phage is selected to be specific for the epitope, the DNA encoded by the specific Fab fragment can be used for genetically engineered cell death (apoptotic) genes and cell proliferation and division. Used by Fab DNA-derived pedigrees. Thus, a locking system is created that delivers genes that induce apoptosis or the proliferation of clumps of cells. Gene lines that kill cells or avoid cell proliferation cause destructuring of the target tissue. The present invention utilizes immunology and genetic engineering technology to generate a single antibody and a virus (phage) exhibiting an antibody Fab fragment. The antibody Fab fragment is selected to react with midgut microvilli cells after imported fire ants, but not with the present Paper size applies Chinese National Standard (CNS) A4 specification (210X297 mm) -10- 585921 A7 ____B7 V. Description of the invention (8) (Please read the precautions on the back before filling this page) Midgut cells after native fire ants reaction. These monoclonal antibodies and phage systems displaying Fab fragments are combined with toxins, such as colloidal, ribosome-inactivating proteins (which have no mechanism of entering the cell and are non-toxic, unless specifically delivered to the inside of the cell) to deliver the venom quickly. To the digestive part of imported fire ants. In order to further understand the features and technical contents of the present invention, please refer to the following detailed descriptions, examples, and drawings of the present invention. However, the following examples are for reference and description only, and are not intended to limit the present invention. Example 1 Immunization of mice with midgut microvilli cells B a 1 b / c mice were immunized with the midgut obtained from eggs hatched after imported fire ants. Use the antigen-composite minced midgut for immunization, and then perform a dual selection procedure (please refer to Example 4) and an immunohistochemical procedure (please refer to Example 5) to select the microvilli cells imported by the fire queens Sexual F ab fragment. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs Using spleen cells from immunized mice, standard cell culture procedures are used to generate monoclonal antibodies of specific species. Monoclonal antibodies have specificity for the intestinal sparingly soluble hair antigen of imported fire ants. The immunohistochemical procedure described in Example 5 was used to select the specificity of the effusion on the resulting fusion tumor. In addition, species-specific antibody phages exhibiting scFv and Fab fragments and midgut microvilli cells were generated. A step-by-step procedure for developing a Fab fragment displaying phage is shown in FIG. The size of this paper is applicable. National Standard (CNS) A4 (210 X 297 mm) 585921 A7 __B7________ V. Description of Invention (9) Example 2 Preparation, amplification and purification of all RNA obtained from the spleen of immunized mice (please (Read the notes on the back before filling out this page)
c D N A 將全部的R N A從三隻免疫老鼠的脾臟萃取並純化。 使用設備（Beohdnger Mannheim)及二組引物——個特定針 對I gG重鏈而另一個特定針對/c輕鏈（4，1 2) -將 RNA反轉錄（RT)成cDNA。CDNA係使用 I g G重鏈及/c輕鏈特定引物分別藉由聚鏈反應（P C R )程序及先前所述的熱循環條件（4，5，6，1 2 )放 大。經P C R放大的產物係根據大小由膠體電泳分離。 實施例3 展現在絲狀噬菌體表面上之抗體片段組合族群的構築 經濟部智慧財產局員工消費合作社印製 組合抗體片段族群係藉由如實施例4所述的二步驟序 配位程序及得自Stratagene公司的ImmunoZAP設備在絲狀 噬菌體表現載體裡構築。簡言之，經膠體純化的P C R產 物係以酵素劈開並將P C R產物配位進入重鏈載體-1 H c 2，及輕鏈載體-1 L c。解構的重及輕鏈族群包 括s c F ν片段。接著，將重及輕鏈結構合倂以產生 F a b組合結構。然後將义細胞以噬菌體族群變形並 侵染輔助弒噬菌體（VCSM13，Stratagene公司），產生足以 篩選及選擇目的之數量的展現噬菌體的抗體片段（4)。 來自免疫重及輕鏈的抗體V -區域特定片段係作爲對目標 細胞具高鍵結親和力之單鏈V -片段（s c F ν )或作爲 $紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐) ~ 585921 A 7 __B7_ 五、發明説明（10 ) 對目標細胞具高度鍵結親和力的組合重/輕鏈V -片段（ F a b ) 〇 (請先閱讀背面之注意事項再填寫本頁) 實施例4 辨識具來自進口火蟻后之中腸微絨毛抗原特定性的抗體 s c Fv或F a b片段的二步驟選擇法 第一步驟包括將整個展現抗體s c F v或F a b片段 的噬菌體族群與打開露出微絨毛細胞的中腸反應。將無法 結合的噬菌體沖掉，而將展現噬菌體的s c F v或F a b 片段-局度親和力地結合進口火犠中腸抗原-沖提並保存 。第二次選擇係使用來自本土火蟻后的中腸製劑。只收集 展現噬菌體的s c F v或F a b片段—沒有與本土火蟻后 的中腸膜抗原反應-。所選擇的弒菌（與進口火蟻后的中 腸膜抗原反應，但不與本土火蟻后的反應）並在万.裡 生長及無性生殖。 實施例5 經濟部智慧財產局員工消費合作社印製 經選擇展現噬菌體之抗體s c F v或F a b片段及單株抗 體與進口火蟻中腸微絨毛細胞反應的證實 經選擇展現噬菌體之抗體s c F v或F a b片段（來 自上述實施例4 )與各種中腸細胞膜抗原，包括中腸微絨 毛細胞的抗原反應。截切中腸組織的免疫組織化學染色係 用來辨識專門與進口火蟻后中腸微絨毛細胞反應之無性生 殖的展現噬菌體S c F v或F a b片段。中腸截切組織係 -13- 本紙張尺度適用中.國國家標準（CNS ) A4規格（210X 297公釐） 585921 A7 B7 五、發明説明（彳彳） (請先閲讀背面之注意事項再填寫本頁) 與無性生殖的s c F v或F a b片段反應，並使用敏感性 免疫過氧酶 ECTASTAIN Elite ABC 系統（Vector Laboratories )評估菌體存在與否。將與進口火蟻微絨毛 細胞行陽性反應而且不法與本土火蟻微絨毛細胞反應的的 無性生殖系在及co//裡放大並如實施例6和7所述使用。 爲了選擇單株抗體，係將中腸組織切片與融合瘤上淸液反 應，沖洗，再與對老鼠I g之鹼性磷酸酯酶共軛的兔子二 級抗體反應。然後將基質加入檢查載玻片看看中腸微絨毛 抗原是否有陽性反應。 實施例6 測試噬菌體體過境進口火蟻后活體 建立進口火蟻之群落，使得每一個群落含有至少5隻 或多於5隻之蟻后（自單一土墩聚集處建立每一個群落） 。建立該群落後，令可溶性Μ 1 3噬菌體體置入葡萄糖水 (1 0%葡萄糖w/v )中，且令該火蟻無限制地攝食該 葡萄糖-噬菌體體混合物。在攝食該噬菌體體溶液2 4， 經濟部智慧財產局員工消費合作社印製 4 8及7 2小時後，收集火蟻后。分離中腸，將其置於 P B S中加以均質化，隨後藉由離心以除去碎屑。將所生 成之混合物經不同之稀釋後置入L B瓊脂中，該L B瓊脂 含有40/zl X — ga 1 (SOmg/j於二甲基甲醯 胺中）溶液及 4//1 IPTG(20〇mg/m£)，其 中該I P T G係倒入至L B瓊脂盤中。覆蓋該盤且令該瓊 脂硬化。令該盤倒置並於3 7 t下培育。於塗覆後1 2和 本紙張尺度適用中.國國家標準（CNS ) A4規格（21〇X 297公釐） -14- 585921 A7 ____B7 _ 五、發明説明（12 ) (請先聞讀背面之注意事項再填寫本頁) 2 4小時出現菌落。淡藍色斑於4小時之短時間內開始形 成’並於8至1 2小時內完全形成。於4 °C下培育數小時 以增強其顏色。此結果顯示，藉由攝食，可將展現F a b 片段之噬菌體體成功地導入至進口火蟻后活體之中腸中。 經選擇展現噬菌體之抗體片段，及單株抗體活體內有效的 證實 將選擇對進口火蟻微絨毛具有特定性及高親和力結合 力的單細胞抗體及噬菌體進行測試在口服時由微絨毛細胞 內化的能力。在控制的環境中建立螞蟻群體（進口及本土 )，每一個群落包括一隻孵蛋的進口火蟻后，50隻未交 配的火蟻后及大約5 0 0 0工蟻。將群落餵混合在大豆餐 食中的單株抗體或顯示噬菌體之s c F v或F a b片段。 將每一群落的孵蛋蟻后和未交配蟻后殺死並取出中腸。將 中腸做成冷凍的組織切片，並藉由上述免疫組織化學染色 將這些切片分析噬菌體是否存在及內化。 經濟部智慧財產局員工消費合作社印製 實施例7 經選擇展現噬菌體之抗體片段及單株抗體活體對於目標解 構進口火蟻而言是有效輸送.系統的證實 藉由使用以半胱胺酸殘基修飾之膠洛尼類似物，利用 先前所述的程序（1 1 )將核糖體不活化蛋白質，膠洛尼 (1 0 - 1 2 )係連接至單株抗體和展現抗體噬菌體之 F a b片段。 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） -15- 585921 A7 B7 五、發明説明（13) 以下爲本案之參考資料： (請先閱讀背面之注意事項再填寫本頁) 1. Holldobler，B. & E. 0. Wilson. 1990· Tans· The Belknap Press, Cambridge, Mass. 2. Barbas, C. & R. Lerner. 1991. Combinatorial immunoglobulin libraries on the surface of phage (phabs): rapid selection of antigen-specific. Fab· Methods: Comp. Met. Enzym.2:119. 3. Ames, R., M. Tornetta, C. Jones & P. Tsui. 1994. Isolation of neutralizing anti-C5a antibodies from a filamentous phagemono valent Fab display library. J. I mm un. 152:4572. 4.Ames, et al (15 個作者）.1 995. Neutralizing murinemonoclonal antibodies to human IL-5 isolated from hy bridomas & afilamentous phage Fab display library. J· Immunol. 1 54:6355. 5. Winter, G., A. D. Griffiths, R. E. Hawkins & H. R. Hoogenboom.l994.Making antibodies by phage display 經濟部智慧財產局員工消费合作社印製 technology. Ann. Rev. Immunol. 1 2:433. 6. Vaughan，T. J., et al., 1996. Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phagedisplay library. Nature Biotech. 14: 309. 7. Kruif, J. de, et al., 1 996. New perspectives on recombinantuman antibodies. Immunology Today 17:453. 8. Kruif, J · de, & T. Logtenberg. 1996. Leucine zipper 本紙張尺度適用中國國家標準（cns ) A4規格（210x297公釐） -16- 585921 經濟部智慧財產局員工消費合作社印製 Λ7 B7五、發明説明（14) dimerized bivalent & bispecific scFv antibodies from a semi-synthetic antibody phage display library. /. Bio. Chom. 271:7630. 9. Davies, J. & L. Riechmann. 1 995. Antibody VH domains as small recognition units. Biotechnology 1 3:475. 10. French, R, C. Penney, A. Browning, F. Stirpe, A. George & M. Glennie. 1 995. Delivery of the ribosomeinactivating protein, gelonin, to lymphoma cells via CD22 & CD38 using bispecific antibodies. Brit. J· Cancer 71:986. 11. Better, M., S. Bernhard, D. Fishwild, P. Nolan, R. Bauer, A. Kung, & S · Carroll. 1994. Gelonin analogs with engineered cysteineresidues form antibody immunoconjugates with unique properties. J.BioL Chem. 269:9644. 12. Analytical Biochemistry 1 62:156- 1 59, 1987. 上述之具體實施例係用來詳細說明本發明之目的、特 徵及功效，對於熟悉該項技藝人士而言，可能根據上述說 明而對該具體實施例作部分變更或修改，卻不脫離出本發 明之精神範疇，因此，本發明之專利範閫僅由附錄之申請 專利範圍加以說明之。 (請先閱讀背面之注意事項再填寫本頁) -裝· 、11 ▼線 本紙張尺度適用中.國國家標隼（CNS ) A4規格（210X297公釐） -17-c D N A The entire R N A was extracted from the spleens of three immunized mice and purified. Using equipment (Beohdnger Mannheim) and two sets of primers-one specific for the IgG heavy chain and another specific for the / c light chain (4, 12)-reverse transcription (RT) the RNA into cDNA. CDNA was amplified using Ig G heavy chain and / c light chain specific primers, respectively, by the polymer chain reaction (PCR) procedure and the thermal cycling conditions (4, 5, 6, 12) described previously. The P C R amplified product was separated by colloidal electrophoresis according to size. Example 3 Construction of a combination group of antibody fragments displayed on the surface of filamentous bacteriophages Printed by the Consumer Property Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs The combination antibody fragment family was produced by a two-step sequential coordination procedure as described in Example 4 and obtained from Stratagene's ImmunoZAP device was built in a filamentous phage expression vector. Briefly, the colloidally purified PCR product was cleaved with an enzyme and the PCR product was coordinated into the heavy chain carrier-1Hc2 and the light chain carrier-1Lc. The deconstructed heavy and light chain groups include the s c F ν fragment. Next, the heavy and light chain structures are combined to produce a F a b combined structure. The sense cells are then deformed by the phage population and infect the helper phage (VCSM13, Stratagene) to produce a sufficient number of phage-expressing antibody fragments (4) for screening and selection purposes. V-region specific fragments of antibodies from immunoheavy and light chains are single-chain V-fragments (sc F ν) with high binding affinity for target cells or as Chinese paper standards. Applicable to China National Standard (CNS) A4 (210X297) (Mm) ~ 585921 A 7 __B7_ V. Description of the invention (10) Combined heavy / light chain V-fragment (F ab) with high binding affinity for target cells 〇 (Please read the precautions on the back before filling this page) Example 4 Two-step selection method for identifying sc Fv or F ab fragments of antibodies specific to midgut microvilli antigens from imported fire ants The first step involves the entire phage population displaying antibody sc F v or F ab fragments and Open the midgut response exposing microvilli. The unbound phages are washed away, and the scFv or Fab fragments that display the phages—locally bound to the imported fire salamander midgut antigen—are washed and stored. The second selection used a midgut formulation from a native fire queen. Only scFv or Fab fragments displaying phages were collected—without reacting to the midgut membrane antigens of native fire queens. The selected maggots (reactive with the midgut membrane antigens of imported fire queens but not the native fire queens) were grown in Wanli and asexually reproduced. Example 5 Printing of the sc F v or F ab fragment of an antibody selected to exhibit phage and the detection of the response of a single antibody to imported fire ant midgut microvilli cells by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs The v or Fab fragment (from Example 4 above) reacts with various midgut cell membrane antigens, including those from the midgut microvilli. Immunohistochemical staining of cut midgut tissue is used to identify asexually displayed phage S c F v or Fa b fragments that specifically react with midgut microvilli cells after imported fire ants. Department of Midgut Cut Tissue-13- This paper is applicable in China. National Standard (CNS) A4 (210X 297 mm) 585921 A7 B7 V. Description of the Invention (彳 彳) (Please read the notes on the back before filling This page) reacts with asexual sc F v or F ab fragments and assesses the presence or absence of bacteria using the sensitive immunoperoxidase ECTASTAIN Elite ABC system (Vector Laboratories). The clonal germline that reacted positively with imported fire ant microvilli cells and failed to react with native fire ant microvilli cells was amplified in and co // and used as described in Examples 6 and 7. In order to select individual antibodies, the midgut tissue sections were reacted with the effusion from the fusion tumor, washed, and then reacted with rabbit secondary antibody conjugated to alkaline phosphatase of rat Ig. The matrix is then added to the test slide to see if the midgut microvilli antigen is positive. Example 6 Testing of bacteriophage crossing imported fire ants in vivo. Establishing imported fire ant colonies so that each community contains at least 5 or more queens (each community is established from a single mound gathering place). After the community was established, the soluble M13 phage was placed in glucose water (10% glucose w / v), and the fire ant was allowed to ingest the glucose-phage mixture without restriction. After ingesting the phage solution 24, it was printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs for 48 and 72 hours, and the fire queen was collected. The midgut was separated, homogenized in P B S and then centrifuged to remove debris. The resulting mixture was diluted into different LB agars. The LB agar contained 40 / zl X-ga 1 (SOmg / j in dimethylformamide) solution and 4 // 1 IPTG (20. mg / m £), where the IPTG was poured into LB agar plates. Cover the plate and harden the agar. The dish was inverted and incubated at 37 t. After coating 1 2 and this paper size is applicable. National National Standard (CNS) A4 specification (21〇X 297 mm) -14- 585921 A7 ____B7 _ V. Description of the invention (12) (Please read the first Note: Please fill out this page again) 2 4 hours, colonies appear. The pale blue spots started to form 'within a short time of 4 hours and completely formed within 8 to 12 hours. Incubate at 4 ° C for several hours to enhance its color. This result shows that, by feeding, a phage displaying a Fab fragment can be successfully introduced into the living midgut of an imported fire ant. Single-cell antibodies and phages that have specificity and high affinity for imported fire ant microvilli will be selected for selection of antibody fragments that display phages and effective verification in vivo of individual antibodies. Internalization of microvilli cells by oral administration Ability. Establish ant colonies (imported and native) in a controlled environment. Each community includes an imported fire queen that hatches eggs, 50 unmated fire queens, and approximately 5000 worker ants. The colonies are fed with monoclonal antibodies mixed in soy meals or scFv or Fab fragments showing phage. The hatched and unmated queens of each community were killed and the midgut was removed. The midgut was made into frozen tissue sections, and these sections were analyzed for the presence and internalization of phage by the above-mentioned immunohistochemical staining. Printed in Example 7 by the Consumers ’Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. The antibody fragments and individual antibody living organisms selected to exhibit phage were effectively transported for the target deconstructed imported fire ants. Modified gelloney analogs, using the previously described procedure (1 1) to ribosome-inactivated proteins, gelloney (1 0-1 2) were linked to the monoclonal antibody and the F ab fragment displaying antibody phage. This paper size applies Chinese National Standard (CNS) A4 specification (210X297 mm) -15-585921 A7 B7 V. Description of invention (13) The following is the reference for this case: (Please read the precautions on the back before filling this page) 1. Holldobler, B. & E. 0. Wilson. 1990 · Tans · The Belknap Press, Cambridge, Mass. 2. Barbas, C. & R. Lerner. 1991. Combinatorial immunoglobulin libraries on the surface of phage (phabs ): rapid selection of antigen-specific. Fab · Methods: Comp. Met. Enzym. 2: 119. 3. Ames, R., M. Tornetta, C. Jones & P. Tsui. 1994. Isolation of neutralizing anti- C5a antibodies from a filamentous phagemono valent Fab display library. J. I mm un. 152: 4572. 4. Ames, et al (15 authors). 1 995. Neutralizing murinemonoclonal antibodies to human IL-5 isolated from hy bridomas & afilamentous phage Fab display library. J. Immunol. 1 54: 6355. 5. Winter, G., AD Griffiths, RE Hawkins & HR Hoogenboom.l994.Making antibodies by phage display Printed technology. Ann. Rev. Immunol. 1 2: 433. 6. Vaughan, TJ, et al., 1996. Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phagedisplay library. Nature Biotech. 14: 309 7. Kruif, J. de, et al., 1 996. New perspectives on recombinantuman antibodies. Immunology Today 17: 453. 8. Kruif, J. de, & T. Logtenberg. 1996. Leucine zipper Applicable to paper sizes China National Standard (cns) A4 size (210x297 mm) -16- 585921 Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs Λ7 B7 V. Description of the invention (14) dimerized bivalent & bispecific scFv antibodies from a semi-synthetic antibody phage display library. /. Bio. Chom. 271: 7630. 9. Davies, J. & L. Riechmann. 1 995. Antibody VH domains as small recognition units. Biotechnology 1 3: 475. 10. French, R, C. Penney, A. Browning, F. Stirpe, A. George & M. Glennie. 1 995. Delivery of the ribosomeinactivating protein, gelonin, to lymphoma cells via CD22 & CD38 using bispecific antibodies. B rit. J. Cancer 71: 986. 11. Better, M., S. Bernhard, D. Fishwild, P. Nolan, R. Bauer, A. Kung, & S · Carroll. 1994. Gelonin analogs with engineered cysteineresidues form Antibody immunoconjugates with unique properties. J. BioL Chem. 269: 9644. 12. Analytical Biochemistry 1 62: 156- 1 59, 1987. The specific examples described above are used to illustrate the purpose, characteristics and efficacy of the present invention in detail. For those skilled in the art, some changes or modifications may be made to the specific embodiment according to the above description without departing from the spirit of the present invention. Therefore, the patent scope of the present invention is only explained by the scope of patent application in the appendix. Of it. (Please read the precautions on the back before filling out this page) -Packing ·, 11 ▼ Lines The paper size is applicable. National Standards (CNS) A4 (210X297 mm) -17-