TW209245B - - Google Patents
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- TW209245B TW209245B TW81102989A TW81102989A TW209245B TW 209245 B TW209245 B TW 209245B TW 81102989 A TW81102989 A TW 81102989A TW 81102989 A TW81102989 A TW 81102989A TW 209245 B TW209245 B TW 209245B
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2 Ο Λ 6 η 6 經濟部中央樣準局IS;工消费合作社印製 五、發明説明() 本發明闋係於一棰組合物,包含著用重組DNA技術製 成的多呔序列及其載剤,作為對抗慢性病毒肝炎的治療劑 。本發明也關係於编碼該多呔序列的DNA順序及表現該順 序的轉移感染細胞。 至少有五棰不同的病毒,亦即,A,B,C,D和E 等五型肝炎病毒,會引起急性肝炎的臨床症狀。經由腸傳 染的A型和E型肝炎係經常可治癒者,而B型和C型肝炎 (M前稱為非經腸肝炎Non-A,和Non-B),及D型肝炎,則 可能發展成為慢性發炎狀態,並可能轉而導致肝硬化及初 级肝細胞癌。 有鼸C型和D型肝炎,其診斷和治療方法及其俚別的 病毒等的可得數據相當少。D型肝炎病毒是已知為不完全 型RNA病毒。所Μ,需要幫助者病毒才能在患者體内發育 ,且只在被HBV感染過的人體内發現過。直到最近· C型 肝炎病毒才被偵檢出來,並開發出可以診斷慢性C型肝炎 感染的抗體檢驗(抗-HCV)。不過,對於逭種疾病的治療致 使痊癒,仍有逐增地緊急需要存在著。 對於慢性Β型肝炎,有關免疫學方法進行確認,其 肇病病毒及病毒生命循環和DNA順序等皆有較佳硏究過的 這棰疾病,也有相同的情況存在。若患者醱内有病毒DNA 存在超過十週,HBe-抗原(HBeAg)存在超過12遇,或若Β 型肝炎表面抗原(HBsAg)存在超過六値月等,則該患者即 為B型肝炎病毒的長期載原者。 粗略估計,有三億人係被認爲患有慢性B型肝炎者, 其中大部份是住在遠東地區者。 對於這些人,主要的被感染危機是出現在生產期間或 剛生產後,原因在於患慢性感染的母親將病毒傳染給她的 (請先間讀背而之注意事項再場寫本頁) 本紙5IL尺度遑用中Β Β家標準(CNS) Τ4規格(210X297公*) 3 81. 4. 10,〇〇〇張(Η) ς〇9:^〇 Λ 6 Π 6 經濟部中央櫺準局β工消仲合作杜印製 五、發明説明() 新生兒。經由逭棰方式被感染的孩童中有百分之九十會在 其Μ後的生活也變成慢性感染。於西方世界中,感染最常 發生於生命後期,於孩童時代或甚至成人期都可能,主要 是經由腸或性而傳遞。於這些生後感染Β型肝炎的病例中 ,只有百分之五到十的被感染者會變成長期載原者。不遇 ,被移轉的病毒並不會促成被感染者不論是消除掉病毒或 终生保留在體内等任一種明顯的反應。因此之故,決定未 來生理狀況者似乎是免疫學狀態的鼷係。 ΗΒ-病毒粒子(Dane粒子)係由不同的結構蛋白霣所構 成,包括核心蛋白質和表面蛋白質。後者是由具有三餡S-型域的编碼順序所構成的開放密碼之轉譯產物,該三鴒S-型域都是Μ可Μ在醱内起始轉譯的ATG參聯駸(ATG triplet) 作為開頭的。這三個域分別稱為pre-Sl,pre-S2和S ,係 依分子的到端順序依次命名的。從逭悃ORF可衍生 出六種蛋白質:只從S域轉譯出的糖苷化與非糖苷化形式 之主要蛋白質(gp27和p24〉(226艏胺基酸 > ;具有一條或兩 條多醣侧鐽(分別爲gp33和gp36)的中間蛋白質(281個胺基 酸〉,係由preS2-域和S-域所编碼者;Μ及,糖苷化形式 (gp42)和非糖苷化形式(Ρ39>的大蛋白質(389-400艏胺基 酸,決定於病毒的血型>,係由preSl,preS2和S等三域轉 譯形成者。其核心蛋白質爲HBcAg和HBeAg ,後者據信為 HBcAg的處理產物。 作為傅染性病毒原粒的Dane粒子包含著核心蛋白質和 表面蛋白質,而絲體則為六棰表面抗原的混合物所構成, (請先閲讀背而之注意事項#填寫本頁) 本紙張尺度遑用中國圉家楳準(CNS) «Ρ4規格(210X297公;») 4 81. 4. 10,000張(||) Λ 6 Β6 20924ο 五、發明説明() S呔本身組合而形成所謂的20η·粒子,條完全非傳染性者 (請先闓讀背而之注意I項洱项寫本頁) Ο 被ΗΒ病毒感染的患者在被認為俤長期HBV-感染者之前 ,會經遇幾龆肝炎階段。緊接在感染之後,會有一艏傳染 性階段,其特徹爲血澝中含有HBeAg。難有抑制住的HBV複 製,仍發生連鑛的HBs抗原血症,顯示病毒DNA序列已整 合到患者的細胞基因體(genome)之內。整合後的病毒序列 並不能促使寄主細胞合成完整的病毒。不過,具有整合HB V-序列的肝細胞則只能產生HBsAg ,其轉而可在患者血清 中偵檢出而作為慢性B型肝炎的指示物。最可能的情形為 ,轉形後的肝細胞不會被胞毒性T-細胞所胞溶,反而會滋 長而誘導出長期存在的肝炎(CPH)或長期活性肝炎(CAH), 之後可能進展到肝硬化或初期肝細胞癌,促成患者早死。 經濟部中央標準局只工消费合作社印製 最近已確定慢性HBV感染患者會顯示出内源干擾素生 產缺陷之症狀(Abb et al., 1985:J. Med. Virol 17卜176)。這是用干擾素a(IFNa )治療血淸中含有HBeAg 和HBV-DNA的慢性B型肝炎患者之理論基礎。用大數目的 患者進行控制試驗的結果顯示服用干播素a者,與對照組 比較起來,B型肝炎病毒的消除程度有明顯地增加。不過 ,在出生時或生產之後的期間被感染者對這棰治療未顯示 血清轉化效果。這棰現象不幸地使得約70%載原者不能從 IFNa治療獲得效益。 到目前為止,干擾素a對慢性B型肝炎的正確作用方 本紙》•尺度遑用中Ββ家榣準(CNS)T4規格(210x297公釐) -5 - 81. 4. 10,_張⑻ C09:4〇 A 6 Π6 經济部屮央標準局A工消费合作社印製 五、發明説明() 式仍未清楚。其抗病毒活性可能爲保護被感染細胞免於傳 染或者減少病毒在HBV-感染細胞内的轉錄,轉譯和複製。 干播素可經由將T-細胞,巨噬菌體和NK-細胞等活化及誘 導MHC I類蛋白質的表現等而另具免疫調制效應。 另一種治療慢性B型肝炎的方法所依據的概念為抑制 病毒的複製,因而將其防禦力減損到足Μ使宿主免疫条統 能消滅該病毒。這種方法導致嘗試用抗病毒藥物,如,腺 _昤阿糖苷及腺曜昤阿糖苷-磷酸酯等來治療慢性HBV感染 患者。不過,不超過一半的患者會對逭棰治療有反應,不 論是持續地或短暫的血清轉化都有(HBeAg+到抗-HBeM。 這些抗病毒藥物的另一項負面事項為其所具之免疫壓抑性 質。 曾被試驗用Μ治療慢性載原者的其他藥物包括干擾素 /8 |無環鳥糞瞟昤核苷,間白素2,類固醇,如脫氫皮甾 醇,及上述之組合物等。但是其中沒有一種可提供出比干 擾素α治療更為好的結果。只有一棰組合式治療法,包括 初始服用類固酵,接著服用IFNa ,可Μ增加所遘患者的 回應速率。 從既有技術可知,慢性HBV感染遇的小猩猩既不能用 HBsAg (結合到破傷風類毒素)也不能用抗-HBs抗體予Μ治 癒。此外,也有嘗試經由服用S肽類Μ使慢性HBV感染患 者免疫者。這種處理甚至於不能促使這些患者形成抗-HBs 抗體。 此外,依據定義,B型肝炎病毒長期載原者的特擞在 (請先閲讀背而之注意事項再塥寫本頁) 裝_ 線· 本紙張尺度遑用中國β家榣準(CNS) T4規格(210X297公*) 6 81. 4. 10,0U0張(Η) 209.4ο Λ 6 ΙΪ6 經濟部中央標準局貞工消费合作社印製 五、發明説明() 於其血清中可探測到HBsAg 。因此,絕對意料之外者,本 發明包含箸T-細胞對病毒S呔的活化抗原決定基之組合物 竟能誘導B型肝炎病毒長期載原者的免疫作用且最終逢到 痊癒。 考慮上面所討論過的技術狀態之下,本發明的目的即 為提出有效的治療劑,係可導致完全回應而用Μ治療病骞 慢性肝炎疾病者(亦即,可導致持續地抑制HBV-複製,減 少HBV DNA和DWA聚合酶,及使患者血清所含HBeAg和HBsAg 減少且最後消失等)。 依據本發明,達到這項目的的方式為將U)至少一個 可促成一種或多棰抗原決定基的抗原性之多肽序列,與<b> 可將該抗原決定基序列(a>呈出的載剤組合而成,其中, 該多呔序列(a)可經由吸附,任何棰化學鍵結或二级價鍵 等方式結合到載劑(b>上。 本發明另外關係於将這棰組合用來生產供治療慢性病 赛肝炎的翳藥之用途。 本發明邏闢係於處理慢性病毒肝炎的方法,係將上述 含有下列成份的组合物使患者服用而成:U〉至少一傾可 促成一棰或多種抗原決定基的抗原性之多呔序列,及<b) 可將該抗原決定基序列(a>呈出的載劑,其中,該多呔序 列(a>可經由吸附,任何棰化學鍵結或二级價鍵等方式結 合到載劑(b)上。 重要的是,該可為一棰或多棰相異多呔的多呔序列<a> 係Μ直接方式或間接方式促成T細胞活化抗原決定基之抗 原性。依據本發明,多肽序列(a>可爲任何羼型的Β型肝 (請先閲讀背而之注意事項再場寫本頁) 裝· 訂- 本紙張尺度遑用中β國家樣準(CNS)甲4規格(210x297公*) 81. 4. ΐο,υοο張(Η) 20934ο Λ 6 Π 6 五、發明説明() 炎病毒,待別是adw,ayw,adr和ady等的一播多呔或其兩 棰或多種多呔的組合。從B型肝炎病毒衍生出的埴些呔可 為HBV呔preS卜preS2或S,或為HBV核心原。 可用爲多呔序列(a)者除了前述任何種多肽外 > 另有 經由胺基酸刪除而改質過的兩種或多種多呔之組合,但其 中至少要保留一餹含有至少六鶴連繙胺基酸餘_抗原決 定基,或經由在N-端,或C-端加上其餘的胺基或在多 呔序列U)内插置其他的基酸等方式改質過的兩棰或多 棰多呔之組合。這些慵$論如何皆為必要者為必須維持 住生物活性。 較適當者,多呔序列u)係經肉豆爸酿化者。 爲了顯示出恰當的蕖學活性,本發明組合物中的多呔 序列U)必須在載劑(b〉上呈現。此檯載爾是由特殊物質所 構成者,例如,可潙疏水性聚合物粒子,無機粒子,或爲 多醣粒子等。較適當者,載»(b>為於分泌時卽形成粒子 的第二種多呔序列,該粒子較適當者具有至少l〇nB的直徑 (請先閲讀背而之注意事項再塡寫本頁) 經濟部中央標準局貝工消费合作社印製 較適當者,形成粒子的多呔序列(t〇為可萑自:HBV #·-. 3肽,抑¥核心抗原,》^抜>心抗原,|^^表面抗原,111乂表 面抗原和HIV核心抗原Μ及脊鰱灰質類病毒表面抗原等的 多呔所具完整胺基酸序列之實質部份。較適當的粒子形成 性載劑(b>為HBV S呔和/或核心呔。 用為載劑序列(b)時,上述多呔可經由隨意地蜊除胺 基酸,將一備或多個胺基酸取代掉或在N-端,或C-端添加 本紙尺度遑用中《明家楳準(CNS) ^規格(210X297公使) 8 81. 4. 10,000張(Ji) ς〇9 ,4ό 經濟部屮央櫺準局貝工消t合作杜印Μ 五、發明説明() 一僱或多傾胺基酸,或在多肽序列(b)中揷置一健或多鵪 胺基酸等方式予Μ改霣,但必須維持住其粒子形成能力。 較適當者,多呔序列(b)係經肉豆蔻釀化者。 若載薄<b>爲一種多肽序列時,則序列(a)舆(bh兩者 可經由γ種'或多播下述交互作用予以聯結起來,疏水性固 著(由肉豆薄Ρ所促成),二破橋鍵形成等;或者逭兩種序 列可經由呔鍵達接而形成融合肽。於後述方式中,可在多 呔序列U)舆多肽序列(b)之間插置間隔質序列,其係經由 呔鍵聯結到該兩多呔上者。 本發明另提出一種编碼一棰組合物的重組DNA分子, 該組合物係可用以生產治療慢性病毒肝病之轚藥者。該重 组DNA分子包含著至少一種第一DNA序列,適意地包含第二 ,第三和/或第四DNA序列,其中 <1 >該至少一種第一 DNA序列»碼著至少一棰上文定義 過之多呔序列(a), (u>該第二DNA序列編碼著依上文粒子形成性呔定義所 定之多肽序列(b〉, (m)該第三DNA序列编瑪著間隔質序列,及 (iv>該第四DNA序列鏞碼著灌擇檷誌,且其中逭些DNA序 列係被表現時必具的DNA元素所控制者,且遽當地 具有一段共同密碼。 基於許多種胺基酸可由一棰Μ上的參聯臞所編定,因 此,本發明包含着幾種DNA序列,»可鼷瑪上文定義的呔 序列(a)和(b>。除此之外,本發明另Μ含著重組DNA分子 (請先W讀背而之注意事項孙填寫本頁) 裝· 訂· 本紙尺度遑用中a國家標準(CNS)T4規格(210x297公龙) 9 81. 4. 10,000¾ (II) Λ 6 Η 6 經濟部屮央橾準局员工消费合作社印製 五、發明説明() ,其異於上文定義過的重组DNA分子之處在於有多逋30% 的核苷酸可經置捵過。 本專利申請的另一項目的為提出被编碼上述組合物的 重組DNA分子所轉移运染遇之宿主細胞;其中該組合物係 可用Μ治療慢性HBV-感染患者的。逭棰宿主細胞可為晡乳 動物,除母或細饉等之細胞。基於本發明之目的,這棰細 胞較適當者爲不會產生除了上述組合物所包含的多呔之外 的其他任何人類血淸蛋白質或任何»長類血淸蛋白質。 本發明所用“ HBV S呔”一詞指的是被整鹤HBV基因 艚區域所编碼的肽。本發明所用“HBV ?1^-52呔"一詞指 的是被HBV基因體的整掴pre-S2和S兩域所缠碼的呔。本發 明所用“HBV pre-Sl呔” 一詞指的是被HBV基因釀的整偭 pre-Sl ,pre-S2和S等域所编碼的多肽。本發明所用“抗 原決定基”一詞指的是被所纊定基因龌域所编碼,含有至 少六徧達繙胺基酸之序列(例如,“ HBV pre-S2抗原決定 基”指的是被HBV基因讎的Pre-S2域所编碼,含有至少六 餾胺基酸之序列 >。本發明所用“T-細胞抗原決定基”一 詞指的是可與T-細胞表面上的受醱交互作用Μ促進或者促 成免疫反應之抗原決定基。 本發明所用“抗原性”意爲剌瀲免疫反應的能力(亦 即,具抗原之作用),促成抗賺產生的能力(亦邸,具抗原 之作用> 和/或與細胞表面受讎交互作用Μ促進免疫反應 或抗黼產生之能力等。 “HBV” 一詞意為文獻中述及的任何屬型的該棰病毒 (請先閲讀背而之注意事項#填寫本頁) 裝- 訂- 線. 冬紙張尺度遑用中8 Β家«毕(CNS)甲4規格(210X297公*) 81. 4. 10,000張(Π) 五、發明説明 lie ,it 另!Iadw, ayw » adrfO ayr等(Ρ· Valenzuela, Nature Vol. 280, p.815 (1979), Gerlich EP-A-85 111 361, Neurath, EP-A-85 102 250>。構成多呔序列(a>,可引起 一種或多棰epitopes的抗原性,之呔序列例子列於、M序列 表”中(SEQ ID No. 17-20, 22>。 本發明的較佳實施例包括下列组合物: 一 HB S-抗原粒子輿pre-Sl呔,pre-S2呔和/或核心呔 等的特殊抗原決定基(決定因子>; -HB核心抗原粒子與pre-S1呔,pre-S2呔,S-呔,和/ 或核心抗原等的持殊抗原決定基(決定因子>; 一 A型肝炎抗原粒子與B型肝炎S呔,pre-Sl呔,pre-S2呔,和/或核心呔等的特殊抗原決定基(決定因子〉 (請先閲讀背而之注意事項再塡寫本頁) 經濟部屮央標準局员工消费合作社印製 較適合用於本發明的重組DNA分子之特撖為含有序列 可编碼多呔序列(a),其係可引起一棰或多種T-細胞抗原 決定基的抗原性者,及编碼多呔序列<b),其係在分泌時 可形成直徑1〇η·或更大的粒子者,而谙兩者都受適當的啟 動因子所控制。對U)編碼的序列例子可爲“序列表”中 ID编號1到24的任一序列。對多呔序列(b)编碼的DNA序列 例子可用“序列表”中ID编號25到27的任一序列表之。 上述24個序列(ID编到24)中任何一艏皆可與序列 表中ID纗號25到27所揭示的任何序列相組合,包括a-b和 b-a兩種次序。 本發明的特別較佳資施例係由抗原決定基序列ID No. 本紙5k尺度边用中S困家標準(CNS)甲4規格(210X297公;¢) 11 81. 4.〗0,000» (11) A 6 Η 6 五、發明説明() 28 (對醮於HBV-S1序列中的胺基酸9到28〉與作為粒子形成 者的序列I D编號26和/或27所組合而成者。 本發明重組DNA構成物所用的肝炎病毒序列可利用包 括:限制片段的離析與建接,用合成器(Cyclon , Bio-Search)進行寡聚核苷酸之化學合成,及用PCR方法(T. J. White, N. Arnleim, Η. E. Erlich, 1989: The Polymerase Chain Reaction, Technical Focus 5<6)> 等的任何手段 予M形成或難析出來。 較適當的重組DNA分子係經由將合成的寡聚核苷酸逋 接到取自HBV基因釀S域的5 ' Xbal I -Bgl Κ 3 '片段(ID纗 號27 > ,該片段是從包括整値pre-S卜pre-s2-S-域的 BglF-BglE HBV片段衍生而得者;或連接到整艟S-域等 方式而形成者。用Μ製造這種構成物的寡聚核苷酸摘列於 下面的表I中。 (請先閲讀背而之注意事項再填寫本頁) 經濟部屮央櫺準局员工消費合作杜印製 本紙張尺度遑用中國困家«準(CNS) Τ4規格(210x297公龙) 12 8i. 4. 1(),UU0張(Η) η 62 Ο Λ 6 η 6 Central Prototype Bureau of the Ministry of Economic Affairs IS; Printed by the Industrial and Consumer Cooperatives 5. Description of the invention () The present invention is in a composition consisting of multiple sequences made by recombinant DNA technology and its contained It is used as a therapeutic agent against chronic viral hepatitis. The present invention also relates to the DNA sequence encoding the multiple sequence and the transfer of infected cells expressing the sequence. There are at least five different viruses, namely A, B, C, D, and E, which cause clinical symptoms of acute hepatitis. Hepatitis A and E transmitted through the intestine are often curable, while Hepatitis B and C (formerly known as non-enteric hepatitis Non-A, and Non-B), and hepatitis D may develop It becomes chronically inflamed and may lead to cirrhosis and primary hepatocellular carcinoma. There are eel hepatitis C and D, and there are relatively few data available on the diagnosis and treatment methods and other viruses. Hepatitis D virus is known as an incomplete RNA virus. Therefore, the virus that needs helpers can develop in patients, and it has only been found in people infected with HBV. It was only recently that the hepatitis C virus was detected and an antibody test (anti-HCV) was developed that can diagnose chronic hepatitis C infection. However, there is still an increasing need for urgent treatment of various diseases. Regarding chronic hepatitis B, the relevant immunological methods have been confirmed, and the onset virus and the virus life cycle and DNA sequence have been better investigated. This disease also has the same situation. If the patient has viral DNA present for more than ten weeks, HBe-antigen (HBeAg) exists for more than 12 times, or if hepatitis B surface antigen (HBsAg) exists for more than six months, etc., the patient is hepatitis B virus Those who have been in the original for a long time. It is roughly estimated that 300 million people are considered to have chronic hepatitis B, and most of them are people living in the Far East. For these people, the main infection crisis occurs during or immediately after the birth, because the mother with the chronic infection transmitted the virus to her (please read the back and the precautions before writing this page) This paper 5IL Standards are used in the China National Standards (CNS) Τ4 specifications (210X297 public *) 3 81. 4. 10, 〇〇〇 Zhang (Η) ς〇9: ^ 〇Λ 6 Π 6 Ministry of Economic Affairs Central Bureau of Standardization Beta Engineering Du Zhong's cooperation with Du Du. V. Description of invention () Newborn. Ninety percent of children who are infected by way of infection will become chronically infected after their lives. In the Western world, infections most often occur late in life, and may be in childhood or even adulthood, mainly through the gut or sex. Of these cases of hepatitis B infection after birth, only 5 to 10 percent of infected people will become long-term carriers. Unexpectedly, the transferred virus does not cause any obvious reaction whether the infected person eliminates the virus or remains in the body for life. For this reason, those who decide the future physical condition seem to be the immune system. The HB virus particles (Dane particles) are composed of different structural proteins, including core proteins and surface proteins. The latter is a translation product of an open cipher composed of a coding sequence with three stuffed S-type domains. The three-ridge S-type domains are all ATG triplet (ATG triplet) whose translation can be initiated within the first time. As the beginning. These three domains are called pre-S1, pre-S2 and S, which are named in order according to the end-to-end order of the molecules. Six proteins can be derived from the ORF: the main protein in glycosylated and non-glycosylated forms (gp27 and p24> (226 p-amino acids); with one or two polysaccharide side chains only translated from the S domain (Respectively gp33 and gp36) intermediate proteins (281 amino acids), which are encoded by the preS2-domain and S-domain; Μ and, glycosylated form (gp42) and non-glycosylated form (Ρ39>) The large protein (389-400 ketoamino acid, which depends on the blood type of the virus>, is formed by the translation of preSl, preS2, and S domains. Its core proteins are HBcAg and HBeAg, which is believed to be the treatment product of HBcAg. The Dane particles, which are the primary particles of the infectious virus, contain the core protein and the surface protein, while the silk body is composed of a mixture of six surface antigens. (Please read the back and the precautions # fill out this page) Using the Chinese Marshmallow (CNS) «P4 specification (210X297 ;;)) 4 81. 4. 10,000 sheets (||) Λ 6 Β6 20924ο V. Description of the invention () S combined itself to form the so-called 20η particles , Article completely non-infectious (please read back and pay attention to item I Item write this page) Ο Patients infected with HB virus will experience several stages of hepatitis before being considered as a long-term HBV-infected person. Immediately after infection, there will be an infectious stage, which is specifically bloody Contains HBeAg. It is difficult to suppress the replication of HBV, and the continuous HBs antigenemia still occurs, showing that the viral DNA sequence has been integrated into the patient's cell genome (genome). The integrated viral sequence does not promote the host cell Synthesize a complete virus. However, hepatocytes with integrated HB V-sequence can only produce HBsAg, which can be detected in the patient's serum and used as an indicator of chronic hepatitis B. The most likely case is that The shaped hepatocytes will not be lysed by cytotoxic T-cells, but will grow and induce long-term hepatitis (CPH) or long-term active hepatitis (CAH), and then may progress to cirrhosis or early hepatocellular carcinoma, Promotes premature death of patients. Printed by the Consumer Labor Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs. It has recently been determined that patients with chronic HBV infection will display symptoms of endogenous interferon production defects (Abb et al., 1985: J. Med. Virol 17 Bu 176). This is the theoretical basis for the treatment of chronic hepatitis B patients with HBeAg and HBV-DNA in blood plasma using interferon alpha (IFNa). The results of control trials with a large number of patients showed that those taking dry somatosin a Compared with the control group, the degree of elimination of hepatitis B virus has increased significantly. However, the infected people did not show seroconversion effects on this treatment at birth or during the period after birth. This phenomenon unfortunately makes about 70 % Of the original carriers cannot benefit from IFNa treatment. So far, the correct effect of interferon a on chronic hepatitis B prescription paper "• The standard is used in the home beta (CNS) T4 specification (210x297 mm) -5-81. 4. 10, _ 张 ⑻ C09 : 4〇A 6 Π6 Printed by the A-Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 5. The description of the invention () is still unclear. Its antiviral activity may be to protect infected cells from infection or reduce the transcription, translation, and replication of viruses in HBV-infected cells. Dry somatostatin can have another immunomodulatory effect by activating T-cells, macrophages and NK-cells, and inducing the expression of MHC class I proteins. Another method for treating chronic hepatitis B is based on the concept of inhibiting the replication of the virus, thus reducing its defense to a sufficient level so that the host immune system can eliminate the virus. This approach has led to attempts to treat patients with chronic HBV infection with antiviral drugs, such as adenosine glucosinolate and adenosine glucosinolate-phosphate. However, no more than half of the patients will respond to the treatment, whether it is continuous or transient seroconversion (HBeAg + to anti-HBeM. Another negative aspect of these antiviral drugs is their immunosuppression Properties. Other drugs that have been tested to treat chronic carriers with M include interferon / 8 | acyclic guano nucleoside, interleukin 2, steroids such as dehydrocortisol, and combinations of the above. But none of them can provide better results than interferon alpha treatment. Only a combination of treatments, including initial use of steroids followed by IFNa, can increase the response rate of all patients. From the existing The technology shows that the orangutans encountered with chronic HBV infection can neither be cured with HBsAg (bound to tetanus toxoid) nor with anti-HBs antibodies. In addition, there are also attempts to immunize patients with chronic HBV infection by taking S peptide M. This treatment does not even encourage these patients to form anti-HBs antibodies. In addition, according to the definition, the specialty of the hepatitis B virus long-term carrier (please read the precautions before you This page) Installation _ Line · This paper standard adopts China's β Family Standard (CNS) T4 specifications (210X297 g *) 6 81. 4. 10,0U0 sheets (Η) 209.4ο Λ 6 ΙΪ6 Central Standards Bureau of the Ministry of Economic Affairs Printed by the Industry and Consumer Cooperatives 5. Description of the invention () HBsAg can be detected in its serum. Therefore, it is absolutely unexpected that the composition of the present invention containing the activated epitope for the activation of the virus S can be induced by the T-cell Hepatitis B virus has a long-term carrier immune function and eventually recovers. Considering the technical status discussed above, the purpose of the present invention is to propose an effective therapeutic agent that can lead to a complete response and use M to treat the disease People with chronic hepatitis disease (ie, can lead to continuous inhibition of HBV replication, reduction of HBV DNA and DWA polymerase, and reduction and final disappearance of HBeAg and HBsAg in patient serum, etc.) According to the present invention, this objective is achieved The method is to combine U) at least one antigenic polypeptide sequence that can contribute to one or more epitopes, and < b > can combine the epitope sequence (a > The multiple sequence (a) By means of adsorption, any chemical bond or secondary valence bond is bound to the carrier (b >. The present invention is also related to the use of this combination in the production of a panacea for the treatment of chronic disease Hepatitis. It is a method for treating chronic viral hepatitis, which is made by taking a patient with the above composition containing the following ingredients: U> At least one tilt can contribute to the antigenicity of one or more epitopes, and < b ) The epitope sequence (a > presented carrier, wherein the multiple sequence (a > can be bound to the carrier (b) via adsorption, any chemical bond or secondary valence bond, etc. Importantly, the multi-sequence, which may be one or more different, is a direct or indirect method that contributes to the antigen resistance of T cell activation epitopes. According to the present invention, the polypeptide sequence (a > can be any type B liver (please read the precautions first and then write this page) Binding and ordering-this paper uses the National Beta Standard (CNS) A 4 specifications (210x297 g *) 81. 4. Ιο, υοο Zhang (Η) 20934ο Λ 6 Π 6 V. Description of invention () Inflammation virus, wait for it to be adw, ayw, adr and ady etc. A combination of two or more of them. Some derived from hepatitis B virus can be HBV, preS, preS2 or S, or the core of HBV. Can be used as multiple sequences (a) except any of the foregoing In addition to peptides> There are also combinations of two or more polyammoniums modified by amino acid deletion, but at least one of them must contain at least six amino acid residues_antigenic determinants, or via N-terminal, or C-terminal plus the remaining amine groups or a combination of two or more chitosans modified in such a way as to insert other base acids in the chimeric sequence U). All these quarrels are necessary to maintain biological activity. The more appropriate, the more sequence u) is the one brewed by Peas. In order to show proper pharmacological activity, the multiple sequence U) in the composition of the present invention must be presented on the carrier (b>. This carrier is composed of a special substance, for example, a hydrophobic polymer Particles, inorganic particles, or polysaccharide particles, etc. Where appropriate, the second type of multi-sequences that form particles during secretion is contained. (B > The particles which are more suitable have a diameter of at least 10 nB (please first Read the back-to-back notes and write this page) Printed more appropriately by the Beigong Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs to form a multi-sequence sequence of particles (t〇 can be taken from: HBV # ·-. 3 peptide, suppress ¥ Core antigen, "^ 抜> Heart antigen, | ^^ surface antigen, 111% surface antigen and HIV core antigen M and poliomyelitis gray surface virus surface antigen, etc. The substantial part of the complete amino acid sequence The more suitable particle-forming carrier (b > is HBV S and / or core. When used as the carrier sequence (b), the above-mentioned multi-cracker can be freely removed by removing amino acids, and one or more Amino acids are substituted or added at the N-terminal or C-terminal Standard (CNS) ^ Specifications (210X297 Minister) 8 81. 4. 10,000 copies (Ji) ς〇9, 4ό Cooperation with the Ministry of Economic Affairs, the Central Bureau of the Ministry of Economic Affairs, Beigongxiao, Du Yinmu V. Invention description () One or more Pour the amino acid, or put a healthy or polyquaternary amino acid in the polypeptide sequence (b) to change the M, but it must maintain its particle forming ability. The more appropriate, the multi-sequence (b) system It is brewed by nutmeg. If the loaded thin < b > is a polypeptide sequence, the sequence (a) and (bh) can be linked together through the following interactions of γ species or multicast, and the hydrophobic fixation (Contributed by the thin bean flesh), the formation of two broken bridges, etc .; or the two sequences can be connected via a bond to form a fusion peptide. In the manner described below, the polypeptide sequence (b) ) Intervening a spacer sequence, which is linked to the two polytopes via a bond. The invention also proposes a recombinant DNA molecule encoding a composition, which can be used to produce a treatment for chronic viral liver disease The drug. The recombinant DNA molecule contains at least one first DNA sequence, suitably contains the second, third and / Or a fourth DNA sequence, wherein < 1 > the at least one first DNA sequence »encodes at least one of the above-defined sequences (a), (u > the second DNA sequence encodes The particle formation is defined by the defined polypeptide sequence (b>, (m) the third DNA sequence encodes the spacer sequence, and (iv > the fourth DNA sequence assembles the selection sequence, and some of them The DNA sequence is controlled by the DNA elements that must be present when expressing, and it has a common code locally. Based on many kinds of amino acids can be compiled by the ginseng on a ΜΜ, therefore, the present invention includes several DNA sequence, »Keoma's above defined sequences (a) and (b >. In addition, the present invention also contains recombinant DNA molecules (please read the precautions before filling in Sun's page). Binding · Order · This paper uses the National Standard (CNS) T4 specification (210x297 male dragon) ) 9 81. 4. 10,000¾ (II) Λ 6 Η 6 Printed by the Employee Consumer Cooperative of the Ministry of Economic Affairs, Pyongyang quasi-bureau V. Invention description (), which differs from the recombinant DNA molecule defined above in how many 30% of the nucleotides can be passed through. Another item of this patent application is to propose host cells that are transfected by recombinant DNA molecules encoding the above-mentioned composition; wherein the composition can be used to treat chronic HBV-infected patients with M. The host cell may be a mammal, a cell other than a mother or a larvae. For the purpose of the present invention, these cells are more suitable as they do not produce any human hemoglobin protein or any other elder hemoglobin protein other than the polyamine contained in the above composition. The term "HBV S 呔" used in the present invention refers to the peptide encoded by the stern region of the HBV gene of the whole crane. The term "HBV 1 ^ -52 呔" used in the present invention refers to the entanglement coded by the pre-S2 and S domains of the HBV genome. The term "HBV pre-Sl 呔" used in the present invention It refers to the polypeptide encoded by the pre-S1, pre-S2, and S domains of the HBV gene. The term "antigenic determinant" used in the present invention refers to the encoded domain of the designated gene. , A sequence containing at least six peddopamine amino acids (for example, "HBV pre-S2 epitope" refers to the sequence encoded by the Pre-S2 domain of the HBV gene, containing at least six amino acids sequence> The term "T-cell epitope" used in the present invention refers to an epitope that can promote or contribute to an immune response by interaction with M on the surface of T-cells. "Antigenicity" used in the present invention means Ability to suppress immune response (i.e., having the role of an antigen), and the ability to promote anti-revenue production (Yi Di, having the role of an antigen) and / or interact with the cell surface by M Ability, etc. The term "HBV" means any type of the virus described in the literature (please read first And the precautions # fill in this page) Binding-Bookbinding-Line. Winter paper standard use 8 Β 家 «Bi (CNS) A 4 specifications (210X297 public *) 81. 4. 10,000 sheets (Π) V. Description of invention Lie, it another! Iadw, ayw »adrfO ayr et al. (Ρ · Valenzuela, Nature Vol. 280, p.815 (1979), Gerlich EP-A-85 111 361, Neurath, EP-A-85 102 250>. Composition Multiple sequences (a > can cause one or more epitopes to be antigenic. Examples of sequences are listed in the "M Sequence Listing" (SEQ ID No. 17-20, 22 >. Preferred embodiments of the present invention include The following composition: a HB S-antigen particle and pre-Sl, pre-S2, and / or core-specific special epitopes (determining factors> -HB core antigen particle and pre-S1, pre- Special antigenic determinants for S2, S-, and / or core antigens (determinant> Hepatitis A antigen particles and Hepatitis B S-, pre-Sl, pre-S2, and / Or core special determinants (determinants) (please read the precautions before writing this page) printed by the Employee Consumer Cooperative of the Bureau of Standards of the Ministry of Economic Affairs is more suitable for use in this issue The specificity of the recombinant DNA molecule contains a sequence that can encode a multiple sequence (a), which is an antigenic substance that can cause one or more T-cell epitopes, and encodes a multiple sequence <b), It is one that can form particles with a diameter of 10 n · or larger when secreted, and both are controlled by appropriate activation factors. An example of the sequence encoding U) may be any sequence with ID numbers 1 to 24 in the "Sequence List". An example of the DNA sequence encoding the multiple sequence (b) can be any of the sequence lists with ID numbers 25 to 27 in the "Sequence List". Any of the above 24 sequences (ID numbers from 24 to 24) can be combined with any sequence disclosed by ID numbers 25 to 27 in the sequence table, including a-b and b-a. The particularly preferred embodiment of the present invention is based on the epitope sequence ID No. The 5K scale of the paper is used in the S-S sleep standard (CNS) A 4 specification (210X297 public; ¢) 11 81. 4.〗 0,000 »(11 ) A 6 Η 6 5. Description of the invention () 28 (The amino acids 9 to 28 in the HBV-S1 sequence are combined with the sequence ID numbers 26 and / or 27 as the particle generator. The hepatitis virus sequence used in the recombinant DNA construct of the present invention can be used to include: isolation and connection of restriction fragments, chemical synthesis of oligonucleotides with a synthesizer (Cyclon, Bio-Search), and PCR method (TJ White , N. Arnleim, Η.E. Erlich, 1989: The Polymerase Chain Reaction, Technical Focus 5 < 6) > and other means to form or difficult to precipitate out of M. The more suitable recombinant DNA molecule is through the synthesis of oligo The polynucleotide was connected to the 5 'Xbal I-Bgl Κ 3' fragment (ID No. 27 >) taken from the S domain of the HBV gene. The fragment was included from the entire value pre-Sb pre-s2-S- BglF-BglE HBV fragment derived from the domain; or formed by connecting to the whole S-domain. The oligo nucleus of this structure is made with Μ The acid picks are listed in the following Table I. (Please read the precautions before filling in this page) Employee consumption cooperation of the Ministry of Economic Affairs of the Ministry of Economic Affairs of the Ministry of Economic Affairs. The paper is printed using the standard of China ’s poor home «quasi (CNS) Τ4 specifications (210x297 male dragon) 12 8i. 4. 1 (), UU0 Zhang (Η) η 6
五、發明説明() 表I 經濟部中央標準局员工消伢合作社印製 功 能 定 義 序列ID缠號 core(adw) aa * 59-87 6 core(adw) aa 2-28 7 core(adw) aa -10-28 8 core (adw) aa 29-58 9 core (adw) aa 1-87 10 core (adw) aa -10-87 11 core (adw) aa 70-110 12 core (adw) aa 80-125 13 core (adw) aa 88-120 15 SI(ayw) aa 9-28 17 SI (ayw) aa 83-103 18 SI(ayw) aa 20-40 19 S1(ayw) aa 59-94 20 S1 (ayw) aa 94-114 21 SI (ayw) aa 70-105 22 S2 (ayw) aa 2-21 23 S2(ayw) aa 14-33 24 胺基酸 其他的較適DNA分子係經由將用PCR方法所製備成编碼 本紙張尺度遑用中國國家標準(CNS) Τ4規格(210x297公釐) 13 81. 4. 10,000張(Η) (請先閲讀背而之注意事項再填寫本頁) Λ 6 η 6_ 五、發明説明() τ-細胞抗原決定基的核心序列連接到HBV的核心序列(SEQ ID NO 25,作為多呔序列B)而形成者。 用以製備這些構成物的寡聚核苷酸皆列於表1-1中。 表 H -1 ' 功 能 定 義 SEQ ID编號 核心 完全,bp 190卜2500 1 核心 C-端_除,bp 190卜2405 2 核心 C-端刪除且嵌入停止密碼 3 (stop codon) > bp 1901-2450 核心/前核心 10 aa前核心(precore),C-端 4 刪除 bp 1871-2405 核心/前核心 10 aa前核心,C-端刪除且嵌 5 入停止密碼,bp 1871-2405 核心 aa (-10-120) 16 核心/前核心 10 aa前核心,完全核心, 35 bp 1871-2500 經濟部中央櫺準局员工消费合作社印製 表Π -2列出幾個例子,其中编碼T-細胞抗原決定基的 DNA序列係經由將HBV基因體予以限制片段化後離析出來並 連接到對上文所定義多呔序列(b)编碼的DNA序列上。 81. 4_ 10,000張(11) (請先閲讀背而之注意事項存填寫本頁) 本紙張尺度边用中國困家標準(CNS)肀4規格(210X297公;») ς〇9 五、發明説明() 轰.1:2 功 能 定 義 SEQ ID编號 核心/前核心 完全,bp 1403-31 «Μ S2 ay/ad Μ Ν S2 (K) ay/ad S2-S, 7儀密碼被刪除掉,起 14 始密碼ATG改成ΑΤΑ ""該序列係Galibert, F· et al (1979; Nature 281, 646-650>與Ono, Υ· et al. (1983; NuCl. Acid Res· 11(6〉, 1747-1757)等所發表者。 表I-3列出特殊的重組DNA分子。其構成程序將在實 施例中予以更詳細地說明。 (請先閲讀背而之注意事項#填寫本頁) 經濟部中央櫺準局员工消赀合作社印製 本紙51尺度遑用中a困家標準(CNS) f 4規格(210X297公釐) 15 81. 4.丨0,000張(11) 五、發明説明() 表1-3 Λ 6 It 6 最終構成物 T-細胞抗原決定基 粒子形成物 選擇基因 町-核心(-10-120) + SAg + neo 核心(aa-10-120) S adw/ayw 或 S/Xbal/Bgl I neo MT-Sl(aa 9-28)-S + egpt Sl(aa a-28)ay S adw/^yw S/Xl»l /BglE V.,k egpt# MT~ 核心-neo 核心/前核心 bp 1403-31 核心adw neo MT-核心(1-87) + HBsAg - neo 核心(aa/-87) S adw/ayw 或 S/Xba I /Bgl E neo (請先閲讀背而之注意事項洱填寫木頁) 經濟部中央標準局员工消费合作社印製 #egpt =大腸菌黃花色精鳥糞賺呤磷醢核耱基移轉酶。 本發明的較適重組DNA分子中,除了编碼多呔(a)和(b) 的域外,邇包含著编碼著選擇檷誌的額外DNA序列。此外 ,也包含著供表現的所有一般必要元素,例如,啟動基因 序列,起始密碼和一摘聚腺苷酸化訊號等。 適當的啟動子例子有:使用哺乳動物細脃作為宿主細 胞時的methallothionein (MT〉,U2和H2K啟動子等。若使 用酵母或細菌等的細胞時,可以分別使用適當的酵母啟動 本紙張尺度边用中B因家標準(CNS)肀4規格(210X297公;it) 81. 4. Η),000張(Η) ^09^40 Λ 6 Π 6 五、發明説明() 基因,例如GCN4-啟動子和GAL 1/10啟動子,及適當的細 菌啟動子,如,原核細胞trp-和tac啟動子等。 為了產生本發明的多呔(a>和多呔(b)組合,要将重組 DNA分子以轉移感染(用哺乳動物細胞時 >,轉形(用醉母和 細醣等的細胞時),或其他方法而嵌入宿主細胞中,任何 生物的細胞只要能夠轉錄和轉譯該重組DNA分子,例如, 哺乳動物,細豳和酵母等的細胞都可作爲宿主細胞。 可用於本發明的適當哺乳動物細胞例子有VER0細胞( 一種猴子腎細胞糸),3T3-,C127和L等細胞(鼠科纖維母 細胞糸),和CHD (中囲田鼠卵聚> 細胞,其爲脫氫葉酸逦原 酶跚性或陰性者。 依據本發明的一特殊實施例,分別编碼著多呔序列 (a)和多呔序列(b),上文所定義之第一 DNA序列和上文定 義之第二DNA序列,有可能分別存在於不同的重組DNA分子 内,在這種情形下,要用逭兩棰重組DN A分子同時轉移感 染宿主細胞。 (請先閱讀背而之注意事項洱塡寫本頁) -裝· 訂_ 經濟部中央橾準局貝工消费合作杜印製 本紙張反度遑用+ β國家楳準(CNS) T4規格(210X297公*) 81. 4. 10,000張(II) Π Γ) 五、發明説明() 經濟部中央標準局貝工消费合作社印製 ΜΙ<οι· (1-8?) « H8sAg -neo MT-cote-neo 蔼嫌 a 3游 ΜΤΛΙ2/ϋ2 ΜΤΛΪ2Λί2 ΜΤΛΙ2Λ12 MMI2/U2 mnm2 MT/II2AJ2 MT/H2/U2 MT/1I2AJ2 MT/M2AJ2 MT/lt2AJ2 MT/1I2AJ2 '興子… • ί___ 核 i{>(M29-5n) 核心(Μ-10·βη) 核心(ΛΛ 2 - 2ΒΪ 核心(M 59 - B7) ! 1 i Μ 2 bp 1871-2405 IS? * + ws +孩 bp 1B7I -2405 核心+前核心 Ι0ΛΛ; C除 核心與前核心 ΙΟΛΛ bp 1901-2405 (=f ·热 IS? 理铕 WM i热 £ bp 1901-2405 -铒 a亡 〇ir 難1 '铒 bp 1901 -2500 核心,無荊核心 -細触原縱基 rcn I tip ί-103 *31 核心與前核心 \ m m entire S aciw/nyw 5/Xba(^〇II| entire S adft/Ttfw 5/Xb3l/B〇lll entlie S adwA-iyvr S/Xb3l/B〇lll enliie S adw/nyw 5/Xbat/Bglll eniiie S adw/nyw S/Xbal/Oglll 核心{ndw} 核心…㈣ 核心㈣ 核心(arfw) 核心(ndw) 核心㈣w) 粒子形成物 neo/egpt neo/e〇pt neo/eypt neo/egpt neo/egpl neo/egpt 1 neo/egpl neo/egpl neo/e〇p| 丨…――_ neo/egpt neo/egpl 細基因 材料與方法 ! : ....................· !#二!·^ *(請先閲讀背而之注意事項#填寫本頁) 本紙5lc尺度遑用中《«家樣準(CNS)〒4規格(2〗0父297公货) 81. 4. 10,000張(H) 209 4〇 Λ 6 Π 6 五、發明説明() ϋ : 一眯雜碱薪宝323薛 S. 3 辟理屮书 m a lens» a 3躲祕w»薪漥 . 經濟部屮央榣準局员工消伢合作杜印製 MT-S1 (AA9-28)-S *egpt MT<ore (-10-+120) ♦ SAq ♦ neo mmm MT/H2AJ2 MTyH2AJ2 -ΜΤΛΙ2Λί2 MT/H2AJ2 MT/H2AJ2 MT/H2AJ2 MTM2AJ2 MTAI2/U2 ΜΤΛΪ2ΛΙ2 ΜΤΛΙ2ΛΙ2 MTM2AJ2 MT/H2/U2 MTVH2/U2 j ΜΤΛ42ΛΙ2 ΜΤΛΪ2Λί2 ΜΤΛΙ2Λβ MTA12/U2 啟動子i- 應接者 S2-(M14-33)(ay) S2-(M2-21)(ay) Sl-(M9-28) (ay) SI-{ΑΛ 70-105) (ad) SI-(ΛΛ 94-114) (ad) S1-(AA59-9-1)(3y) SI-(ΛΛ 20- 40) (ay) S1*-{ΛΛ S3-103) (ay) S1-(M9-2B) (ay) j ^4>(M-10*SArG4 ΑΛ2-87) 核心(ΛΑ 88- 120). 核心(ΛΛ 80-125) 核心(M 70-110) 核心(ΛΛ-10-·87) T SYN | 核心(M-10-+120) -細ISftM決定基 rCR | i S2 ayw m h S ayw/adw S ayw/adw entire S adw/ayw S/Xbal/Bglll entire S adw/ayw S/Xbal/B〇ili core adw entire S adw/ayw 5/Xbal/B〇m · entire S adw/ayw S/Xbal/BQlll entire S adw/ayw S/Xbal/Bglll · entire S adw/a^rw S/Xbal/Bglti entire S adw/ayw SAbaUBglll . entire S adw/ayw S/Xbal/OgHI entire S adw/ayw S^baUBgtil · entire S adw/ayw S^bai^gtl! entire S adw/ayw S/Xbal/BgHI entire S adw/ayw S/Xbai/QQlll entire S adw/^rw. S/XbaUB〇iti . entire S adw/ayw S/Xbal/B〇HI 粒子形成物 neo/egp! neo/egpt neo/egpt neo/egpt neo/egpt neo/egpt neo/egpt neo/eopt ...... neo/?gpt neo/egpt neo/egpt neo/e〇pt neo/egpt neo/egpt neo/egpt neo/egpt neo/egpt 材料與方法 cr 谢B i ) ·········-····'···········裝· · · ·,ν.6· · · ·線··(請先閲讀背而之注意事項再塡寫本頁) 本紙張尺度遑用中a B家標準(CNS)T4規格(210X297公放) 81. 4. 10,000張(II) 經濟部中央標準局貝工消费合作社印製 Λ 6 _Π_6__ 五、發明説明() 表HI綜列出將適當DNA序列組合以得到本發明DNA構成 物之各棰可能組成。要特別提及者,該表所掲示的任何組 成份都可組合成DNA序列,於轉移感染到宿主細胞内,可 用以產生組合物(含有多呔<a)和供作@治療慢性病 毒肝炎的醫藥。對T-細胞抗原決定基序列緬碼的DNA序列 可以經由用Biosearch Cyclon合成器予Μ合成製得(SYN) ,或用PCR程序(PCR)產生,或將病毒基因體利用限制酵素 切成片段等方式製備而得。 對於慢性病毒肝炎患者的治療,可将多呔序列U>和 載剤(b〉的組合調配成任何棰醫藥组合物,其中可另含適 當的稀釋劑或藥用載體材料,如缓衝溶液等。 其給藥作業可用任何方法來完成,例如,非經腸方式 (如,靜脈内或肌肉内),或口服給蕖(如,用傷寒桿鯖細 胞將活性物質包囊起來)等。 ..、 該藥用製劑含有足夠濃度的上述組合物Μ在給蕖後誘 f: ΛV. Description of invention () Table I. Function definition serial number printed by the employee consumption cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs. Core ID (core), aa * 59-87 6 core (adw) aa 2-28 7 core (adw) aa- 10-28 8 core (adw) aa 29-58 9 core (adw) aa 1-87 10 core (adw) aa -10-87 11 core (adw) aa 70-110 12 core (adw) aa 80-125 13 core (adw) aa 88-120 15 SI (ayw) aa 9-28 17 SI (ayw) aa 83-103 18 SI (ayw) aa 20-40 19 S1 (ayw) aa 59-94 20 S1 (ayw) aa 94-114 21 SI (ayw) aa 70-105 22 S2 (ayw) aa 2-21 23 S2 (ayw) aa 14-33 24 Amino acid Other suitable DNA molecules are prepared by PCR method The codebook paper standard uses the Chinese National Standard (CNS) Τ4 specification (210x297 mm) 13 81. 4. 10,000 sheets (Η) (please read the precautions before filling in this page) Λ 6 η 6_ V. Invention Description () The core sequence of the τ-cell epitope is connected to the core sequence of HBV (SEQ ID NO 25, as multiple sequence B). The oligonucleotides used to prepare these constructs are listed in Table 1-1. Table H -1 'Function definition SEQ ID number core complete, bp 190 bu 2500 1 core C-terminal_exclude, bp 190 bu 2405 2 core C-terminal deleted and embedded stop code 3 (stop codon) > bp 1901- 2450 core / pre-core 10 aa precore (precore), C-end 4 delete bp 1871-2405 core / pre-core 10 aa precore, C-end delete and embed 5 stop password, bp 1871-2405 core aa (- 10-120) 16 core / pre-core 10 aa pre-core, complete core, 35 bp 1871-2500 The Ministry of Economic Affairs Central Bureau of Employee Employee Consumer Cooperatives printed table Π -2 lists a few examples, which encode T-cell antigens The DNA sequence of the determinant is isolated by restriction fragmentation of the HBV gene body and connected to the DNA sequence encoding the multiple sequence (b) defined above. 81. 4_ 10,000 sheets (11) (please read the precautions and fill in this page first) The paper size is based on the Chinese Standards (CNS) 4 specifications (210X297; ») ς〇9 V. Description of the invention () Boom. 1: 2 Function definition SEQ ID number Core / Front core complete, bp 1403-31 «Μ S2 ay / ad Μ Ν S2 (K) ay / ad S2-S, 7 instrument password is deleted, from 14 The initial code ATG was changed to ΑΤΑ " " the sequence was Galibert, F. et al (1979; Nature 281, 646-650 > and Ono, Υ · et al. (1983; NuCl. Acid Res. 11 (6> , 1747-1757), etc. Table I-3 lists special recombinant DNA molecules. Its composition procedures will be described in more detail in the examples. (Please read the back to the precautions # fill in this page) The Ministry of Economic Affairs, Central Bureau of Pricing and Employee's Cooperative Society printed this paper at 51 scales, and used the Standards (CNS) f 4 specifications (210X297 mm) 15 81. 4. 丨 0,000 sheets (11) V. Description of invention () Table 1-3 Λ 6 It 6 final construct T-cell epitope particle former selection gene-core (-10-120) + SAg + neo core (aa-10-120) S adw / ayw or S / Xbal / B gl I neo MT-Sl (aa 9-28) -S + egpt Sl (aa a-28) ay S adw / ^ yw S / Xl »l / BglE V., k egpt # MT ~ core-neo core / front Core bp 1403-31 core adw neo MT-core (1-87) + HBsAg-neo core (aa / -87) S adw / ayw or S / Xba I / Bgl E neo (please read the notes beforehand (Fill in the wooden page) #egpt = Escherichia coli yellow flower colored guano guanosine nucleophosphoryl nuclear transferase printed by the Employee Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs. In addition to coding for more than one in the more suitable recombinant DNA molecule of the present invention ( Outside the domains of a) and (b), it contains additional DNA sequences that encode selection records. In addition, it also contains all the generally necessary elements for performance, such as the starter gene sequence, the start code, and a polygland. Glycosylation signals, etc. Examples of suitable promoters are: methallothionein (MT> when using mammalian larvae as host cells, U2 and H2K promoters, etc. If yeast or bacteria cells are used, appropriate yeast can be used respectively Activate the B-size standard (CNS) 4 specifications (210X297 g; it) 81. 4. Η), 000 sheets (Η) ^ 09 ^ 40 Λ 6 Π 6 Specify () genes, such as the GCN4-promoter and GAL 1/10 promoter, and appropriate bacterial promoters, such as prokaryotic trp- and tac promoters. In order to produce the polyoxa (a > and polyoxo (b) combination of the present invention, recombinant DNA molecules are to be transferred for infection (when using mammalian cells>, transformed (when using cells such as drunk mother and fine sugar), Or other methods to embed in the host cell, as long as the cells of any organism can transcribe and translate the recombinant DNA molecule, for example, cells of mammals, shrubs and yeast can be used as host cells. Appropriate mammalian cells that can be used in the present invention Examples are VER0 cells (a monkey kidney cell strain), 3T3-, C127, and L cells (murine fibroblast cell strain), and CHD (Zhongluo vole oocytes> cells, which are dehydrofolate enzymatic enzymes) Sexual or negative. According to a special embodiment of the present invention, it encodes multiple sequences (a) and multiple sequences (b), the first DNA sequence defined above and the second DNA sequence defined above , It may exist in different recombinant DNA molecules, in this case, it is necessary to transfer the infected DNA with two recombinant DNA molecules at the same time. (Please read the precautions to write this page first)- Binding · Order _ Ministry of Economic Affairs The Central Bureau of Standards and Technology's Beigong Consumer Cooperation Co., Ltd. printed the paper in reverse. + Β National Standard (CNS) T4 specification (210X297) * 81. 4. 10,000 sheets (II) Π Γ) V. Description of invention () (1-8?) «H8sAg-neo MT-cote-neo A 3 games ΜΤΛΙ2 / ϋ2 ΜΤΛΪ2Λί2 ΜΤΛΙ2Λ12 MMI2 / U2 mnm2 MT / II2AJ2 MT / H2 / U2 MT / 1I2AJ2 MT / M2AJ2 MT / lt2AJ2 MT / 1I2AJ2 'Kingko… • ί ___ core i {> (M29-5n) core (Μ-10 · βη) core (ΛΛ 2-2ΒΪ core (M 59-B7) ! 1 i Μ 2 bp 1871-2405 IS? * + Ws + child bp 1B7I -2405 core + front core Ι0ΛΛ; C except core and front core ΙΟΛΛ bp 1901-2405 (= f bp 1901-2405-erbium a dead 〇ir hard 1 'erbium bp 1901 -2500 core, no wattle core-fine touch original vertical base rcn I tip ί-103 * 31 core and front core \ mm entire S aciw / nyw 5 / Xba (^ 〇II | entire S adft / Ttfw 5 / Xb3l / B〇lll entlie S adwA-iyvr S / Xb3l / B〇lll enliie S adw / nyw 5 / Xbat / Bglll eniiie S adw / nyw S / Xbal / Ogl ll core {ndw} core… ㈣ core㈣ core (arfw) core (ndw) core㈣w) particle formation neo / egpt neo / e〇pt neo / eypt neo / egpt neo / egpl neo / egpt 1 neo / egpl neo / egpl neo / e〇p | 丨… ――_ neo / egpt neo / egpl Fine genetic materials and methods!: .........................!! # 2! · ^ * (Please read the back and the precautions # fill in this page) the 5lc standard of this paper is used in "« Home Sample Standard (CNS) 〒 4 specifications (2〗 0 father 297 public goods) 81. 4. 10,000 sheets ( H) 209 4〇Λ 6 Π 6 V. Description of the invention () ϋ: a scorpion zaobao 323 Xue S. 3 Bi Lei book ma lens »a 3 hide secret w» salary 漥. Ministry of Economic Affairs MT-S1 (AA9-28) -S * egpt MT &ore; (-10- + 120) ♦ SAq ♦ neo mmm MT / H2AJ2 MTyH2AJ2 -ΜΤΛΙ2Λί2 MT / H2AJ2 MT / H2AJ2 MT / H2AJ2 MTM2AJ2 MTAI2 / U2 ΜΤΛΪ2ΛΙ2 ΜΤΛΙ2ΛΙ2 MTM2AJ2 MT / H2 / U2 MTVH2 / U2 j ΜΤΛ42ΛΙ2 ΜΤΛΪ2Λί2 ΜΤΛΙ2Λβ MTA12 / U2 promoter i-responder S2- (M14-33) (ay14) -(M9-28) (ay) SI- (ΑΛ 70-105) (ad) SI- (ΛΛ 94-114) (ad) S1- (AA59-9-1) (3y) SI- (ΛΛ 20- 40 ) (ay) S1 *-(ΛΛ S3-103) (ay) S1- (M9-2B) (ay) j ^ 4> (M-10 * SArG4 ΑΛ2-87) core (ΛΑ 88- 120). core ( ΛΛ 80-125) core (M 70-110) core (ΛΛ-10- · 87) T SYN | core (M-10- + 120)-fine ISftM determinant rCR | i S2 ayw mh S ayw / adw S ayw / adw entire S adw / ayw S / Xbal / Bglll entire S adw / ayw S / Xbal / B〇ili core adw entire S adw / ayw 5 / Xbal / B〇m · entire S adw / ayw S / Xbal / BQlll entire S adw / ayw S / Xbal / Bglll · entire S adw / a ^ rw S / Xbal / Bglti entire S adw / ayw SAbaUBglll. entire S adw / ayw S / Xbal / OgHI entire S adw / ayw S ^ baUBgtil · entire S adw / ayw S ^ bai ^ gtl! Entire S adw / ayw S / Xbal / BgHI entire S adw / ayw S / Xbai / QQlll entire S adw / ^ rw. S / XbaUB〇iti. Entire S adw / ayw S / Xbal / B〇HI Particle formation neo / egp! Neo / egpt neo / egpt neo / egpt neo / egpt neo / egpt neo / egpt neo / eopt ...... neo /? Gpt neo / egpt neo / egpt neo / e〇pt neo / egpt neo / egpt neo / egpt neo / egpt neo / egpt materials and methods cr Xie B i) ······························ · · · · · · · · · · · · · · · · · · · · · · · · (Please read the precautions first and then write this page) This paper standard is not used in a B standard (CNS) T4 specifications ( (210X297 public release) 81. 4. 10,000 sheets (II) Printed by Beigong Consumer Cooperative of Central Bureau of Standards, Ministry of Economic Affairs Λ 6 _Π_6__ V. Description of invention () Table HI The appropriate DNA sequence listed combined to give each problem, the DNA construct of the present invention may be comprised. In particular, any of the components shown in this table can be combined into a DNA sequence, which can be used to produce a composition (containing Duo < a) and used for @treatment of chronic viral hepatitis when transferred into a host cell. Medicine. The DNA sequence of the T-cell epitope sequence can be prepared by synthesizing (SYN) with Biosearch Cyclon synthesizer (SYN), or generated by PCR program (PCR), or the viral genome can be cut into fragments using restriction enzymes, etc. It is prepared in a way. For the treatment of patients with chronic viral hepatitis, the combination of the multi-sequence U > and the carrier (b>) can be formulated into any medicinal composition, which can additionally contain a suitable diluent or pharmaceutical carrier material, such as buffer solution The drug administration can be accomplished by any method, for example, parenteral (eg, intravenous or intramuscular), or given orally (eg, encapsulating the active substance with typhoid fever mackerel cells), etc. , The medicinal preparation contains a sufficient concentration of the above-mentioned composition Μ After giving it, it induces f: Λ
繪騙之餹酪說明 圖I 顯示一 DNA構成物,编碼著一值啟動基因,一匍粒 子形成物序列和一掴遘擇基因(於賣施例3/4中 說明之)。 圖Π 顯示一棰DNA-基因構成物,含有一個啟動基因,一 個具有整値HB-S-Ag的抗原決定基及一儘選擇基因 (於實施例3 / 18中說明之〉。 圖K 顯示一種DNA構成物,呈出一艏啟動基因,一値具 本紙張尺度逍用中a Η家標準(CNS)甲4規格(210X297公;tt) 81. 4. 1〇,〇〇〇張(π) (請先閲讀背而之注意事項外填寫本頁) 裝- 線- 經濟部中央櫺準局员工消费合作杜印製 Λ 6 __ _ Π 6_ 五、發明説明() 有粒子形成物餘鼸的τ-細胞抗原決定基,及一匍遘 擇基因(於實施例3/21中說明之)。 豳IV 列出黑埋猩1在Hepa-Care治療期間的AST值(於實 施例10/ 1中說明之>。 、 圏V 顯示出黑猩猩1在Hepa-Care治療期間的抗原值(於 實施例10/ 1中說明之〉。 圏VI 顯示出黑猩猩1在用Hepa-Care加強治療三次後, 其肝酵素ALT和GGT兩者的值(於實施例10/2中說明 之)。 . 圖V» 顯示出黑猩猩2在Hepa-Care治療期間,其肝酵素 ALT,AST,和GGT,及其抗原等的值。(於實施例10 /3中說明之)。 團VI 列出未接受治療的對照組黑猩猩經澜得之肝》素值 (¥實施例10/3中說明之)。 圖IX&X 顯示出患者1在Hepa-Care治療期間的抗原力 價與抗羅力價(於實施例11中說明之)。 園XI&XI 顯示出患者2在Hepa -Care 治療期間的抗 原力價與抗腥力價(於實施例11中說明之>。 圖XI & XIV 顯示出患者2在Hepa-Care治療期間的抗 原力價與抗鼸力價(於實施例11中說明之〉。 本發明播Μ下列實施例予以更特別地說明之。 奮旃例1 1.用聚乙二醇(PEG)進行分段沈澱 從產生HBV蛋白質的培養物收集得上澄液後分開成各 (請先閲讀背而之注意事項#填寫本頁) 裝. 本紙張尺度边用中困國家楳準(CNS) Ή規格(210X297公;¢) 81. 4. 10,000張(H) 209>4〇 Λ 6 116 經濟部中央梂準局貝工消费合作社印¾ 五、發明説明() 為2400毫升的數部份。於每一部份,加入144克PEG 6000 (Serua〉並於室溫攪拌20分鏟予以溶解後,再於4C鳙攪拌 6小時。然後,裝於500毫升瓶中,在GS 3轉盤内,於10¾ ,以9,000rpn( 15,000xg)離心30分鐘予以分離。收集所 得上澄液,加入144克PEG 6000並依上述溶解之。將所得 溶液置於41C攪拌3小時。依上述方式收集得該溶液之沈澱 物,不同處在於其離心操作係持續60分鐮。 2. 凝膠層析分雛法 將PEG沈澱所得物質再溶解於20毫升PBS中,然後在A -5m(Biora<〇上進行凝膠層析分離。管柱尺寸爲25X 1000毫 米,床體積為480毫升。典型分液分離操作中,係載入10 到15毫升含有1,000 m g PEG沈濺出的HBV蛋白質之溶液, 並M6滴/分<18毫升/小時)之速率用PBS洗提。 質係在第一層析峰被洗提出來。将收集到的分液進彳tj» CsCl梯度沈降分離。 3. CsCl梯度沈降分離 將A-5·管柱層析分離第一層析峰所函蓋的含有純化 HBV蛋白質之30份左右分液收集成約100毫升。將該溶液用 CsCl調整到1.30克/立方厘米之密度,然後移置於配合到 SW 27/28轉盤内之PolyalloBer管子中。梯度形成方式爲 在管子底層放置4毫升密度1.35克/立方厘米的CsCl溶液 ,接箸覆置4毫升密度1.25克/立方厘米的CsCl溶液,最 後覆置4毫升密度1.20克/立方厘米的CsCl溶液。將此梯 度M 28,000rp·在101C操作50小時。之後,將此梯度分段 (請先閲讀背而之注意事項再填寫本頁) 裝- 線. 本紙*尺度遑用中B國家«準(CNS) T4規格(2】0χ297公*) 81. 4. 10,000張(Η) 209H〇 Λ 6 Π 6 經濟部屮央標準局貞工消赀合作杜印製 五、發明説明() 分離,收集出浮置於1.20克/立方厘米密度層内的純化 HBV蛋白質。將此溶液置於透析袋中對水透析三次Μ脫除 鹽份。 奮掄例2 、 HBV蛋白質之定量測定 1. 用放射免疫分析法 於AUSRIA 11-125“夾心式’’放射免疫分析法中(其設 備可購自Abbot),係將塗佈著天竺鼠對抗Β型肝炎抗原的 抗醱(抗-HBs)之珠粒去和血淸,血漿,或純化蛋白質及適 當的對照樣等進行接觸。内含的任何HBsAg都會结合到該 固相抗體。將未結合物抽吸出並洗滌珠粒之後,用人類 iaeT-抗-HBs與珠粒上的抗臞-抗原複合物進行反應。然後 ,洗滌該珠粒以脫除未結合的18βΤ-抗-HBs。 )-抗-HBs HBsAg )-抗-HBs · HBsAg 1ββΤ-抗-HBs )-抗-HBs · HBsAg ♦…T-抗-HBs 殘留在珠粒上的放射性則用《I嗎(7>閃嫌計數器予M計數 出。 2. 用 ELISA 於Enzygnost HBsAg撤“夾心式”分析中(其設備可購 自Behring〉,各洞皆Μ抗>HBs塗佈。然後將血清,血漿或 純化蛋白質及適當的對照樣等分別加到洞内並予K接注。 於洗滌之後,Μ過氧化酶-檫示的對抗HBsAg之抗龌醣即用 來與剩餘抗原決定因子進行反應。然後,將未結合的酵素 (請先WJ讀背而之注意事項#填¾本頁) 裝< 訂· 線< 本紙張尺度逍用中國國家«準(CNS) T4規格(210x297公*) 81. 4. 10,000張(II) 1° ** 9 ο 經濟部中央標準局员工消费合作杜印製Description of the deception case Figure I shows a DNA construct encoding a value-initiating gene, a stoichiometric sequence and a slap selection gene (explained in Example 3/4). Figure Π shows a DNA-gene construct, containing a promoter gene, an epitope with an integer HB-S-Ag, and an exhaustive selection gene (explained in Example 3/18). Figure K shows a DNA construct, presenting a prokaryotic gene, a copy of the paper standard for easy use in a family standard (CNS) A 4 specifications (210X297 public; tt) 81. 4. 1〇, 〇〇〇 Zhang (π) (Please read the notes before filling in and fill out this page) Outfit-Line-Printed by the Employee Consumption Cooperation of the Central Bureau of Economic Affairs of the Ministry of Economic Affairs Λ 6 __ _ Π 6_ V. Description of the invention () τ with particle formation -Cell epitope, and a selective gene (explained in Example 3/21). Bin IV lists the AST value of Black Budgie 1 during Hepa-Care treatment (explained in Example 10/1 Of>. 圏 V showed the antigen value of chimpanzee 1 during Hepa-Care treatment (explained in Example 10/1). 圏 VI showed that chimpanzee 1 had liver after three intensive treatments with Hepa-Care The values of both enzymes ALT and GGT (explained in Example 10/2). Figure V »shows chimpanzee 2 during Hepa-Care treatment The values of liver enzymes ALT, AST, and GGT, and their antigens. (Explained in Example 10/3). Mission VI lists the non-treated control chimpanzee's hepatic liver "vegetarian value (¥ (Explained in Example 10/3). Figure IX & X shows the antigenic valence and anti-rollet valence of Patient 1 during Hepa-Care treatment (explained in Example 11). Yuan XI & XI shows the patient 2 Antigen potency and anti-odor valence during Hepa-Care treatment (explained in Example 11). Figure XI & XIV shows patient 2's antigen potency and anti-manual force during Hepa-Care treatment Price (described in Example 11). The following examples of the present invention will be described more specifically. Example 1 1. Staged precipitation with polyethylene glycol (PEG) from HBV protein-producing cultures After collecting the clear liquid, separate it into pieces (please read the back-end precautions # fill out this page) and install it. This paper is used in the national standard (CNS) Ή specifications (210X297; ¢) 81. 4. 10,000 sheets (H) 209> 4〇Λ 6 116 Printed by the Beigong Consumer Cooperative of the Central Bureau of Economic Affairs of the Ministry of Economic Affairs It is part of 2400 ml. In each part, add 144 g of PEG 6000 (Serua> and stir at room temperature for 20 minutes to dissolve, then stir in 4C bighead for 6 hours. Then, put it in a 500 ml bottle , Centrifuged at 9,000rpn (15,000xg) for 30 minutes at 10¾ in a GS 3 turntable. Collect the resulting clear solution, add 144 grams of PEG 6000 and dissolve as described above. The resulting solution was placed at 41C and stirred for 3 hours. The precipitate of the solution was collected in the above manner, except that the centrifugation operation continued for 60 minutes. 2. Gel chromatography separation method The material obtained by PEG precipitation is redissolved in 20 ml of PBS, and then subjected to gel chromatography separation on A-5m (Biora < 〇. The column size is 25X 1000 mm, and the bed volume is 480 ml. In a typical liquid separation operation, 10 to 15 ml of a solution containing 1,000 mg of PEG in HBV protein was poured, and eluted with PBS at a rate of M6 drops / min (<18 ml / h). The mass system was eluted at the first chromatographic peak. The collected liquid fraction was separated into tj »CsCl gradient sedimentation. 3. Separation of CsCl gradient sedimentation Separate about 30 mL fractions containing purified HBV protein covered by the first chromatographic peak of A-5 · Column Chromatography Separation into about 100 mL. The solution was adjusted with CsCl to a density of 1.30 g / cm3, and then transferred to a PolyalloBer tube fitted into a SW 27/28 turntable. The gradient is formed by placing 4 ml of CsCl solution with a density of 1.35 g / cm3 at the bottom of the tube, then covering with 4 ml of CsCl solution with a density of 1.25 g / cm3, and finally coating 4 ml of CsCl solution with a density of 1.20 g / cm3 . This gradient M 28,000rp · was operated at 101C for 50 hours. Afterwards, divide the gradient into sections (please read the precautions before filling in this page) to install-line. This paper * standard is used in the B country «quasi (CNS) T4 specification (2) 0χ297 public *) 81. 4 . 10,000 sheets (Η) 209H〇Λ 6 Π 6 Printed in collaboration with Zhengong Consumers, Bureau of Standards, Ministry of Economic Affairs. V. Invention description () Separate, collect purified HBV floating in 1.20 g / cm3 density layer protein. This solution was placed in a dialysis bag and dialyzed against water three times to remove salt. Example 2: Quantitative determination of HBV protein 1. Using radioimmunoassay in AUSRIA 11-125 "sandwich" radioimmunoassay (the equipment can be purchased from Abbot), the guinea pig will be coated against type B Beads of anti-hepatitis (anti-HBs) of hepatitis antigens are brought into contact with blood plasma, plasma, or purified proteins and appropriate controls, etc. Any HBsAg contained will bind to the solid-phase antibody. Extract unbound substances After aspirating and washing the beads, human iaeT-anti-HBs are used to react with the anti-hepatite-antigen complex on the beads. Then, the beads are washed to remove unbound 18βΤ-anti-HBs.)-Anti -HBs HBsAg) -anti-HBs · HBsAg 1ββΤ-anti-HBs) -anti-HBs · HBsAg ♦… T-anti-HBs The radioactivity remaining on the beads is counted by M 2. Use ELISA in Enzygnost HBsAg withdrawal "sandwich" analysis (the equipment can be purchased from Behring), each hole is coated with anti-HBs. Then serum, plasma or purified protein and appropriate control samples, etc. Add to the hole separately and give K to receive the injection. After washing, M peroxidase-sassaying pair HBsAg's anti-glucose is used to react with the remaining antigenic determinants. Then, the unbound enzyme (please read the WJ back first and note #fill ¾ this page) is installed < Order · Line < This paper size Use the Chinese National Standard (CNS) T4 specifications (210x297 g *) 81. 4. 10,000 sheets (II) 1 ° ** 9 ο Printed by the employee consumption cooperation of the Central Bureau of Standards of the Ministry of Economic Affairs
五、發明説明() 聯結抗齷洗除,再測定留在固相上的酵素活性。遇氧化氫 與發色康之間的酵素催化反應係經由添加稀硫酸予Μ終止 。其顔色強度係正比於樣品的HBsAg濃度,且係將未知樣 的顔色強度輿伴隨的陰性與隱性對照血淸的顔色強度Μ光 度計比較而拥定者。 窗掄Μ 3 本發明含有啟動子,所欲抗原序列和遘擇基因等的基 因構成物之製備 1. ΜΤ-啟動基因之離析 將質釀 pBPV-342-12(ATCC 37224)用核酸内切 llBgll 和Ba«H g號轉分解。產生三棰DNA分子。所欲片段含有 νΐ' Θ methalloiliionein啟動基因及具有4.5kb的PBR322序列, 很容易從其他片段中探測出來(2.0kb和7.6kb)。 該反應是在緦腥稹200 U1反豳級衝液,最後濃度0.5 /i g//i 1 DNA及100單位各棰限制酵素等之溶液中進行的。 於37它溫置3小時後,用瓊脂糖凝謬霣泳法M0.8%瓊脂 糖凝膠檢核分解反應的完成情形。該反應係經加入4/il 0.5M EDTA 予 Μ 停止。 接著,用製備型1.2%瓊脂耱凝膠«泳法將該4.5kb片 段與其他片段分離開來。將該段瓌脂耱凝膠置於DE-81 Whatman濾紙上,以高鹽级衝液將該DNA洗提出來。該DNA 再用酚-氣仿萃取及兩次乙酵沈澱予K純化。 2. 連接编碼著HBV-核心抗原的1.8kb片段 從含HBV的DNA中将含有HBV-核心纊碼域的1.8kb BamH I (請先閲讀背而之注意事項#填窍本頁) 裝- 線· 本紙»尺度逍用中Β釅家標準(CNS)甲4規格(210X297公;《:) 81. 4. 10,000¾ (II) -09:4〇 Λ 6 116 經濟部屮央標準局员工消费合作社印製 五、發明説明() Bam-HI片段離析出來。然後將此片段與含有MT-啟動子和 PBR餘段的4.5kbH段(於1中說明者)連接在一起。 將2w 1的1.8kb片段與3« 1該4.5kb片段混合在一起, 並於含有2單位T4-DNA連接_和2·Μ ATP的1〇</ 1連接播衝 液中在14¾反應隔夜而建接在一起。 接著,將該連接反臁混合物加到150/ul恰當的細菌細 胞懸浮液中以攝取DN A。在DNA攝取後,将細豳細胞Μ每板 50到300 «丨細胞懸浮液的黼積比散佈到含有50/i 1/»1氨苄 青徽素的LB瓊脂板上。然後将該瓊脂板置於37t:隔夜。最 後對單離細鯖菌落進行篩選以求得含有所欲片段的質鼸。 3. 篩選含有所欲質黼之細_菌落 將單離菌落用牙籤挑起胃置於裝有LB-氨苄青徽素 培餐基的試管中(5毫升>。If;試f置於37C,快速霉盪環 境中溫育隔夜。之後,對每個$長後的細鷓懸浮液進行小 型質體製備。各種不同的DNA則用限制核酸内切酶Bgll 予以分解證明。預期的對象有400bP片段和5.9kbii|H段 兩種分子。該分解反臁係用瓊脂耱凝膠«泳法予W分ft。 如此,從細囍細胞中離析出質鰻DNA。 4. 嵌入新徽素灌擇檷誌 將上面(3)中得到的質醱用限制酵素EcoRI分解而使之 線形化。該反應係M50//1的總羅積與1/ig/w 1質醱DNA的 最後濃度進行者。加入50單位的EcoRr後,在37t溫置3小 時,再Μ璜脂耱凝膠霄泳法驗證該分解反應。然後,加入 1/u 1 0.5Μ EDTA中止該反應,並Μ最後溅度0.3Μ的釀酸納 (請先閲讀背而之注意事項#填寫本頁) 裝· - 線· 本紙張尺度遑用中B國家樣準(CNS)甲4規格(210X297公¢) 81, 4. 10,000張(II) 2〇9 Λ 6 Π 6 經濟部+央橾準局员工消费合作社印製 五、發明説明() 及3-4醍積的乙酵加入,置於-80t,30分鳙以沈澱出DNA 。接著Λ將沈澱出的DNA溶解於50W 1蒸籣水中。 将1線形化的霣艚與3// 1含有 nethallothionein啟 動基βΗΓ新徽素逸擇基因〔將質讎PMT-ne〇-E(可得自ATCC /Exogene)用核酸内切酶EcoRI分解,離析得者,為 3.9kb之片段〕等的DNA片段混合並連接在一起。對單離細 醣菌落進行筛SM求得含有所欲質鑛者。 5. 離析出含有U2啟動子序列的Η段 將質饈pUC-8-42(得自Exogene)用限制核酸内切酶EcoR I 和Apal予以分解。產生出兩種DNA分子。含有U2-啟動子 的所欲片段具有340bp且易於從另一片段<3160bp)探拥|出 來。該分解反應係在200« 1總臁稹的反鼸级衢液中,以 0.5Wg//il DNA的最後濃度,及含有100單位的鹤別限制 酵素等條件下進行的。於371D S置三小時後,用0.7%瓊 脂耱凝膠以瓊脂糖凝謬電泳法檢定分解反鼸的完全性。該 反應係經由添加4W 1 0.5M EDTA予Μ停止。然後,用製備 型1.2%瓊脂糖凝膠霣泳法從質«DNA中分離出340bp片段 。接著將含該片段的瓌脂糖凝膠置於DE-81 hlhaUan濾紙 上,用高鹽份级衝液洗提出該DNA。最後,用酚/氯仿萃 取與兩次乙酵沈»鈍化該DNA。 6. 将含有啟動子序列的片段鑲嵌到多聯結者霣釀中 將含有多聯結者序列(polylinker sequence)^含有下 列限制部位:EcoRI ,Sac I ,S^a I > Aoa I > BaiH I , Bgl I,Sal I,Pst I,Hindi > 的質黼 pSP165 (可購自 (請先WI讀背而之注意事項#填寫本w) 裝- 訂 線· 本紙張尺度遑用中e a家榣準(CNS) T 4規格(210X297公*) 81. 4. 10,000張(H) 經濟部屮央樑準局员工消费合作社印製 五、發明説明() Pro^ega Biotec >用限制酵素予Μ線形化。該反醮係在 50«1總醴積舆lwg/« 1質鼸DNA最後濃度的溶液中進行者 。加入50單位的EcoRI,並於371C溫置三小時後,Μ璉脂 耱凝膠霄泳法檢定該分解反醮。反醮係經由添加1於1 0.5Μ EDTA予Μ停止,再Μ0.3Μ最後濃度的ϋ酸納和3-4體積的 乙醇,於-801C,經30分鐘,將DNA沈澱出來。然後,将沈 澱出來的DNA溶解於50//丨蒸«水中。 将2w 1質體DNA與10« 1含U2啟動基因的片段DNA混合 ,再於含有2單位T4-DNA達接酶和2·Μ ATP,總黼稹25w 1 的連接缓衝液中,於14Ϊ:,經隔夜,而連接在一起。之後 ,用酚/氯仿萃取接著兩次乙酵沈濺将DNA純化後,溶解 1〇Μ 1蒸《水中。所得EcoRI與MI黏端必須轉化成鈍端 \r:' 、 後再連接。該黏端係經由用心!^豆被酸分解酶進行反應而 轉化成鈍端者,如下所述:於25 m 1含DNA(1« g/w 1灌度) 的反應缠衝液中,加入20單位的酵素,使其含有1%最後 濃度的甘油及35W1最後反應體積。於30C溫置30分鐘後 *用酚-氮仿萃取接著兩次乙酵沈澱而純化出DNA。再將 該DN A溶解於5« 1蒸皤水中。將所得鈍端於含有比上面所 用多10倍的T4連接酶與2mM ATP等之15wl反應溶液中,在 14TC隔夜後連接在一起。 接著,將該連接反應混合物加到150 μ 1適當的細鰌細 胞懸浮液中Μ進行DNA攝取。於DNA攝取後,將細醣細胞Μ 每板50到300 w 1的釅積塗佈在含有50w 8/·1氨苄青徽素的 LB瓊脂板上。然後,將該瓊脂板在371D溫置隔夜。之後, (請先閲讀背而之注意事項再填窍本頁) 裝- 訂· -線- 本紙張尺度逍用中Β «家標準(CNS)甲4規格(210X297公龙) * 21 —· 81. 4. 10,000張(Η) 經濟部中央橾準局β工消费合作杜印製V. Description of the invention () The anti-galvanized washing is removed, and the enzyme activity remaining on the solid phase is measured. The enzyme-catalyzed reaction between hydrogen oxide and Fasikang is terminated by adding dilute sulfuric acid to M. The color intensity is proportional to the HBsAg concentration of the sample, and it is the one who compares the color intensity of the unknown sample with the negative intensity of the hidden sample and the color intensity M photometer of the hidden control blood volume. Window M3 The present invention contains a promoter, a desired antigen sequence, and the preparation of a gene construct such as a selective gene 1. Isolation of the MT-promoter gene The quality pBPV-342-12 (ATCC 37224) is endonucleated with nucleic acid llBgll And Ba «H g number to decompose. Generate three DNA molecules. The desired fragment contains the νl 'Θ methalloiliionein promoter gene and the PBR322 sequence with 4.5 kb, which can be easily detected from other fragments (2.0 kb and 7.6 kb). The reaction was carried out in a solution containing 200 U1 anti-bin-grade flushing liquid with a final concentration of 0.5 / i g // i 1 DNA and 100 units of restriction enzymes. After being left at 37 ° C for 3 hours, the completion of the decomposition reaction was checked by agarose gel swimming method M0.8% agarose gel. The reaction was stopped by adding 4 / il 0.5M EDTA to M. Next, the 4.5 kb fragment was separated from other fragments using a preparative 1.2% agarose gel «swimming method. Put this piece of jellyfish gel on DE-81 Whatman filter paper, and elute the DNA with high-salt grade liquid. The DNA was then purified by K-phenol extraction and two ethanol precipitations. 2. Connect the 1.8kb fragment encoding HBV-core antigen from the DNA containing HBV to the 1.8kb BamH I containing the HBV-core code domain (please read the back-end notes #fill this page) to install- Thread · Original paper »Standard Xiaozhong Standard (CNS) Grade 4 (210X297);": "81. 4. 10,000¾ (II) -09: 4〇Λ 6 116 Employee Consumption of the Central Standards Bureau of the Ministry of Economic Affairs Printed by the cooperative. V. Description of invention () Bam-HI fragments are isolated. This fragment was then ligated with a 4.5 kbH segment (explained in 1) containing the MT-promoter and the rest of the PBR. Mix 2w 1 of the 1.8kb fragment with 3 «1 of the 4.5kb fragment, and react in 14¾ reaction overnight in 1〇 < / 1 ligation solution containing 2 units of T4-DNA ligation and 2.M ATP Build together. Next, the ligation mixture was added to 150 / ul of the appropriate bacterial cell suspension to ingest DNA. After DNA uptake, the ratio of cell volume of fine cell M to 50 to 300 per cell plate was spread on LB agar plates containing 50 / i 1 / »1 ampicillin. The agar plate was then placed at 37t: overnight. Finally, the colonies of solitary mackerel were screened to obtain the pluton with the desired fragment. 3. Screen the fine _ colony containing the desired texture. Isolate the colony to pick up the stomach with a toothpick and place it in a test tube (5 ml) containing LB-Ampicillin-based food base. If; test f is placed at 37C , Incubate overnight in a rapid moldy environment. Afterwards, a small plastid preparation was performed for each partridge suspension. A variety of different DNAs were decomposed using the restriction endonuclease Bgll. The expected target was 400bP fragments And 5.9kbii | H-segment molecules. The decomposition reaction is carried out by agarose gel «swimming method to give W minutes ft. In this way, the quality eel DNA is isolated from the fine cells. The mass fraction obtained in (3) above was decomposed with the restriction enzyme EcoRI to linearize it. This reaction was performed by the total product of M50 // 1 and the final concentration of 1 mass fraction DNA of 1 / ig / w. Add 50 After the unit of EcoRr, let it stand at 37t for 3 hours, and then verify the decomposition reaction by the method of gel skimming. Then, add 1 / u 1 0.5M EDTA to stop the reaction, and the final splash of 0.3M brewing acid Accept (please read the back and attention matters # fill in this page) to install ·-line · The paper size is not in the national B standard (CNS) A 4 specifications (210X297 public ¢) 81, 4. 10,000 sheets (II) 2〇9 Λ 6 Π 6 Printed by the Ministry of Economics + Central Central Bureau of Employee Consumer Cooperative Fifth, the description of invention () and the fermentation of 3-4% of the yeast added, Put it at -80t, 30 minutes of bighead carp to precipitate DNA. Then Δdissolve the precipitated DNA in 50W 1 distilled water. Dissolve 1 linearized cockroach with 3 // 1 containing nethallothionein promoter βΗΓ 新 徽 素 逸 择DNA fragments (such as PMT-ne〇-E (available from ATCC / Exogene), which is decomposed with endonuclease EcoRI, and the isolated one is a 3.9 kb fragment) are mixed and ligated together. Fine sugar colonies were screened by SM to obtain those containing the desired minerals. 5. Isolate the H segment containing the U2 promoter sequence and use pUC-8-42 (available from Exogene) with restriction endonucleases EcoR I and Apal Decompose. Two types of DNA molecules are produced. The desired fragment containing the U2-promoter has 340 bp and is easy to probe from another fragment (<3160 bp). The decomposition reaction was carried out in 200 «1 total staghornis, at a final concentration of 0.5Wg // il DNA, and containing 100 units of Hebei restriction enzyme, etc. Three hours after leaving at 371D S, the completeness of the decomposition reaction was checked by agarose gel electrophoresis using 0.7% agar gel. The reaction was stopped by adding 4W 1 0.5M EDTA to M. Then, a 340 bp fragment was isolated from the genomic DNA by preparative 1.2% agarose gel electrophoresis. Next, the oligosaccharide gel containing the fragment was placed on DE-81 hlhaUan filter paper, and the DNA was extracted with a high-salt-level flush solution. Finally, the DNA was inactivated by phenol / chloroform extraction and two ethyl acetate precipitations. 6. Mosaic the fragment containing the promoter sequence into the multi-linker fermented brew will contain the polylinker sequence (polylinker sequence) ^ contains the following restriction sites: EcoRI, Sac I, S ^ a I > Aoa I > BaiH I , Bgl I, Sal I, Pst I, Hindi > the quality of pSP165 (available from (please read the first WI back and the precautions # fill out this w))-Threading · This paper size is not used in the ea family 榣Standard (CNS) T 4 specifications (210X297 g *) 81. 4. 10,000 sheets (H) Printed by the Employee Consumer Cooperative of the Ministry of Economic Affairs, Gwangyang, Ministry of Economic Affairs V. Description of invention () Pro ^ ega Biotec > Use restricted enzymes for M Linearization. The anti-condensation system is carried out in a solution of 50 «1 total product and the final concentration of lwg /« 1 qualitative DNA. Add 50 units of EcoRI and place at 371C for three hours. The decomposition reaction was verified by the gel-swimming method. The reaction was stopped by adding 1 to 1 0.5 M EDTA to M, and then M 0.3 M sodium hydroxide at a final concentration and 3-4 volumes of ethanol at -801C for 30 minutes. Precipitate the DNA. Then, dissolve the precipitated DNA in 50 // 丨 distilled «water. 2w 1 plastid DNA and 10« 1 fragment DN containing U2 promoter gene Mix A, and then ligate them together in a ligation buffer containing 2 units of T4-DNA ligase and 2 · M ATP, 25% of total tares, at 14Ϊ :, and connect them together overnight. After that, extract with phenol / chloroform Then, after the DNA was purified by two ethyl acetate precipitations, dissolve 10 μM in distilled water. The sticky ends of EcoRI and MI must be converted into blunt ends \ r: 'and then connected. The sticky ends are carefully prepared! ^ 豆Those reacted by acid-decomposing enzymes and converted into blunt ends are as follows: add 25 units of enzyme to 25 m 1 of the reaction entanglement solution containing DNA (1 «g / w 1 irrigation) to make it contain 1% The final concentration of glycerol and the final reaction volume of 35W1. After 30 minutes at 30C, the DNA was purified by extraction with phenol-nitroform followed by two ethyl acetate precipitations. The DNA was then dissolved in 5 «1 distilled water. The resulting blunt ends were ligated together in a 15wl reaction solution containing 10 times more T4 ligase and 2 mM ATP, etc. than used above, after 14TC. Next, the ligation reaction mixture was added to 150 μ 1 of appropriate fine cells DNA ingestion of M in the suspension. After DNA ingestion, apply a volume of 50 to 300 w 1 of fine sugar cells M per plate on LB agar plate containing 50w 8 / · 1 ampicillin. Then, place the agar plate at 371D overnight. Afterwards, (please read the precautions before filling in this page) Installation-Order ·- Thread-This paper standard is used in beta «Family Standard (CNS) A 4 specifications (210X297 male dragon) * 21 — 81. 4. 10,000 sheets (Η) Printed by the Central Bureau of Industry and Commerce β Industrial Consumption Cooperative Print
五、發明説明() 對單離細皤菌落進行篩蘧以求得含有所欲U2-啟動基因Η 段的質黼。最後,從細醻細胞tt析出所得質體並用限制酵 素分析法予Μ钂定。 7. 合成寡-DNA-核苷醚89(SEQ ID Νο.:30)與ΜΤ-啟動基因 片段(4.5kb〉連接在一起 將含有MT-啟動子舆pBR餘段的4.5kb片段(述於上面1 中〉舆合成寡聚核苷酸89(SEQ ID No.:30>連接在一起。将 該連接反應混合物加到150/il適當的細_細胞懸浮液中進 行DNA攝取。然後*從單雛細菌囍落中篩S出含有所欲質 鱧者。該新質鼸係經限制核酸内切酶EcoRI和Xbal予Μ 分解後證明者。預期有兩種分子,一為2.Okb者,另一 為 2.6kb。 8. 將合成寡核苷酸101(5£1110^〇.:32)與質||(上面7中 所述者)連接在一起 将質齷(7中所述者)用Bgll和BaiHI分解除去含有 13_核苷酸的片段(1中述及者 >。将含有第一餹寡核苷酸 89(SEQ ID Νο·:30)的片段與寡核苷酸 l〇l(SEQ ID No.:32 ),係一棰Bgl Π -BamH I片段,連接在一起。在DNA攝取後 ,篩S出含有所欲幕錶的單離菌落細胞。該新貿讎係經核 酸內切酶EcoR I和,或EcoR I和Bgin分解證明通者 〇 、 9. 將合成DNA-寡核苷酸99(SEQ ID Νο·:31>連接到4.5kb片 段(1中述及者) 將 4. 5kb片段(Bgl E -Ba^H I )與 DNA寡核苷酸 99 (SEQ (請先閲讀背而之注意事項再填寫本頁) 本紙》尺度遑用中a β家《準(CNS) Τ4規格(210x297公货) 81. 4. 10,000張(Π) 五、發明説明()V. Description of the invention () Sieve the single-isolated scallion colonies to obtain the plutonium containing the desired H2-promoting gene H segment. Finally, the resulting plastids were precipitated from the fine cells and determined by the restriction enzyme analysis method. 7. Synthesize oligo-DNA-nucleoside ether 89 (SEQ ID No .: 30) and MT-promoter gene fragment (4.5 kb) to link together the 4.5 kb fragment containing the MT-promoter and pBR remainder (described above 1 Middle> Synthetic oligonucleotide 89 (SEQ ID No .: 30> ligated together. The ligation reaction mixture was added to 150 / il appropriate fine cell suspension for DNA uptake. Then * from single chick The bacteria are screened to find out the ones with the desired quality. The new quality mule is proved by the restriction endonuclease EcoRI and Xbal decomposed by M. It is expected that there are two kinds of molecules, one is 2.Okb, the other It is 2.6kb. 8. Link the synthetic oligonucleotide 101 (5 £ 1110 ^ 〇.: 32) with the quality || (the one described in 7 above). Connect the quality Qiang (the one described in 7) with Bgll Decompose with BaiHI to remove the 13-nucleotide-containing fragment (as described in 1>. The first fragment oligonucleotide 89 (SEQ ID No: 30) and the oligonucleotide l〇l ( SEQ ID No .: 32), which is a fragment of Bgl Π-BamH I, ligated together. After the DNA is taken up, the isolated colony cells containing the desired screen are screened out. The new trade is endonucleated Enzymes EcoR I and, or EcoR I and Bg in decomposition proof passer. 9. Connect the synthetic DNA-oligonucleotide 99 (SEQ ID No .: 31> to the 4.5 kb fragment (described in 1). 4. The 4.5 kb fragment (Bgl E -Ba ^ HI ) And DNA oligonucleotide 99 (SEQ (please read the precautions before filling out this page) This paper "standard is used in a β home" quasi (CNS) Τ4 specifications (210x297 public goods) 81. 4. 10,000 Zhang (Π) 5. Description of the invention ()
經濟部屮央標準局员工消费合作社印M ID No. :31〉連接在一起。對含有不同DNA的單離細菌菌落 進行篩選之後,MEcoRI分解,得到1.9kb和2.7kb兩片段 ,並用Bgl II或BamHI進行陽性線形化分析而鑑定出所欲 質體。 然後用Pst I和Ba«HI分解該新質體。預期者有兩棰 分子,一爲2.6kb ,含有pBR餘段,MT-啟動子和該寡核苷 酸等的片段,另一爲2.Okb含pBR餘段的片段。離析出該 2. 6kb片段。 10.將9中所述2.6kb的質體片段與質體離析出來的片段( «I 8中所述者)連接在一起 將含有DNA寡核苷酸89和1〇1<分別為SEQ ID No.:30與 32)用PstI與BglE予以分解。預期有兩掴Η段。一為含 有PBR餘段和ΜΤ-啟動子的2.5kb片段•及另一含pBR餘段和 兩個寡核苷酸之2.2kb片段。 將其中2.2kb片段舆8中所述含有PBR餘段,MT-啟動 與寡核苷酸99<SEQ ID No.:31)等的2.6kb片段連接在一起 〇 於篩遘出所欲的質醱後,用Bgl Π -Xbal限制核酸内 切酶分解進行鑑定。預期有兩個片段,一為寡-DNA-核话 酸的270bpj=j段及另一含MT-啟動子和pBR餘段的4.5kb。 11.2.3kb HBV BglE-Bglll 片段之連接 從含HBV的DNA離析出含有HBV pre-Sl,Pre-S2和S等 编碼域的2.3kb Bgl E -Bgl E片段。將該2. 3kb片段與含有 methollathionein啟動子的4.5kbH段(1中所得者)連接 (請先Ml讀背而之注意事項洱堝寫本頁) 本紙»尺度遑用中B因家«準(CNS)肀4規格(210X297公*) 81. 4. 1(),()00張(||) Λ 6η 6 經濟部屮央榣準局貝工消费合作杜印製 五、發明説明() 在一起。 将2/i 1的2.3kb片段與3« 1的4.5kbH段混合後,在總 讎稹l〇/ul)i接反應緩衢液中,於含有2單位T4-DNA連接酶 和2·Μ ATP之情況下,在14TC隔夜而連接在一起。、 然後,將該連接反醮混合物加到150/ul適當的細豳細 胞懸浮液中進行DNA攝取。於DNA攝取後,将細齠細胞以每 板50到300« 1的體積散佈在含有50/z g/«l氨苄青徽素的LB 瓊脂板上。將該瓊脂板在37C溫育隔夜。之後,對單離细 菌菌落進行筛選以求出含所欲片段之霣體。 12_將部份HBV-基因序列用HBV-核心抗原決定基予以轉化 將上面11所得霣臁用核酸内切酶Bgll舆Xbal予Μ分 解。預期有兩種分子,一為550bp片段及另一6.25kb片段 ,於瓊脂糖凝膠霣泳後離析出來。 将其中的6.25kb片段與10中所述對HBV-核心基因抗原 決定基部份缠碼的270bP質體Η段的建接在一起(在上文所 述用Bgll和Xbal分解與片段離析之後>。 将連接混合物加到150 適酋的細醣細胞懸浮液中進 行DNA攝取。然後,對單離細薗菌落篩選以求得所欲之質 體。該新質《係經Ba«HI分解予Μ證明過。預期有三種分 子:950bp,450bp 和 5150bp 等三片段。 13. “媒介”質黼的製備 將11中述及之質鱧用EcoRI和Xbal予Μ分解。預期 有兩棰分子,一為2450bP片段及另一 4,350bp片段,於凝 腰轚泳處理後離析出來。 (請先閲讀背而之注意事項#填寫本頁) 本紙ΪΙ尺度逍用中β B家«毕(CNS) T4規格(210X297公*) 81. 4. 10J00張(II) ;:4〇 Λ 6 Η6 五、發明説明 經濟部中央榣準局员工消费合作社印製 將其中的4.35bpH段與對整鶴HBV-S-基因中ATG到Xba I部位的DNA序列编碼之寡-DNA-核苷酸39(SEQ ID No.:29 ,其中ATG改換成ΑΤΑ)連接在一起。 14. 整個HBV-S基因的核心-抗原決定基上游段 、 然後,將所得“媒介”質釀用Pst I和Xbal分解,預 期有兩棰分子,一為600bp質讎餘段和另一 3,850bp片段。 後者於離析出來後與10中所述質鼸分解鐮析出之2,800bp ( 2,700bp)PstI -Xbal 片段連接在一起。 於篩S出所欲質體後,用EcoR I和Xba I ,EcoR I和 Bgll和BaiHI等限制核酸内切酶予Μ分解鑑定。 15. 選擇檷誌之嵌入 將14所得質«用EcoR I予以線形化。該反應係在50 wl.# 總《和1/Z g/tf 1質黼DNA最後獷度下進行的。加入50單位 的EcoR I,於37P#置3小時後,用瑷脂耱凝膠電泳法檢 定該分解反_。 t 之後,加入lwl的0.5MEDTA而停止該反應,並用 灌度0.3M的»酸納及3-4錶積的乙酵在-801,30分鐘等條 件下沈澱出DNA。將沈澱出之DNA溶解於50/i 1蒸籣水中。 取2/il線形化質齷與 3//丨含騰ethallothionein啟動子 新徽素遘揮基因的DNA片段(4中所述者)混合並連接在 =起。對單離細醣阌落篩逋所欲質匾,並予Μ離析,純化 和鑑定。 ---¾ 上面述及的每一棰基因構成物也可用U2-啟潘\_予 以構成,藉此,在同EcoRI和Bgin分解後,將含MT-啟動The MID No.: 31 of the Employee Consumer Cooperative of the Bureau of Standards of the Ministry of Economic Affairs is linked together. After screening isolated bacterial colonies containing different DNA, MEcoRI was decomposed to obtain two fragments of 1.9 kb and 2.7 kb, and positive linearization analysis was performed with Bgl II or BamHI to identify the desired plastid. Then use Pst I and Ba «HI to decompose the new plastid. The anticipator has two molecules, one is 2.6 kb, which contains the pBR residue, the MT-promoter and the oligonucleotide, and the other is 2.0 kb, which contains the pBR residue. The 2.6 kb fragment was isolated. 10. The plastid fragment of 2.6 kb described in 9 and the fragment isolated from plastid (the one described in «I 8) are linked together. DNA oligonucleotides 89 and 10 are contained <SEQ ID No. .: 30 and 32) Decompose with PstI and BglE. It is expected to have two slaps. One is a 2.5 kb fragment containing the PBR residue and MT-promoter, and the other is a 2.2 kb fragment containing the pBR residue and two oligonucleotides. Among them, the 2.2 kb fragment and the 2.6 kb fragment containing the PBR remainder, MT-initiator, and oligonucleotide 99 < SEQ ID No .: 31) are linked together. After the desired quality has been screened out , Bgl Π -Xbal restriction endonuclease digestion for identification. Two fragments are expected, one for the 270bpj = j segment of the oligo-DNA-nucleic acid and the other 4.5kb containing the MT-promoter and the rest of the pBR. 11.2.3kb Ligation of HBV BglE-Bglll Fragments The 2.3kb Bgl E-Bgl E fragments containing HBV pre-Sl, Pre-S2 and S coding domains were isolated from HBV-containing DNA. Connect the 2.3 kb fragment to the 4.5 kbH segment containing the methollathionein promoter (the one obtained from 1) (please read the notes before Ml and write this page on this page) CNS) 4 specifications (210X297 g *) 81. 4. 1 (), () 00 sheets (||) Λ 6η 6 Produced by the Beigong Bureau of Economics, Ministry of Economic Affairs, Beigong Consumer Cooperation Co., Ltd. V. Description of invention () in together. After mixing the 2.3kb fragment of 2 / i 1 with the 4.5kbH segment of 3 «1, in a total of 10 mL / ul) reaction mixture, containing 2 units of T4-DNA ligase and 2. In the case of ATP, they are connected together at 14TC overnight. Then, add the ligation reaction mixture to 150 / ul of appropriate cell suspension for DNA uptake. After DNA uptake, the cells were spread on LB agar plates containing 50 / z g / «l ampicillin at a volume of 50 to 300« 1 per plate. The agar plate was incubated overnight at 37C. After that, the isolated bacterial colonies were screened to find the ear body containing the desired fragment. 12_ Part of the HBV-gene sequence is transformed with HBV-core epitope. The haze obtained in 11 above is decomposed with M by the endonuclease Bgll and Xbal. Two molecules are expected, one is a 550 bp fragment and the other is a 6.25 kb fragment, which is isolated after dipping on an agarose gel. Connect the 6.25 kb fragment to the 270bP plastid H segment of the HBV-core gene epitope partially described in 10 (after Bgll and Xbal decomposition and fragment isolation as described above) ;. The ligation mixture is added to the fine sugar cell suspension of 150 suitable for DNA uptake. Then, the single-isolated barley colony is screened to obtain the desired plastid. The new substance is decomposed by Ba «HI Μ proved that. There are three kinds of molecules expected: 950bp, 450bp and 5150bp and other three fragments. 13. Preparation of "medium" quality scorpion will decompose the quality snakehead mentioned in 11 with EcoRI and Xbal. M is expected to have two molecules, one It is a 2450bP fragment and another 4,350bp fragment, which is isolated after condensing and swimming. (Please read the back to the precautions # fill out this page) This paper is used in the β B home «Bi (CNS) T4 specification (210X297 public *) 81. 4. 10J00 Zhang (II) ;: 4〇Λ 6 Η6 V. Invention description Printed by the Employee Consumer Cooperative of the Central Bureau of Economics of the Ministry of Economic Affairs. The 4.35bpH section and HBV-S- The oligo-DNA-nucleotide 39 encoded by the DNA sequence from ATG to Xba I in the gene (SEQ ID No .: 29, of which ATG Replaced by ΑΤΑ) connected together. 14. The entire core-epitope upstream segment of the HBV-S gene, and then, the resulting "medium" is brewed with Pst I and Xbal to decompose, it is expected that there are two molecules, one is 600bp quality The remaining segment of 雠 and another 3,850 bp fragment. The latter after isolation is connected with the 2,800 bp (2,700 bp) PstI -Xbal fragment precipitated by the decomposition of the mass muslin described in 10. After sieving out the desired plastid, use Restriction endonucleases such as EcoR I and Xba I, EcoR I and Bgll and BaiHI are decomposed and identified by M. 15. Selecting the embedding of 溷 志 之 to linearize the resulting substance 14 with «EcoR I. The reaction is at 50 wl. # Total 1 / Z g / tf 1 quality DNA was performed at the final temperature. After adding 50 units of EcoR I and set at 37P # for 3 hours, the decomposition reaction was detected by A. lucidum gel electrophoresis. T Afterwards, the reaction was stopped by adding 0.5MEDTA of lwl, and the DNA was precipitated with -300M sodium acetate and 3-4 surface of the yeast under the conditions of -801, 30 minutes, etc. The precipitated DNA was dissolved in 50 / i 1 steamed water. Take 2 / il linearized masses and 3 // 丨 DNA slices containing the gene of the new ethenothionein promoter of Teng ethallothionein (The ones described in 4) Mix and connect them together. Separate the fine plaques from the single-fine sugar sieve, and isolate, purify, and identify them. --- ¾ Each of the genes mentioned above The object can also be constructed with U2-Qipan \ _, by which, after decomposition with EcoRI and Bgin, it will contain MT-start
本紙張尺度边用中BS家標準(CNS)T4規格(210X297公;tt) (請先閲讀背而之注意事項再填窝本頁) 裝- 線· Ψ 81. 4. 10,000張(11) 09. Λ 6 η 6 經濟部屮央標準局员工消费合作社印製 五、發明説明() 基因@ΝΑ片段,可用EcoRI和Bgll分解離析所得含U2-啟DNA片段予以取代。 16. 大腸菌黄花色精烏糞瞟呤磷酿核糖基移轉酶(egpt>選 擇基因之離析 、 將質鶄PMSG用Ba*H I和BglH分解離析所得含egpt選 擇基因之片段(1.8kb)與含MT-啟動子之4.5kb片段(Bgl E -BanHI,1中所逑者)達接在一起。 於篩灌出所欲質鼸後,将其離析,純化並於最後將其 BaaiHI部位轉化成EcoRI部位。 17. 用PCR方法離析出所欲DNA序列 於凝膠電泳處理後離析出一 DNA片段(400bp)。係用特 定的寡核苷酸131和132(SEQ ID No.:33和34>作爲引導者( ?|^«61*)以?0!^方法(實施例5所述者)產生出來者。 将該DNA片段用核酸内切酶Ba«H I和Xba I予Μ分解後 ,以凝腰«泳法純化之。離析所得PCR-片段再與13所述質 鼸用Bgll和Xbal分解離析所得6.25kb片段建接在一起。 於DNA^取和細豳轉形後,對單醵菌落篩遘出所欲質齷。 18. 灌擇檷誌之鏟嵌 將17所得質鼸於最後添加入逸擇基因(依15所逑〉。 19. H2K啟動子之離析 H2K啟動子係從 pSP65H2(得自 Exogene>M EcoR I 和 Bgl I 片段(2kb)形式離析出來者。 上面所述及的所有構成物中,所有啟動基因皆可用 EcoRI /Bgll片段方式予K置捵。 (請先閲讀背而之注意事項洱填寫本頁) 裝- 訂- 線· 本紙張尺度適用中a Η家楳準(CNS)T4規格(2丨0X297公址) 81. 4. 10,000張(||) Λ 6 H6 經濟部中央榣準局ΚΧ工消费合作杜印製 五、發明説明() 20. 部份HBV-基因序列之轉化 將上面11所得質鼸用核酸内切酶BglH和Xbal予以分 解。預期有兩種分子,其中之一為6.250kb片段,於瓊脂 糖凝膠轚泳後離析出來。 、 将該6.250kb片段舆寡-DNA-核苷酸23(SEQ ID No.:28> 建接在一起。然後,将連接混合物加到150 μ 1孃當的细齠 細胞懸浮液中進行DNA攝取。接著,對單離細餹菌落篩遘 出所欲質醱。該新質鼸係經核酸内切酶EcoRI和Bgll[分 解證明過。預期有兩棰分子,一為1.9kb及另一 4.450kb者 〇 21. egpt灌揮檷誌之鑛嵌 將20所得質驩用EcoRI予以線形化。該反應係以100// 1 的總鼸稹與0.6Wg/al質體DNA最後漉度等條件進行的。 加入60單位的EcoRI,於3710¾置三小時後,K瓊脂糖凝 膠電泳法驗證該分解反應。之後,添加2wl的0.5MEDTA 停止該反應並用最後濃度0.3M的醣酸納及4讎稹的乙醇在 -80¾經一小時而沈澱出DNA 。將所沈澱得到之DNA再溶解 於50/i 1的蒸皤水中。 將2«1線形化質讎與3以1經£〇:〇111分解所得含此讣&11〇-1^1〇116 111啟動子和68?1^揮基因(16中所述者>的0“-片段 (3.7kb>混合並連接在一起。之後,篩S單離菌落以求出 所欲質龌。表I中列出的每一種基因構成物都可用上面說 明的相同方式予Μ製備得到。 宭榆锎Λ (請先間讀背而之注意事項再填寫本頁) 本紙張尺度逍用中國國家標準(CNS)肀4規格(210X297公*) 81. 4.】0,000張(li) ,09^40 A 6 η 6 五、發明説明() 經濟部屮央榣準局貝工消费合作社印製 用本發明的構成物轉移感染哺乳動物細胞 為了使本發明構成物縝碼的HBV呔能逹到實質量地分 泌,必須用本發明DNA構成物轉移感染晡乳動物細胞。該 共轉移感染係Μ兩步《方式(對每一構成物分別進衍轉移 感染)或單一步*方式 <即用兩棰構成物的製備物進行一次 轉移感染> 。共轉移感染係經由在兩種構成物上使用不同 的選揮檷誌,或者經由用免疫分析法探澜兩棰構成物的表 現產物之分泌等方式予Μ確定。 另外,也可將編碼著HBV呔序列的本發明序列與纗碼 箸整宿S,或核心,或白質等的鹤別序列組合在單一 構成物之內。 啻麻例5 聚合酶鍵型反應(PCR) _ 聚合酶鍵型反臁可用來將已知基因鑛序列的某一^定 域之特定DNA核苷酸序列在臁外(in uitro>擴大超遇百首" 倍(Thoiaas J. White, Nornan Arnlei·,Henry A· Erlich 1989: The Polynerase Chain reaction(聚合酶鍵型反應) ° Technical Focus, Vol. 5. No. 6; S. Kwok and R. Higuchi 1989: Avoiding false positive with PCR. Nature, Vol. 339, pp 237-238〉。 將自細胞分繡出來的DNA或質讎的DNA處理M分開其互 補鐽股。然後將逭些鋪股用過量的,經化學合成以匹配被 X核苷酸所分隔的序列(此處X通常在50至2,000_對之間 〉之兩DNA寡核苷酸(各爲20-25鐮對長度)予以熱融(anneal) (請先閲讀背而之注意事項#填寫本頁) 裝- 訂_ 本紙張尺度边用中《«家楳準(CNS) T4規格(210X297公;it) 81. 4. 10,000張(Η) Λ 6 Η 6 經濟部中央榣準局貝工消#合作杜印製 五、發明説明() 造兩種寡核苷酸係作為特定引導者,用於DNA聚合海 催化的龌外DNA合成,以複製該兩種寡核苷酸所對醮序列 之間的DNA。若該兩種引導者寡核苷酸含有正確順序,躭 可能在和創造出新的分解位置。 、 在多重反臁循環之後,得到大量具所欲長度的DNA片 段,用凝膠霣泳法純化並用限制酵素分解加上瓊脂耱凝腰 電泳予以鐽定。然後用該擴大且純化過的DNA片段舆其他 片段。如,質錶,達接在一起。 PCR-DNA片段係用鈍绱鑛大的。若要得到黏嫌(以進 行連接程序),則該片段必須用必要的核酸内切_予以分 解並再純化。 該PCR反應可操作20到30循環。一偏循環可分成三傾 步驟,各具不同的反應時間與不同的反應狙度,都是用 PCR-熱循環器(PCR-therao-cycler)予以控制者。第一步 驟為基霣-DNA的“變性”(1分鏟-95¾〉,第二步嫌爲基質 DNA與引導者“混成”(1分瞳/55¾ ),接着為“聚合反應 ”(2分鐘/72Ό >。 一次分析的最後齷積為,例如,30 μΐ ,其中含有下 列最後濃度:PCR-级衡液(Κ〉,具有各為200 « Μ的四種核 苷酸之核苷酸混合物,30u 1中各為200ng的兩種引導者, 每30W 1水溶液中含0.5單位Tag-聚合酶。 奮掄例R 轉移感染細胞的培餐Μ分泌蛋白質 將受纳細胞(C127-或CH0-細胞,得自ATCC)接種於正 (請先閲讀背而之注意事項#填寫本頁) 本紙張尺度遑用中B S家«準(CNS) Τ4規格(210x297公龙) 81. 4. 10,000張(Η) Γ Λ 6 Η 6 五、發明説明( 經濟部中央標準局员工消伢合作社印製 常生長培養基(DMEM+ 10%胎牛血淸,葡萄糖和麩醯胺)中 ,時間為第1日,置於淺培餐皿,<1-2Χ10β細胞/皿, Φ 10公分)中。於隔日將培餐基取出(在DNA沈澱物加到細 胞上之前4小時),並用1 X PBS將細胞洗兩次。然後,加入 8毫升不含FCS的DMEM,4小時後,將DNA沈澱物(依下文 所述製得者)加到該细胞之上。同樣地,在4小時後取出 培養基,加入3毫升甘油-混合物<50亳升2X TBS鍰衝液, 30毫升甘油,1?0毫升蒸籣水 >。於37^»置3分鐘後立邸 取出該甘油-混合物並用IX PBS洗該細胞。接著用8毫升 含10%FCS的DMEM培餐該細胞隔夜。 在48小時後,用胰蛋白酶-EDTA-溶液(0.025%胰蛋白 酶+ 1·Μ EDTA)處理以從皿中回收該細胞。之後,用lx PBS 洗滌Μ将胰蛋白酶-EDTA從細胞中除去,再将細胞懸浮於 含10%FCS的DMEM中,並分配到24Costar-洞-板中(一皿中 所含細胞分到四值24-洞-板中)。 當細胞生長良好時,加入選擇培餐基(濃度爲0.5-1毫 克/毫升的新徽素,或:黄花色精(250 wg/Bl〉,亞黃花 色精(15« g/nl>或腺瞟昤(25« g/臞1),胸脒核苷(10w 8/臟1) 基蝶昤(2// g/酿,徽酚酸<25ju g/el> ,用於,如, 者)。該培餐基要每星期更換。於2星期後可看到 第一健生長細胞群落。 於10« g質黼DNA和20// g載劑-DNA (鮭魚精子DNA ,小 牛胸腺 DNA〉中,加入 TE-缓衝液 <10ibM Tris-HCl, leM MDTA, 戸117.05)到最後體積440 ^£1,再與60«12^!〔&(:18混合在 (請先«請背而之注意事項#填¾本頁) 裝. 線. 本紙*尺度遑用中《明家樣準(CNS) T4規格(210x297公:¢) 81. 4. 10,00051c (II) 209.4b Λ 6 II 6 五、 經濟部+央標準局貝工消费合作杜印製 發明説明() 一起。接著,加入相同量的2XTBS (Hepes 50aM, NaCl 280»>1,“11卟〇4 1.5«^,?»17.05)並充分混合。將該沈澱 溶液置於37¾ 30分鐘後,直接加到欲轉移感染的細胞上。 實施例7 、 純化粒子佐劑之製備 在具有所欲濃度,懸浮於無®食鹽水中的抗原液內, 加入1:10,000讎積的Thiaerosol,1/10AK稹的已通濾減豳 之 0.2M KA1(S0«>» ♦ 12Ηβ0。其 pH值用無菌 IN NaOH調整到 5.0後,將該懸浮液在室»攪拌三小時。之後,於2, OOOrpn 離心10分鐘回收明鑄沈澱的抗原,並重懸浮於含有1:10, 〇〇〇 Thi Beroso丨的無豳正常*水中且於無菌狀態下分配。 鬣施例8 B型肝炎核心抗原之純化 將HB-核心抗原分泌性細胞的細胞上澄液收集起來並 Μ超濾法予Μ濃縮。然後,將濺縮液在Becknan SW28離心 機中於4¾以20,000γρ·離心15分鏞予Μ澄淸。 粒子形成是經由在Becknan SW28離心機中,於41C以 28,000rp·進行Μ糖密度離心(0-45%廉糖)24小時予以檢 試者。之後,將該梯度分成數部份,並對,每單一部份進行 Elisa分析。 奮渝例9 下面的表列出本發明免疫原粒子接受E 分析所得 一些結果,如下所述: I 表IV列出本發明任何HBV-序列構成物,包括pre-Sl (請先間讀背而之注意事項再填寫本頁) 裝- 訂· 線- 本紙張疋度逍用中ββ家楳準(CNS)T4規格(2丨0X297公;《:) 81. 4· 10,000張(ΙΪ) 209 Η。 Λ 6 Η 6 五、發明説明() epitopes舆S域等所產生的純化HBs-轉原粒子,用抗-Pre-Si « 單株抗醱ΜΑ 18/7和抗-HBs單株抗嫌G叫2進行Eli sa分析所 vj 得數據。 v 表IV所列爲CsCl密度梯度沈降後收集到的部份(、21)之 結果。 表 IV -1The size of this paper is used in the BS home standard (CNS) T4 specification (210X297 g; tt) (please read the precautions before filling the nest page) Packing-line · Ψ 81. 4. 10,000 sheets (11) 09 . Λ 6 η 6 Printed by the Employee Consumer Cooperative of the Bureau of Standards, Ministry of Economic Affairs V. Invention description () The gene @ ΝΑ segment can be replaced with the U2-kai DNA fragment obtained by the decomposition and isolation of EcoRI and Bgll. 16. Escherichia coli yellow flower essence black dung dung phosphatidyl ribosyl transferase (egpt > segregation of selection genes, the PMSG PM quality is decomposed and isolated by Ba * HI and BglH and the fragment containing the egpt selection gene (1.8kb) and containing The 4.5 kb fragment of the MT-promoter (Bgl E-BanHI, described in 1) is connected together. After the sieve is irrigated with the desired quality, it is isolated, purified and finally converted from its BaaiHI site to an EcoRI site 17. Isolate the desired DNA sequence by PCR method and isolate a DNA fragment (400bp) after gel electrophoresis treatment. The specific oligonucleotides 131 and 132 (SEQ ID No .: 33 and 34> are used as guides (? | ^ «61 *) produced by the? 0! ^ Method (the one described in Example 5). The DNA fragment was decomposed with the endonucleases Ba« HI and Xba I to M, and then condensed waist « It was purified by electrophoresis. The isolated PCR-fragment was then combined with the 6.25kb fragment obtained by the decomposition and isolation of the masses described in 13 with Bgll and Xbal. Quality. 18. Introduce the shovel of the selection of Zhizhi Zhi into 17 and add the quality of the genus to the final selection of the gene (according to 15). 19. H2K start The H2K promoter is isolated from pSP65H2 (available from Exogene > M EcoRI and Bgl I fragments (2kb). In all the constructs mentioned above, all promoter genes can be pre-treated with EcoRI / Bgll fragments K 置 捵. (Please read the precautions and fill in this page first) Binding-Binding-Line · This paper size is applicable to a 漸 楳 楳 准 (CNS) T4 specification (2 丨 0X297 public address) 81. 4. 10,000 sheets (||) Λ 6 H6 Central Ministry of Economic Affairs, Central Bureau of Industry and Commerce, KK industrial and consumer cooperation du printing 5. Description of the invention () 20. Part of the transformation of HBV-gene sequence The endonuclease BglH obtained from the above 11 pawns And Xbal are decomposed. Two molecules are expected, one of which is 6.250kb fragment, isolated after swimming in agarose gel. The 6.250kb fragment is oligo-DNA-nucleotide 23 (SEQ ID No. : 28 > Connect together. Then, add the ligation mixture to 150 μl of fresh larval cell suspension for DNA uptake. Then, sieve the single isolated larval colony to extract the desired substance. The new substance It was proved by the endonucleases EcoRI and Bgll [decomposed. Two molecules are expected, one is 1.9kb Another 4.450kb. 21. Egpt infiltrate the mines of 抷 志 臨. The 20-mass mass was linearized with EcoRI. The reaction was based on 100 // 1 total mole and 0.6Wg / al plastid DNA. Under other conditions. 60 units of EcoRI were added, and after three hours at 3710¾, K agarose gel electrophoresis was used to verify the decomposition reaction. After that, 2 wl of 0.5 MEDTA was added to stop the reaction, and DNA was precipitated out at -80¾ with an final concentration of 0.3 M sodium sugar acid and 4 mg of ethanol at -80¾ for one hour. The precipitated DNA was redissolved in 50 / i 1 of distilled water. The decomposition of 2 «1 linearized chrysanthemum and 3 in 1 by £ 〇: 〇111 contains this obituary & 11〇-1 ^ 1〇116 111 promoter and 68? 1 ^ gene (the one described in 16> ; 0 "-fragment (3.7kb> mixed and ligated together. After that, sieve S isolated colonies to find the desired quality. Each of the gene constructs listed in Table I can be used in the same way as described above Prepared by Μ. 傭 玉 锎 Λ (please read the precautions before filling in this page) The paper size is free to use the Chinese National Standard (CNS) 4 specifications (210X297 public *) 81. 4.] 0,000 sheets ( li), 09 ^ 40 A 6 η 6 V. Description of the invention () Printed by the Ministry of Economic Affairs, Bureau of Economics and Trade, Beigong Consumer Cooperative to transfer HBV infected with mammalian cells using the structure of the invention in order to make the structure of the invention HBV If it can be secreted in a substantial amount, the DNA construct of the present invention must be used to transfer and infect mammalian cells. The co-transfer infection is a two-step method (different transfer infection for each constituent) or a single step * method < i.e., one-time metastasis infection using the preparation of the two-pile composition > Different selections are used to determine the epidemiology, or by immunoassay to detect the secretion of the expression product of the two constituents of the two bridges. It can also be determined by M. In addition, the sequence of the present invention that encodes the HBV sequence can be determined with the code. The Tsurube sequence of whole-bed S, or core, or white matter, etc. is combined in a single structure. Example 5 Polymerase bond reaction (PCR) _ Polymerase bond type inversion can be used to combine known gene sequence The specific DNA nucleotide sequence of a certain localized area is in uitro (in uitro> expansion of the first 100 times) (Thoiaas J. White, Nornan Arnlei ·, Henry A · Erlich 1989: The Polynerase Chain reaction (polymerase Key type reaction) ° Technical Focus, Vol. 5. No. 6; S. Kwok and R. Higuchi 1989: Avoiding false positive with PCR. Nature, Vol. 339, pp 237-238>. Separated from cells DNA or DNA treatment of DNA M separates its complementary strands. Then some of the strands are used in excess, chemically synthesized to match the sequence separated by X nucleotides (here X is usually between 50 and 2,000_ pairs) Between two DNA oligonucleotides (each 20-25 pairs in length) Anneal (please read the back-end precautions #fill in this page) Binding-Order _ This paper is used in the standard "« 極 楳 准 (CNS) T4 specifications (210X297 public; it) 81. 4. 10,000 Zhang (Η) Λ 6 Η 6 The Ministry of Economic Affairs, Central Bureau of Industry and Commerce #Cooperate with Du Yin, V. Description of the invention () Two types of oligonucleotides were created as specific guides for DNA polymerization DNA synthesis to replicate the DNA between the sequences aligned by the two oligonucleotides. If the two guide oligonucleotides contain the correct sequence, the new decomposition site may be created at and. After multiple cycles of inversion, a large number of DNA fragments of desired length are obtained, purified by gel-swimming method and decomposed by restriction enzyme decomposition and agar gel electrophoresis. This expanded and purified DNA fragment and other fragments are then used. For example, the qualitative table can be connected together. The PCR-DNA fragment is made of blunt ore. To get sticky (for ligation procedures), the fragment must be digested with the necessary endonuclease_ and purified. The PCR reaction can be operated for 20 to 30 cycles. One partial cycle can be divided into three tilting steps, each with different reaction time and different reaction degrees, all controlled by PCR-therao-cycler. The first step is the "denaturation" of the base-DNA (1 minute shovel-95¾), the second step is the "mixing" of the matrix DNA and the leader (1 minute pupil / 55¾), followed by the "polymerization" (2 minutes / 72Ό >. The final volume of an analysis is, for example, 30 μl, which contains the following final concentration: PCR-grade balance solution (Κ>, a nucleotide mixture with four nucleotides each of 200 «Μ , Each of the two guides of 200ng in 30u 1 contains 0.5 units of Tag-polymerase per 30W of 1 aqueous solution. Fen case R Transfer of infected cells to the cultured meal M secreted protein will be received cells (C127- or CH0- cells , Obtained from ATCC) inoculated in the positive (please read the back and the precautions # fill out this page) This paper standard is used in the BS home «quasi (CNS) Τ4 specifications (210x297 male dragon) 81. 4. 10,000 sheets (Η ) Γ Λ 6 Η 6 5. Description of the invention (DMEM + 10% fetal bovine blood, glucose and glutamine) printed on the constant growth medium (DMEM + 10% fetal bovine blood) by the Consumers Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs. Shallow culture dishes, <1-2Χ10β cells / dish, Φ 10 cm). Remove the culture dishes on the next day (in the DNA sink) 4 hours before adding the material to the cells), and wash the cells twice with 1 X PBS. Then, add 8 ml of DMEM without FCS, and after 4 hours, add the DNA pellet (prepared as described below) to On top of the cells, similarly, after 4 hours, the medium was removed, and 3 ml of glycerol-mixture < 50 ml of 2X TBS buffer solution, 30 ml of glycerol, 1.0 ml of distilled water was added. After 3 minutes, the glycerol-mixture was removed and the cells were washed with IX PBS. The cells were then cultured overnight with 8 ml of DMEM containing 10% FCS. After 48 hours, trypsin-EDTA-solution (0.025% trypsin + 1 · Μ EDTA) treatment to recover the cells from the dish. Afterwards, wash trypsin-EDTA with lx PBS to remove the trypsin-EDTA from the cells, and then suspend the cells in DMEM containing 10% FCS and distribute to 24Costar- In the hole-plate (the cells contained in one dish are divided into four-value 24-hole-plates). When the cells grow well, add the selected food base (concentration of 0.5-1 mg / ml of neo-embroidin, or: Yellow flower essence (250 wg / Bl>, sub-yellow flower essence (15 «g / nl> or glandular glomerulus (25« g / 臞 1), thymidine (10w 8 / dirty 1) Jidie (2 // g / stuffed, emblemphenolic acid < 25ju g / el >, for, for example). The meal base should be changed every week. After 2 weeks You can see the colonies of the first healthy growing cells. In 10 «g-quality DNA and 20 // g carrier-DNA (salmon sperm DNA, calf thymus DNA>), add TE-buffer < 10ibM Tris-HCl, leM MDTA, 戸 117.05) to the final volume of 440 ^ £ 1, and then mixed with 60 «12 ^! [& (: 18 mixed in (please first« please note #fill ¾ this page) installed. Line. This paper * The standard is used in the "Mingjia Sample Standard (CNS) T4 specification (210x297mm: ¢) 81. 4. 10,00051c (II) 209.4b Λ 6 II 6 V. Ministry of Economic Affairs + Central Standards Bureau Beigong Consumer Cooperation Print the invention description () together. Next, add the same amount of 2XTBS (Hepes 50aM, NaCl 280 »> 1," 11Porpo4 1.5 «^ ,?» 17.05) and mix thoroughly. After putting the precipitation solution at 37¾ for 30 minutes, add it directly to the desired Transferred to infected cells. Example 7: Preparation of purified particle adjuvant In an antigen solution of the desired concentration, suspended in saline-free saline, adding 1: 10,000 thiaerosol, 1/10 AK Zhen has been filtered Reduced 0.2M KA1 (S0 «> 12Hβ0. After adjusting its pH value to 5.0 with sterile IN NaOH, the suspension was stirred in the chamber» for three hours. After that, it was centrifuged at 2, OOOrpn for 10 minutes to recover the cast Precipitated antigen, and resuspended in normal water containing 1:10, Thi Beroso, and distributed under sterile conditions. Example 8 Purification of Hepatitis B Core Antigen HB-core antigen secreting cells The cells were collected and concentrated by M ultrafiltration method. Then, the splashing condensate was centrifuged at 20,000 γρ for 15 minutes in a Becknan SW28 centrifuge at 4¾. The particles were formed via Becknan SW28. In a centrifuge, centrifuge at 28,000 rp · 41 at 41C with an M sugar density (0-45 % Cheap sugar) 24 hours to be tested. After that, the gradient is divided into several parts, and each single part is subjected to Elisa analysis. Fenyu Example 9 The following table lists the immunogenic particles of the present invention obtained by E analysis Some results are as follows: I Table IV lists any HBV-sequence constructs of the present invention, including pre-Sl (please read back and forth before filling in this page) Binding-Binding · Thread-This paper content The ββ family quasi (CNS) T4 specification (2 丨 0X297); ":) 81. 4 · 10,000 sheets (ΙΪ) 209 Η. Λ 6 Η 6. V. Description () Produced by epitopes and S domain etc. The purified HBs-transgenic particles were subjected to Eli sa analysis using anti-Pre-Si «single-anti-anti-MA 18/7 and anti-HBs single anti-G 2 2. v Table IV lists CsCl The result of the part (21) collected after the density gradient settled. Table IV -1
CsU-梯度部份纒號 Elisa澜量(E= 492) 單株抗鼸18/7 13 0.092 14 0.210 15 0.388 16 1.662 17 2.604 18 0.648 19 0.031 (請先閲讀背而之注意事項#堝窍本頁) 裝- 訂- 經濟部中央標準局貝工消费合作社印製 本紙»尺度边用中《«家«準(CNS>甲4規格(210x297公釐) 81. 4, 10,000張(II)CsU-gradient part of the number of Elisa Lan (E = 492) single plant anti-Mian 18/7 13 0.092 14 0.210 15 0.388 16 1.662 17 2.604 18 0.648 19 0.031 (please read the backing notes first ) Binding-Ordering-Printed Paper of Beigong Consumer Cooperatives, Central Bureau of Standards, Ministry of Economics, Standard Side Use «Home» Standard (CNS> A4 Specification (210x297mm) 81. 4, 10,000 sheets (II)
五、發明説明() 表W -2V. Description of Invention () Table W-2
CsH-梯度部份编號 Elisa測量(E= 492) 單株抗鼸G022 、 13 0. 136 14 0.426 15 0.822 16 1.970 17 2.954 18 0.967 19 0.076 (請先間讀背而之注意事項#填寫本頁) 表V所示為本發明任何HB-核心序列構成物所產生的純化 HB-核心-抗原粒子,用對抗HB-核心的多株抗體與對抗HB-S-Ag的單株抗膿G022進行Elisa分析所得數據。 經濟部屮央桴準局员工消费合作社印製 本紙Λ尺度遑用中國B家標準(CNS)T4規格(210x297公*) 81. 4, 10,000張(H) r 五、發明説明() Λ (i Β 6 表V -1 Μ耱梯度部份編號 El isa測量(Ε= 492) 單株抗體 6 0.25 7 0.922 8 1.423 9 1.5 10 1.5 11 1.28 12 0.466 表V -2 (請先閲讀背而之注意事項#堝寫本頁) 裝- 訂_ 經濟部屮央標準局员工消t合作社印製 蔗耱梯度部份編號 El isa測量(Ε= 492) 單株抗體G022 6 0.020 7 0.024 8 0.018 9 0.011 10 0.015 11 0.020 12 0.022 線- 本紙》尺度逍用中國國家樣準(CNS)甲4規格(210X297公龙) 81. 4. 10,000¾ (II) 09.4 ο 經濟部屮央標準局员工消t合作社印製 五、發明説明() 奮掄m 10 黑猩猩服用Hepa-Care的硏究: Hepa-Care為將B型肝炎表面抗原<S1和S)調製成特殊 配方(比例50:50)所製成之粒子,係用Μ治療肝炎病毒長 期載原者。 . 實驗1 將Β型肝炎載原者黑猩埋1在0,4,和8星期之時 用每注射18« g的劑量施以Hepa-Care治療(肌肉内注射>。 治療期間監測其肝酵素(圈IV)及其B型肝炎抗原水平 (圈 V >。 實驗2 依上面所述治療後的黑猩猩1再於30, 34 ,和38等遇 之時給予加強治療。其結果列於圖VI中。 實驗3 將黑猩埋2施MHepa-Care治療,但不同於黑猩猩1 之處在於係採用靜脈内給藥。其_量爲2毫克。其結果列 於圖VI中。 另外也對作為對照組的黑猩猩3進行肝酵素之監測, 其結果列於圓中。 謇渝俐11 用Hepa-Care治療:(其定義說明於實施例1〇中) 患者1 (男,年齡=65歲,已患病2年): B型肝炎參數:HBsAg Μ性 抗-HBs 陰性 (請先W讀背而之注意事項存堝寫本頁)CsH-gradient part number Elisa measurement (E = 492) individual strains of anti-Mule G022, 13 0. 136 14 0.426 15 0.822 16 1.970 17 2.954 18 0.967 19 0.076 (please read back and attention beforehand # fill in this page ) Table V shows purified HB-core-antigen particles produced by any HB-core sequence constructs of the present invention. Elisa was performed with multiple anti-HB-core antibodies and single anti-pus G022 against HB-S-Ag Analyze the data. The paper printed by the Employee Consumer Cooperative of the Central Bureau of Economic Affairs of the Ministry of Economic Affairs is printed on the Λ scale, using the Chinese B-standard (CNS) T4 specifications (210x297 g *) 81. 4, 10,000 sheets (H) r V. Invention description () Λ (i Β 6 Table V -1 Μ 耱 Gradient part number El isa measurement (Ε = 492) Monoclonal antibody 6 0.25 7 0.922 8 1.423 9 1.5 10 1.5 11 1.28 12 0.466 Table V -2 (Please read the precautions first #Pot write this page) Packing-Order _ Gradient part number of the sugarcane gradient printed by the staff cooperative of the Central Standards Bureau of the Ministry of Economic Affairs Elisa measurement (Ε = 492) monoclonal antibody G022 6 0.020 7 0.024 8 0.018 9 0.011 10 0.015 11 0.020 12 0.022 Thread-This Paper "scale is used in China National Standards (CNS) A 4 specifications (210X297 male dragon) 81. 4. 10,000¾ (II) 09.4 ο Printed by the employees of the Central Standards Bureau of the Ministry of Economy 3. Description of the invention () Fen M 10 The study of chimpanzees taking Hepa-Care: Hepa-Care is a particle made by mixing Hepatitis B surface antigen (S1 and S) into a special formula (ratio 50:50), It is a long-term carrier of hepatitis virus treated with M. . Experiment 1 Hepa-Care was administered with a dose of 18 «g per injection at 0, 4, and 8 weeks when the chimpanzee carrier of the hepatitis B carrier was buried 1 (intramuscular injection). The liver was monitored during the treatment Enzymes (circle IV) and their hepatitis B antigen levels (circle V >. Experiment 2 Chimpanzees 1 treated as described above were given intensive treatment at 30, 34, and 38. The results are shown in the graph. In VI. Experiment 3 Chimpanzee 2 was treated with MHepa-Care, but it is different from Chimpanzee 1 in that it is administered intravenously. Its amount is 2 mg. The results are shown in Figure VI. The chimpanzee 3 in the control group was monitored for liver enzymes, and the results are listed in the circle. Jian Yuli 11 treated with Hepa-Care: (The definition is described in Example 10) Patient 1 (Male, age = 65 years old, already 2 years of illness): Hepatitis B parameters: HBsAg Μ-anti-HBs negative (please read the back of the notes and write this page)
本紙張尺度逍用中國困家楳準(CNS)肀4規格(21〇Χ297公*) 81. 4. 10,000張(II) 五、發明説明() HBeAg 陰性 抗HBe 陽性 抗HBc 陰性The size of this paper is easy to use China Sleepless Standard (CNS) 4 specifications (21〇297297 *) 81. 4. 10,000 sheets (II) V. Description of invention () HBeAg negative Anti-HBe positive Anti-HBc negative
於0,1,6,和7等月用Hepa-Care予Μ治療(肌肉内注 射)。其抗原和抗鱷澜董结果列於圏Κ和X之中° 患者2 (女,年龄=48歲,已患病12年> : B型肝炎參數:HBsAg 陽性 HBeAg 陰性 抗-HBs 陰性 抗-HBe 隈性 抗-HBc 隈性 於Ο,1,和6等月用Hepa-Care予以治療(肌肉内注射) 。其抗原和抗钃澜量結果列於園XI和XI之中。 患者3(女,年齡=41歲,已患病5年>: B型肝炎參數:HBsAg 陽性 HBeAg 陰性 抗-HBs 陰性 抗-HBe 曝性 (請先閲讀背而之注意事項#填寫本頁) 裝. -SJ . 線. 經濟部中央櫺準局貝工消伢合作社印製 於0,1,2和5等月用Hepa-Care予以治療(肌肉内注射 )。HBs抗原與抗-HBs抗鼸的澜量值列於^XI和XIV之中 本紙Λ尺度逍用中a困家《毕(CNS) 規格(210X297公*) 81. 4. 10,000張(II) 匕〇9, 五、發明説明() 席列丟 A β It 6 經濟部中央標準局员工消费合作社印製 SEQ ID N0 : 1 SEQ類別: 核苷酸 序列長度: 558bp 鏈股性: 單股 形態: 線形 分子類別: 基因讎DNA 原始來海: HBV核心 直接實驗來源: PCR-攘大Hepa-Care was used for M treatment (intramuscular injection) at 0, 1, 6, and 7 months. The antigen and anti-crocodile antibody results are listed in 圏 Κ and X ° Patient 2 (Female, age = 48 years, has been sick for 12 years>: Hepatitis B parameters: HBsAg positive HBeAg negative anti-HBs negative anti- HBe anti-HBc anti-HBc is treated with Hepa-Care (intramuscular injection) at Ο, 1, and 6 months. Its antigen and anti-medium results are listed in gardens XI and XI. Patient 3 (female , Age = 41 years, has been ill for 5 years >: Hepatitis B parameters: HBsAg positive HBeAg negative anti-HBs negative anti-HBe exposure (please read back to the precautions # fill out this page) installed. -SJ . Line. Printed by Hepa-Care (intramuscular injection) for Hepa-Care printed in the Pei Gong Consumer Cooperative of the Central Bureau of Economics of the Ministry of Economic Affairs. Listed in ^ XI and XIV, the paper is Λ-scale, easy-to-use, and homey, "Bi (CNS) specifications (210X297 g *) 81. 4. 10,000 sheets (II) dagger 〇9, V. Description of invention () A β It 6 SEQ ID N0: 1 SEQ category: nucleotide sequence length: 558bp chain strand: single strand form: linear divide Category: DNA gene feud original to the sea: HBV core direct experimental Source: PCR- big throw into confusion
ATG GAC ATT GAC CCT TAT AAA GAA GGA GCT ACT GTG GAG TTA CTC TCG TTT TTG CCT TCT GAC TTC TTT CCT TCC GTA CGA GAT CTC CTA GAC ACC GCC TCA GCT CTG TAT CGA GAA GCC TTA GAG TCT CCT GAG CAT TGC TCA CCT CAC CAT ACT GCA CTC AGG CAA GCC ATT CTC TGC TGG GGG GAA TTG ATG ACT· CTA GCT ACC TGG GTG GGT AAT AAT TTG CAA GAT CCA GCA TCC AGA GAT CTA GTA GTC AAT TAT GTT AAT ACT AAC ATG GGT TTA AAG ATC AGG CAA CTA TTG TGG TTT CAT ATA TCT TGC CTT ACT TTT GGA AGA GAG ACT GTA CTT GAA TAT TTG GTC TCT TTC GGA GTG TGG ATT CGC ACT CCT CCA GCC TAT AGA CCA CCA AAT GCC CCT ATG TTA TCA ACA CTT CCG GAA ACT ACT GTT GTT AGA CGA CGG GAC CGA GGC AGG TCC CCT AGA AGA AGA ACT CCC TCG CCT CGC AGA CGT AGA TCT CAA . TCG CCG CGT CGC AGA AGA TCT CAA TCT CGG GAA TCT CAA TGT TAG (請先閲讀背而之注意事項#填寫本頁) 裝- 訂_ -線. 本紙張尺度边用中Β B家《準(CNS)甲4規格(210x297公釐) 81. 4. 10,000張(II) 9 ο 40 66 ΛΒ 五、發明説明() SEQ ID N0 : 2 SEQ類別: 核苷醴 序列長度: 504bp 鏈股性: 單股 形態: 線形 分子類別: 基因黼DNA 原始來源: HBV核心 直接實驗來源: PCR-擴大 (請先閱讀背而之注意事項#填窍本頁) 經濟部屮央標準局ts:工消费合作社印製ATG GAC ATT GAC CCT TAT AAA GAA GGA GCT ACT GTG GAG TTA CTC TCG TTT TTG CCT TCT GAC TTC TTT CCT TCC GTA CGA GAT CTC CTA GAC ACC GCC TCA GCT CTG TAT CGA GAA GCC TTA GAG TCT CCT GAG CAT TGC TCA CAT ACT GCA CTC AGG CAA GCC ATT CTC TGC TGG GGG GAA TTG ATG ACTCTA GCT ACC TGG GTG GGT AAT AAT TTG CAA GAT CCA GCA TCC AGA GAT CTA GTA GTC AAT TAT GTT AAT ACT AAC ATG GGT TTA AAG ATC AGG CAA TTG TGG TTT CAT ATA TCT TGC CTT ACT TTT GGA AGA GAG ACT GTA CTT GAA TAT TTG GTC TCT TTC GGA GTG TGG ATT CGC ACT CCT CCA GCC TAT AGA CCA CCA AAT GCC CCT ATG TTA TCA ACA CTT CCG GAA ACT ACT GTT CGA CGG GAC CGA GGC AGG TCC CCT AGA AGA AGA ACT CCC TCG CCT CGC AGA CGT AGA TCT CAA. TCG CCG CGT CGC AGA AGA TCT CAA TCT CGG GAA TCT CAA TGT TAG (please read the notes on the back # fill in this page ) Binding-Order _-Line. This paper is used in the standard BB family "Standard (CNS) A 4 specifications (210x297 mm) 81. 4. 10,000 sheets (II) 9 ο 40 66 ΛΒ V. Description of invention () SEQ ID N0: 2 SEQ category: nucleoside Sequence length: 504bp Stranded strands: Single strand form: Linear molecular category: Gene striated DNA Original source: HBV core direct experimental source: PCR-amplification (please read back to the precautions #fill this page) Ministry of Economic Affairs Standards Bureau ts: Printed by industrial and consumer cooperatives
ATG GAC ATT GAC CCT TAT !AAA GAA ^ »r* m . GGA GCT ACT C-TG GAG TTA CTC TC3 m m TTC- CCT TC? GAC \ T:C TTT CCT TCC GTA CGA GAT CTC CTA GAC ACC ..卜 TCA GCT CTG TAT CGA GAA GCC TTA GAG TCT CCT GAG CAT TGC TCA CC7 CAC CAT ACT GCA CTC AGG CAA GCC ATT CTC TGC GGG GAA mmQ ATG AC? CTA GCT ACC TGG GTG GGT AAT AAT TTG CAA GAT CCA GCA TCC AGA GAT CTA GTA GTC AAT TAT GTT .\AT ACT AAC ATG GGT TTA AAG ATC AGG CAA CTA TTG TGG TTT CAT ATA TCT TGC CTT ACT TTT GGA AGA GAG ACT GTA CTT GAA TAT TTG GTC TCT TTC GGA GTG TGG ATT CGC ACT CCT CCA GCC TAT AGA CCA CCA AAT GCC CCT ATG TTA TCA ACA CTT CCG GAA ACT ACT GTT GTT AGA CGA CGG GAC CGA GGC AGG TCC CCT AGA AGA AGA ACT ccc TCG CCT CGC AGA CGT 本紙Λ尺度边用中Η Η家標準(CNS)f 4規格(210x297公;¢) 81. 4. 10,000張⑻ .0¾ ,4b 五、發明説明() SEQ ID NO : 3 SEQ類別: 核苷酸 序列長度: 504bP 鍵股性: 單股 形態: 線形 分子類別: 基因驩DNA 原始來源: HBV核心 直接實驗來源: PCR-擴大 (請先WI讀背而之注意事項孙填寫本頁) 經濟部中央標準局貝工消费合作社印製ATG GAC ATT GAC CCT TAT! AAA GAA ^ »r * m. GGA GCT ACT C-TG GAG TTA CTC TC3 mm TTC- CCT TC? GAC \ T: C TTT CCT TCC GTA CGA GAT CTC CTA GAC ACC .. Bu TCA GCT CTG TAT CGA GAA GCC TTA GAG TCT CCT GAG CAT TGC TCA CC7 CAC CAT ACT GCA CTC AGG CAA GCC ATT CTC TGC GGG GAA mmQ ATG AC? CTA GCT ACC TGG GTG GGT AAT AAT TTG CAA GAT CCA GCA TCC AGA GAT CTA GTC AAT TAT GTT. \ AT ACT AAC ATG GGT TTA AAG ATC AGG CAA CTA TTG TGG TTT CAT ATA TCT TGC CTT ACT TTT GGA AGA GAG ACT GTA CTT GAA TAT TTG GTC TCT TTC GGA GTG TGG ATT CGC ACT CCT CCA GCC TAT AGA CCA CCA AAT GCC CCT ATG TTA TCA ACA CTT CCG GAA ACT ACT GTT GTT AGA CGA CGG GAC CGA GGC AGG TCC CCT AGA AGA AGA ACT ccc TCG CCT CGC AGA CGT Specification (210x297; ¢) 81. 4. 10,000 sheets ⑻ .0¾, 4b V. Description of the invention () SEQ ID NO: 3 SEQ category: nucleotide sequence length: 504bP bond strand: single strand form: linear molecular category : Gene Huan DNA Original source: HBV core direct experimental source : PCR-Expansion (please read WI's notes beforehand, Sun fills this page) Printed by Beigong Consumer Cooperative, Central Bureau of Standards, Ministry of Economic Affairs
ATG GAC ATT GAC CCT TAT AAA GAA ΤΤΊ* GGA GCi1 ACT gig GAG TTA CTC TCG -nmm TTG CCT TCT GAC TTC fTi m rrt CCT TCC GTA CGA GAT CTC CTA GAC ACC 广 r· TCA 广 <» m Λ. CTG TA? CGA GAA GCC TTA GAG CCT GAG CAT TGC TCA CCT CAC CAT AC? GCA CTC •-•Lrvj CAA C-CC ATT CTC TGC TGG GGG GAA TTG ATG AC? CTA C-CT ACC TGG GTG GGT AAT AAT TTG CAA GAT CCA GCA TCC AGA GAT CTA GTA GTC AAT TAT GTT AAT ACT AAC ATG GGT TTA AAG ATC AGG CAA CTA TTG TC-G TTT CAT ATA TCT TGC CTT ACT TTT GGA AGA GAG ACT GTA CTT GAA TAT TTG GTC TCT TTC GGA GTG TGG ATT CGC ACT CCT CCA GCC TAT AGA CCA CCA AAT GCC CCT ATG TTA TCA ACA CTT CCG GAA ACT ACT GTT GTT AGA CGA CGG GAC CGA GGC AGG TCC CCT AGA AGA AGA ACT CCC TCG CCT CGC- *AGA CGT r- 本紙»尺度遴用中因家標準(CNS)甲4規格(210x297公;Jt) 81. 4. 10,000» (Η) 裝. 訂- 線. 五、發明説明() Λ 6 Π 6 SEQ ID NO : 4 SEQ類別: 核苷酸 序列長度: 534bp 鍵股性: 單股 形態: 線形 分子類別: 基因讎DNA 原始來源: HBV核心 直接實驗來源: PCR-擴大 (諳先閲請背而之注意事項#填寫本頁) 經濟部中央標準局Μ工消#合作社印製ATG GAC ATT GAC CCT TAT AAA GAA ΤΤΊ * GGA GCi1 ACT gig GAG TTA CTC TCG -nmm TTG CCT TCT GAC TTC fTi m rrt CCT TCC GTA CGA GAT CTC CTA GAC ACC Guangr · TCA Guang < »m Λ. CTG TA ? CGA GAA GCC TTA GAG CCT GAG CAT TGC TCA CCT CAC CAT AC? GCA CTC •-• Lrvj CAA C-CC ATT CTC TGC TGG GGG GAA TTG ATG AC? CTA C-CT ACC TGG GTG GGT AAT AAT TTG CAA GAT CCA GCA TCC AGA GAT CTA GTA GTC AAT TAT GTT AAT ACT AAC ATG GGT TTA AAG ATC AGG CAA CTA TTG TC-G TTT CAT ATA TCT TGC CTT ACT TTT GGA AGA GAG ACT GTA CTT GAA TAT TTG GTC TCT TTC GGA GTG TGG ATTGC ACT CCT CCA GCC TAT AGA CCA CCA AAT GCC CCT ATG TTA TCA ACA CTT CCG GAA ACT ACT GTT GTT AGA CGA CGG GAC CGA GGC AGG TCC CCT AGA AGA AGA ACT CCC TCG CCT CGC- * AGA CGT r- Original paper »Standard selection Zhongyinjia Standard (CNS) Grade A 4 specifications (210x297 g; Jt) 81. 4. 10,000 »(Η) binding. Binding-line. 5. Description of invention () Λ 6 Π 6 SEQ ID NO: 4 SEQ category: nuclear Glycine sequence length: 534bp bond strand: single strand morphology: linear molecular category: gene clam DNA Original source: HBV core Direct experiment source: PCR-Amplification (Please read the back-to-back precautions #fill this page) Printed by the Central Standards Bureau of the Ministry of Economic Affairs Μ 工 消 # Cooperative
TCC AAC CTG TGC CX*!* GGG TGG CTT TGG GGC ATG GAC ATT GAC Γ r TAT AAA GAA TTT GGA GCT ACT GTG GAG ^TA CTC TCG m m 上丄 TTG CCT rp广” · GAC TTC r* »r· rr« CCT TCC GTA CGA GAT CTC CTA GAC ACC GCC TCA GCT CTG CGA GAA GCC TTA GAG TCT 广广-1 V·» V·# - GAG CAT TGC TCA CCT CAC CAT AC7 GCA 广 mr*· L泰 AGG CAA GCC ATT CTC TGC GGG GAA TTG ATG AC? CTA GCT ACC TGG GTG <t\ ‘s?V3 丄 AAT AAT TTG CAA GAT CCA GCA TCC AGA GAT CTA GTA GTC AAT TAT GTT AAT ACT AAC ATG GGT TTA AAG ATC AGG ZXk CTA TTG TGG TTT CAT ATA TCT TGC CTT rvC * TTT GGA AGA GAG ACT GTA CTT GAA TAT TTG GTC TCT TTC GGA GTG TGG ATT CGC ACT CCT CCA GCC TAT AGA CCA CCA AAT GCt CCT ATG TTA TCA ACA CTT CCG GAA ACT ACT GTT GTT AGA CGA CGG GAC CGA GGC AGG TCC CCT AGA AGA AGA ACT CCC TCG CCT CGC AGA CGT 本紙張尺度逍用中《Β家標準(CNS)T4規格(210x297公龙) 46 81. 4. 10,000張⑻TCC AAC CTG TGC CX *! * GGG TGG CTT TGG GGC ATG GAC ATT GAC Γ r TAT AAA GAA TTT GGA GCT ACT GTG GAG ^ TA CTC TCG mm 上 丄 TTG CCT rp wide ”GAC TTC r *» r · rr « CCT TCC GTA CGA GAT CTC CTA GAC ACC GCC TCA GCT CTG CGA GAA GCC TTA GAG TCT Guang Guang-1 V · »V · #-GAG CAT TGC TCA CCT CAC CAT AC7 GCA Guang mr * · L Thai AGG CAA GCC ATT CTC TGC GGG GAA TTG ATG AC? CTA GCT ACC TGG GTG < t \ 's? V3 丄 AAT AAT TTG CAA GAT CCA GCA TCC AGA GAT CTA GTA GTC AAT TAT GTT AAT ACT AAC ATG GGT TTA AAG ATC AGG ZXk CTA TTG TGG TTT CAT ATA TCT TGC CTT rvC * TTT GGA AGA GAG ACT GTA CTT GAA TAT TTG GTC TCT TTC GGA GTG TGG ATT CGC ACT CCT CCA GCC TAT AGA CCA CCA AAT GCt CCT ATG TTA TCA ACA CTT CCG GAA ACT ACT GTT GTT AGA CGG GAC CGA GGC AGG TCC CCT AGA AGA AGA ACT CCC TCG CCT CGC AGA CGT This paper standard is used in the "B Family Standard (CNS) T4 specification (210x297 male dragon) 46 81. 4. 10,000 sheets ⑻
SEQ ID NO : 5 SEQ類別: 核苷酸 序列長度: 534bp 鏈股性: 單股 形態: 線形 分子類別: 基因體DNA 原始來源: HBV核心 直接實驗來源: PCR-擴大 (請先閱讀背而之注意事項再填筠本頁) TCC AAC CTG TGC CTT GGG TGG CTT . TGG GGC ATG GAC ATT GAC CCT ΊΑ? AAA GAA GGA GCT ACT C-TG GAG TTA CTC TCG IT? TTG /- r· m TCT GAC TTC CCT TCC QTA CGA GA7 CTC CTA GAC ACC GCC TCA GCT CTG ΊΑ? CGA gaa GCC TTA GAG TCT 广 r· Vw Jr GAG CAT TGC TCA CCT CAC CAT ACT GCA CTC AGG CAA GCC ATT CTC TGC GGG GAA ATG ACT CTA GCT ACC TGG GTG r*r^*r* \S\S X AAT AAT TTG CAA GAT CCA GCA TCC AGA GAT CTA GTA GTC AAT TAT GTT AAT ACT AAC ATG GGT TTA AAG ATC AGG CAA CTA TTG TGG TTT CAT ATA TCT TGC CTT ACT TTT GGA AGA GAG ACT GTA QTT GAA TAT TTG GTC TCT TTC GGA GTG TGG ATT CGC ACT CCT CCA GCC TAT AGA CCA CCA AAT GCC CCT ATG TTA TCA ACA CTT CCG GAA ACT ACT GTT GTT AGA CGA CCG GAC CGA GGC AGG TCC CCT AGA AGA AGA ACT CCC TCG CCT CGC AGA CGT : 裝< 本紙Λ尺度逍用中a明家桴毕(CNS) T4規格(210x297公*) 81. 4. 10,000張(II) 五、發明説明( SEQ ID NO : 6 SEQ類別: 核苷酸 序列長度: 87bp 鐽股性: 單股 形態: 線形 分子類別: 基因體DNA 原始來源: HBV核心 直接實驗來源: 化學合成SEQ ID NO: 5 SEQ category: nucleotide sequence length: 534bp strand strand: single strand morphology: linear molecule category: genomic DNA original source: HBV core direct experimental source: PCR-amplification (please read back to the first note Please fill in this page again) TCC AAC CTG TGC CTT GGG TGG CTT. TGG GGC ATG GAC ATT GAC CCT ΊΑ? AAA GAA GGA GCT ACT C-TG GAG TTA CTC TCG IT? TTG /-r · m TCT GAC TTC CCT TCC QTA CGA GA7 CTC CTA GAC ACC GCC TCA GCT CTG ΊΑ? CGA gaa GCC TTA GAG TCT Guangr · Vw Jr GAG CAT TGC TCA CCT CAC CAT ACT GCA CTC AGG CAA GCC ATT CTC TGC GGG GAA ATG ACT CTA GCT ACC TGG GTG * r ^ * r * \ S \ SX AAT AAT TTG CAA GAT CCA GCA TCC AGA GAT CTA GTA GTC AAT TAT GTT AAT ACT AAC ATG GGT TTA AAG ATC AGG CAA CTA TTG TGG TTT CAT ATA TCT TGC CTT ACT TTT GGA AGA GAG ACT GTA QTT GAA TAT TTG GTC TCT TTC GGA GTG TGG ATT CGC ACT CCT CCA GCC TAT AGA CCA CCA AAT GCC CCT ATG TTA TCA ACA CTT CCG GAA ACT ACT GTT GTT AGA CGA CCG GAC CGA GGC AGG TCC CCT AGA AGA AGA ACT TCG CCT CGC AGA CGT: installed < In this paper, the standard size of the paper is used in the Ming Dynasty (CNS) T4 specification (210x297 g *) 81. 4. 10,000 sheets (II) V. Description of the invention (SEQ ID NO: 6 SEQ category: nucleotide sequence length : 87bp 鐽 Share: single strand form: linear molecular category: genomic DNA Original source: HBV core direct experimental source: chemical synthesis
ATC CTC TGC TGG GGG GAA TGG ACC TGG GTG GGC AAT AAT TTG TCT AGG GAC CTT GTA GTA AAT Λ 6 Η 6 (請先l«請背而之注意事項#填寫本頁) 經濟部中央捣準局员工消费合作社印製 SEQ ID NO : SEQ類別: 序列長度: 鏈股性: 形態: 分子類別: 原始來源: 直接實驗來源: 7 核苷酸 81bp 單股 線形 基因體DNA HBV核心 化學合成 GAC ATT GAC CCT TAT AAA GAA TTT GGA GCT ACT GTG GAG TTA CTC TCG TTT TTG CCT TCT GAC TTC TTT CCT TCC GTC AGG .---, 本紙》尺度逡用中國國家«準(CNS)甲4規格(2丨0x297公*)ATC CTC TGC TGG GGG GAA TGG ACC TGG GTG GGC AAT AAT TTG TCT AGG GAC CTT GTA GTA AAT Λ 6 Η 6 (please first «please note #fill in this page) Employee Consumer Cooperative of the Ministry of Economic Affairs Printed SEQ ID NO: SEQ category: Sequence length: Strand strand: Morphology: Molecule category: Original source: Direct experiment source: 7 nucleotides 81bp Single strand linear genomic DNA HBV core chemical synthesis GAC ATT GAC CCT TAT AAA GAA TTT GGA GCT ACT GTG GAG TTA CTC TCG TTT TTG CCT TCT GAC TTC TTT CCT TCC GTC AGG .---, this paper "standard" uses the Chinese National Standard `` quasi (CNS) A 4 specifications (2 丨 0x297 public *)
81. 4. 10,000» W ........^.....玎…-.線· ^〇9-4b 五、發明説明() Λ 6 Η6 SEQ ID NO : 8 SEQ類別: 核苷醴 序列長度: 114bp 鏈股性: 單股 形態: 線形 分子類別: 基因讎DNA 原始來源: HBV核心 直接實驗來源: 化學合成 (請先閲讀背而之注意事項洱填寫本頁)81. 4. 10,000 »W ........ ^ ..... 玎…-. 线 · ^ 〇9-4b V. Description of the invention () Λ 6 Η6 SEQ ID NO: 8 SEQ category: core Glycoside Sequence Length: 114bp Strand Strand: Single Strand Form: Linear Molecule Type: Gene Chong DNA Original Source: HBV Core Direct Experimental Source: Chemical Synthesis (please read the precautions before filling in this page)
TCC AAC CTG TGC CTT GGG TGG CTT TGG GGC ATG GAC ATT GAC CCT TAT ΆΑΑ GAA TTT GGA GCT ACT GTG GAG TTA CTC TCG TTT TTG CCT TCT GAC TTC TTT CCT TCC GTC AGG 81. 4. 10,〇〇〇張(H) 本紙》尺度逡用中Β β家楳準(CNS)T4規格(210x297公*) 經濟部中央櫺準局β工消费合作杜印製 SEQ ID NO : 9 SEQ類別: 核苷酸 序列長度: 90bp 鋪股性: 單股 形態: 線形 分子類別: 基因鼸DNA 原姶來源: HBV核心 直接實驗來源: 化學合成TCC AAC CTG TGC CTT GGG TGG CTT TGG GGC ATG GAC ATT GAC CCT TAT ΆΑΑ GAA TTT GGA GCT ACT GTG GAG TTA CTC TCG TTT TTG CCT TCT GAC TTC TTT CCT TCC GTC AGG 81. 4. 10, 〇〇〇 Zhang (H ) This paper is used in the standard β Beta Family Standard (CNS) T4 specification (210x297 g *). Printed by the Ministry of Economic Affairs, Central Bureau of Precision Industry, Beta-Consumer Cooperation SEQ ID NO: 9 SEQ Category: Nucleotide Sequence Length: 90bp Spreadability: Single strand morphology: Linear molecular type: Gene mule DNA Original source: HBV core direct experimental source: Chemical synthesis
GAT CTC CTA GAC ACC GCC TCA GCT CTG TAT CGA GAA GCC TTA GAG TCT CCT GAG CTA TGC TCA CCT CAC CAT ACT GCA CTC AGG CAA GGT 五、發明説明() Λ 6 η 6 經濟部屮央標準局员工消奸合作社印製 SEQ ID NO : 10 SEQ類別: 核苷酸 序列長度: 261bp 鐽股性: 單股 形態: 線形 分子類別: 基因鼸DNA 原始來源: HBV核心 直接實驗來源= 化學合成GAT CTC CTA GAC ACC GCC TCA GCT CTG TAT CGA GAA GCC TTA GAG TCT CCT GAG CTA TGC TCA CCT CAC CAT ACT GCA CTC AGG CAA GGT V. Description of invention () Λ 6 η 6 Employee rape co-operatives of the Central Standards Bureau of the Ministry of Economic Affairs Printed SEQ ID NO: 10 SEQ category: nucleotide sequence length: 261 bp strands: single-strand morphology: linear molecule category: gene ray DNA original source: HBV core direct experimental source = chemical synthesis
ATG GAC ATT GAC CCT TAT AAA GAA TTT GGA GCT ACT GTG GAG TTA CTC TCG TTT TTG CCT TCT GAC TTC TTT CCT TCC GTC AGG GAT CTC CTA GAC ACC GCC TCA GCT CTG TAT CGA GAA GCC TTA GAG. TCT CCT GAG CTA TGC TCA. CCT CAC CAT ACT GCA CTC AGG CAA GGT ATC CTC TGC TGG GGG GAA TGG ATG ACT CTA GCT ACC TGG GTG GGC AAT AAT TTG GAA GAT CCA GCA TCT AGG GAC CTT ( Gm GTA AAT (請先閲讀背而之注意事項Λ-填寫本I) -裝- 訂_ -線· 本紙张尺度边用中曲國家標準(CNS)>P4規格(210X297公;it) 81. 4. ΙΟ,ΟΟΟίϋ (II) 209 五、發明説明() SEQ ID NO : 11 SEU類別: 核苷酸 序列長度: 291bp 鏵股性: 單股 形態: 線形 分子類別: 基因體DNA 原始來源: HBV核心 直接實驗來源: 化學合成ATG GAC ATT GAC CCT TAT AAA GAA TTT GGA GCT ACT GTG GAG TTA CTC TCG TTT TTG CCT TCT GAC TTC TTT CCT TCC GTC AGG GAT CTC CTA GAC ACC GCC TCA GCT CTG TAT CGA GAA GCC TTA GAG. TCT CCT GAG CTA TGC . CCT CAC CAT ACT GCA CTC AGG CAA GGT ATC CTC TGC TGG GGG GAA TGG ATG ACT CTA GCT ACC TGG GTG GGC AAT AAT TTG GAA GAT CCA GCA TCT AGG GAC CTT (Gm GTA AAT (please read the precautions first -Fill in this I) -Installation- Order_ -Line · The standard of the paper used in the Chinese standard (CNS) > P4 specification (210X297; it) 81. 4. ΙΟ, ΟΟΟϑ (II) 209 V. Description of invention () SEQ ID NO: 11 SEU category: Nucleotide sequence length: 291bp 铧 strand property: single strand morphology: linear molecule category: genomic DNA Original source: HBV core direct experimental source: chemical synthesis
TCC AAC CTG TGC CTT GGG TGG CTT TGG GGC ATG GAC ATT GAC CCT TAT AAA GAA TTT GGA GCT ACT GTG GAG TTA CTC TCG TTT TTG CCT TCT GAC TTC TTT CCT TCC GTC AGG GAT CTC CTA GAC ACC GCC TCA GCT CTG TAT CGA GAA GCC TTA GAG TCT CCT GAG CTA TGC TCA CCT CAC CAT ACT GCA CTC AGG CAA GGT ATC CTC TGC TGG GGG GAA TGG ATG ACT CTA GCT ACC TGG GTG GGC AAT AAT TTG GAA GAT CCA GCA TCT AGG GAC · .· 6TT GTA GTA AAT (請先閲讀背而之注意事項再填筠本頁)TCC AAC CTG TGC CTT GGG TGG CTT TGG GGC ATG GAC ATT GAC CCT TAT AAA GAA TTT GGA GCT ACT GTG GAG TTA CTC TCG TTT TTG CCT TCT GAC TTC TTT CCT TCC GTC AGG GAT CTC CTA GAC ACC GCC TCA GCT CTG TAT GCC TTA GAG TCT CCT GAG CTA TGC TCA CCT CAC CAT ACT GCA CTC AGG CAA GGT ATC CTC TGC TGG GGG GAA TGG ATG ACT CTA GCT ACC TGG GTG GGC AAT AAT TTG GAA GAT CCA GCA TCT AGG GAC · 6TT GTA GTA AAT (Please read the precautions before filling in this page)
T 經潦部中央榣準庙员工消费合作社印製 本紙張尺度逍用中《國家楳準(CNS) Τ4規格(210x297公*) 81. 4. 10,000張(II) 209^^^五、發明説明() SEQ ID NO : 12 SEQ類別: 核苷酸 序列長度: 123bp 鏈股性: 單股 形態: 線形 分子類別: 基因體DNA 原始來源: HBV核心 直接實驗來源: 化學合成 Λ 6 Π 6 (請先閲讀背而之注意事項#填寫本頁)T printed by the Central Ministry of Education and Consumers Cooperatives of the Central Ministry of Labor and Welfare Cooperative Standards "National Standard (CNS) Τ4 Specification (210x297) *) 81. 4. 10,000 sheets (II) 209 ^^^ V. Description of invention () SEQ ID NO: 12 SEQ category: nucleotide sequence length: 123bp strand strand: single strand morphology: linear molecule category: genomic DNA original source: HBV core direct experimental source: chemical synthesis Λ 6 Π 6 (please first Read the notes to the contrary #fill this page)
ACC TGG GTG GGT AAT AAT TTG CAA GAT CCA GCA TCC AGA GAT CTA GTA GTC AAT TAT GTT AAT ACT AAC ATG- GGT TTA AAG ATC AGG CAA CTA •TTG TGG TTT CAT ΑΤΑ TCT TGC CTT ACT ’TTT 裝- 訂· 經濟部中央榣準局员工消费合作杜印製 SEQ ID NO : 13 SEQ類別: 核苷酸 序列長度: 138bP 鍵股性: 單股 形態: 線形 分子類別: 基因醭DNA 原始來源: HBV核心 直接實驗來源: 化學合成ACC TGG GTG GGT AAT AAT TTG CAA GAT CCA GCA TCC AGA GAT CTA GTA GTC AAT TAT GTT AAT ACT AAC ATG- GGT TTA AAG ATC AGG CAA CTA • TTG TGG TTT CAT ΑΤΑ TCT TGC CTT ACT 'TTT Binding-Order Economy Department DuPont printed by the Central Bureau of Consumer Affairs SEQ ID NO: 13 SEQ category: nucleotide sequence length: 138bP bond strand: single strand form: linear molecule category: gene cDNA DNA original source: HBV core direct experimental source: chemistry synthesis
GCA TCC AGA GAT CTA GTA GTC AAT TAT GTT AAT ACT AAC ATG GGT TTA AAG ATC AGG CAA' CTA TTG TGG TTT CAT ATA TCT TGC CTT ACT TTT GGA AGA GAG ACT GTA CTT GAA TAT TTG GTC TCT TTC GGA GTG TGG 本紙张尺度遑用中《困家標準(CNS) T4規格(2丨0X297公*) 4. 10,000張(II) -線- Λ 6 li 6 五、發明説明()GCA TCC AGA GAT CTA GTA GTC AAT TAT GTT AAT ACT AAC ATG GGT TTA AAG ATC AGG CAA 'CTA TTG TGG TTT CAT ATA TCT TGC CTT ACT TTT GGA AGA GAG ACT GTA CTT GAA TAT TTG GTC TCT TTC GGA GTG TGG To use the "Standard Standards (CNS) T4 Specification (2 丨 0X297) *) 4. 10,000 sheets (II)-line-Λ 6 li 6 5. Description of invention ()
SEQ ID N0 : 14 SEQ類別: 核苷酸 序列長度: 822bp 鏈股性: 單股 形態: 線形 分子類別: 基因醱DNA 原始來源: HBV S2 ayw/adw 直接實驗來源: HBV DNA (請先閲讀背而之注意事項#填寫本頁) 經濟部中央標準局貝工消#合作杜印製 ATG CAG TGG AAT TCC AGA ACC TTC CAC CAA Acr CTG CAA GAT CCC AGA GTG AGA GGC CTG TAT TTC nr'-y' W — C-CT C-GT GGC TCC AGT TCA GGA ACA GTA AAC CCT GTT CTG ACT ACT GCC TCT CCC TTA TCG TCA ATC TTC TCC- AGG ΑΤΑ GAG AAC ATC ACA TCA GGA TTC CTA GGA CCC CTT CTC G?G CAG GCG GGG TTT TTC TTG TTG ACA AGA ATC CTC Λ0Λ n丄A CCC- CAG AGT CTA GAC TCG 二 -GG AC? TCT C?C .-Lrv x TTT CTA GGnJ ACT ACC GT3 ?GT CTT GGC CAA AAT TC3 O.G TCC TCA ACC 7CC ϋ l, CAC TCA CCA ACC rn 广 pn ^ d. TGT CCA ACT TC-? CCT GGT TA? CGC TC-G ATG TC-T CTG CGG CGT ?T? ATC ATC TTC CTC TTC ATC CTG CTG CTA TC-C CTC ATC ΤΤΓ: ^TG GTT cxr CTG GAC ΤΛΤ CAA GGT ATG TTG CCC GTT TGT CCT CTA ATT CCA GGA TCC TCA ACA ACC AGC ACG GGA CCA TGC CGG ACC TGC ATG ACT ACT GCT CAA GGA ACC TC? ATG TAT CCC TCC TGT TC-C TGT ACC ΛΑΑ CCT TCG GAC C-GA AAT TGC ACC TGT ATT CCC ATC CCA TCA TCC TGG GCT TTC GGA ΛΑΑ TTC CTA TGG GAG TGG GCC TCA GCC CGT TTC TCC TGG CTC AGT TTA CTA GTG CCA T-T GTT CAG TGG TTC GTA GGG CTT TCC CCC ACT GTT TGG CTT TCA GTT ATA TGG ATG ATG TGG TAT TGG GGG CCA AGT CTG TAC AGC ATC TTG AGT CCC TTT TTA CCG CTG TTA CCA ATT TTC TTT TGT CTT TGG GTA TAC ATT r 本紙張尺度边用中a國家«準(CNS)T4規格(210X297公龙) 81. 4. 10,〇〇〇張(||) % Λ 6 It 6 五、發明説明() SEQ ID N0 : 15 SEQ類別: 核苷酸 序列長度: 99bp 鏈股性: 單股 形態: 線形 分子類別: 基因醱DNA 原始來源: HBV核心 直接實驗來源: 化學合成SEQ ID N0: 14 SEQ category: nucleotide sequence length: 822bp strand strand: single strand morphology: linear molecule category: gene DNA original source: HBV S2 ayw / adw direct experiment source: HBV DNA (please read the back first Note (fill in this page) Central Ministry of Economic Affairs Bei Gong Xiao # Cooperative Du Printed ATG CAG TGG AAT TCC AGA ACC TTC CAC CAA Acr CTG CAA GAT CCC AGA GTG AGA GGC CTG TAT TTC nr'-y 'W — C-CT C-GT GGC TCC AGT TCA GGA ACA GTA AAC CCT GTT CTG ACT ACT GCC TCT CCC TTA TCG TCA ATC TTC TCC- AGG ΑΤΑ GAG AAC ATC ACA TCA GGA TTC CTA GGA CCC CTT CTC G? G CAG GCG GGG TTT TTC TTG TTG ACA AGA ATC CTC Λ0Λ n 丄 A CCC- CAG AGT CTA GAC TCG II-GG AC? TCT C? C .-Lrv x TTT CTA GGnJ ACT ACC GT3? GT CTT GGC CAA AAT TC3 OG TCC TCA ACC 7CC ϋ l, CAC TCA CCA ACC rn wide pn ^ d. TGT CCA ACT TC-? CCT GGT TA? CGC TC-G ATG TC-T CTG CGG CGT? T? ATC ATC TTC CTC TTC ATC CTG CTG CTA TC-C CTC ATC ΤΓΓ: ^ TG GTT cxr CTG GAC ΤΛΤ CAA GGT ATG TTG CCC GTT TGT CCT CTA ATT CCA GGA TCC TCA ACA ACC AGC ACG GGA CCA TGC CGG ACC TGC ATG ACT ACT GCT CAA GGA ACC TC? ATG TAT CCC TCC TGT TC-C TGT ACC ΛΑΑ CCT TCG GAC C-GA AAT TGC ACC TGT ATT CCC ATC CCA TCA TCC TGG GCT TTC GGA ΛΑ TTC CTA TGG GAG TGG GCC TCA GCC CGT TTC TCC TGG CTC AGT TTA CTA GTG CCA TT GTT CAG TGG TTC GTA GGG CTT TCC CCC ACT GTT TGG CTT TCA GTT ATA TGG ATG ATG TGG TAT TGG GGG CCA AGT CTG TAC AGC ATC TTG CCC TTT TTA CCG CTG TTA CCA ATT TTC TTT TGT CTT TGG GTA TAC ATT r This paper scale is used in the national «quasi (CNS) T4 specification (210X297 male dragon) 81. 4. 10, 〇〇〇 sheets (|| )% Λ 6 It 6 V. Description of the invention () SEQ ID N0: 15 SEQ category: nucleotide sequence length: 99bp strand strands: single strand form: linear molecule category: gene DNA original source: HBV core direct experimental source : Chemical Synthesis
TAT GTT AAT ACT AAC ATG GGT TTA AAG ATC AGG CAA CTA TTG TGG TTT CAT •ATA TCT TGC CTT ACT TTT GGA AGA GAG ACT GTA CTT GAA TAT" \ttg GTC (請先閲讀背而之注意事項#填寫本頁) 裝· 線· 經濟部中央榀準局员工消费合作社印製 本紙張尺度逍用中a Η家標準(CNS) Ή規格(210x297公;¢) 81. 4. ]0,000張(Η) 五、發明説明() SEQ ID NO : 16 SEQ類別: 核苷酸 序列長度: 390bp 鐽股性: 單股 形態: 線形 分子類別: 基因體DNA 原始來源: HBV核心 直接實驗來源: PCR-擴大 (請先閲讀背而之注意事項再填寫本頁) 經濟部屮央標準局貝工消t合作社印製TAT GTT AAT ACT AAC ATG GGT TTA AAG ATC AGG CAA CTA TTG TGG TTT CAT • ATA TCT TGC CTT ACT TTT GGA AGA GAG ACT GTA CTT GAA TAT " \ ttg GTC (please read the notes on the back # fill in this page) Installation · Thread · Printed on the paper standard for easy use by the Consumer Cooperatives of the Central Bureau of Economy, Ministry of Economic Affairs a Η Family Standard (CNS) Ή Specification (210x297 g; ¢) 81. 4.] 0,000 sheets (Η) V. Description of invention () SEQ ID NO: 16 SEQ category: Nucleotide sequence length: 390bp 鐽 strand property: single strand morphology: linear molecular category: genomic DNA Original source: HBV core direct experimental source: PCR-amplification (please read the back side first Please pay attention to this matter and fill out this page) Printed by Beigongxiaot Cooperative Society, Bureau of Standards, Ministry of Economic Affairs
TCC AAC CTG TGC CTT GGG TGG CTT TGG GGC ATG GAC ATT GAC CCT TAT AAA GAA TTT GGA GCT ACT GTG GAG TTA CTC TCG TTT TTG CCT TCT GAC TTC TTT CCT TCC GTA CGA GAT CTC CTA GAC ACC GCC TCA GCT CTG TAT CGA GAA GCC TTA GAG TCT CCT GAG CAT TGC TCA CCT CAC CAT ACT GCA CTC AGG CAA GCC ATT CTC TGC TGG GGG GAA TTG ATG ACT CTA GCT ACC TGG GTG GGT AAT AAT TTG CAA GAT CCA GCA TCC AGA GAT CTA GTA GTC AAT TAT GTT AAT ACT AAC ATG GGT TTA AAG ATC AGG CAA CTA TTG TGG TTT CAT ATA TCT TGC CTT ACT TTT GGA AGA GAG ACT GTA CTT GAA" 'TAT TTG GTC 裝- 本紙張尺度边用中B國家«準(CNS) T4規格(210x297公*) 81. 4. 10,000張(11) 五、發明説明() SEQ ID NO : 17 SEQ類別: 核苷酸與對應的蛋白質 序列長度: 60bp 鐽股性: 單股 形態: 線形 分子類別: 基因齷DNA 原始來源: HBV SI ay 直接資驗來源: 化學合成 AAT CC? CTG GGA TTC TTT ccc GAT CAC CAG TTG GAT CCA GCC TTC AGA C-CA AAC ACC GCA Asn Pro Leu Gly ?he Phe Pro Asp His Gin Leu Asp Pro Ala ?he Arg Ala Asn Thr Ala V、.:.〆 (請先閱讀背而之注意事項洱填寫本頁) 裝- 訂- 線- 經濟部中央標準局KX工消费合作杜印製 本紙張尺度逍用中a國家樣準(CNS)T4規格(210X297公釐) 81. 4. 10,000^(1!) 五、發明説明 Λ (i Β 6 SEQ ID Ν0 : 18 SEQ類別: 核苷酸與對應的蛋白質 序列長度: 63bp 鐽股性: 單股 形態: 線形 分子類別: 基因體DNA 原始來源: HBV SI ay 直接實驗來源: 化學合成 (請先閲讀背而之注意事項再填寫本頁) CC7 TCC ACC AAT CAG TCA GGA Λ广— .-LVjvy CAG - CCZ ACC CCG 匕· θ CCA »·ρ Ff« AOS. AAC Pro Ala Ser Thr Asn Arg Gin Ser C-ly Arg Gin Pro Thr Pro He Ser Pro Pro Leu Asn * 81. 4. 10,000張(Η) 本紙張尺度边用中國國家標準(CNS)Τ4規格(210><297公址) 經濟部屮央櫺準局ΚΧ工消费合作社印製 Λ 6 Π 6 五、發明説明() SEQ ID NO : 19 SEQ類別: 核苷酸與對應的蛋白質 序列長度: 63bp 鍵股性: 單股 形態: 線形 分子類別: 基因體DNA 原始來源: HBV SI ay 直接資驗來源: 化學合成 (請先閲讀背而之注意事項#填寫本頁) GAT CCA GCC TTC AGA GCA ?ΛΟ ACC C-CA ΛΑΤ CCA GAT TGG GAC TTC CCC AAC AAG GAC Λ /"•r* AS? Pro Ala Phe Arg Ala Asn Thr Ala Asn Pro Asp Trp Asp Phe Asn Pro Asn Lys Asp Tiir 81. 4. 10,000張(Η) 本紙張尺度逍用中a國家樣準(CNS)T4規格(2丨0X297公;») 經濟部中央標準局员工消"合作社印製 五、發明説明() Λ 6 It 6 SEQ ID N0 : 20 SEQ類別: 核苷酸與對應的蛋白質 序列長度: 108bp 鐽股性: 單股 形態: 線形 分子類別: 基因驩DNA 原始來源: HBV SI ay 直接實驗來源: 化學合成 CCG CAC 广产' CTT TTG r· m « AC-C CCT CAG 广一 CAG ΑΤΑ CTA ΟΛΑ ACT TTG CCA GCA AAT rr·^ v〇 v*» CCT C-CC TCC ACC ^ > rn CC-C CAG TCA AGG * CAG CCT Pro His Gly Gly Leu Leu Gly .Trp Ser Pro Gin Ala Gin Gly lie Leu Glu Thr Leu Pro Ala Asn Pro Pro Pro Ala Ser Thr Asn Arg Gin Ser Gly Arg Gin Pro Λ " (請先閲讀背而之注意事項#填寫本頁) 裝· 線- 經濟部中央標準局员工消t合作杜印製 本紙張尺度边用中《8家標準(〇阳)肀4規格(210父297公;«:) 81.夂 10,000張(H) 五、發明説明() SEQ ID NO : 21 SEQ類別: 核苷酸 序列長度: 63bP 鏈股性: 單股 形態: 線形 分子類別: 基因體DNA 原姶來源: HBV SI ay 直接實驗來源: 化學合成 Λ 6η 6 (請先閱讀背而之注意事項#塡寫本頁)TCC AAC CTG TGC CTT GGG TGG CTT TGG GGC ATG GAC ATT GAC CCT TAT AAA GAA TTT GGA GCT ACT GTG GAG TTA CTC TCG TTT TTG CCT TCT GAC TTC TTT CCT TCC GTA CGA GAT CTC CTA GAC ACC GCC TCA GCT CTG TAT GCC TTA GAG TCT CCT GAG CAT TGC TCA CCT CAC CAT ACT GCA CTC AGG CAA GCC ATT CTC TGC TGG GGG GAA TTG ATG ACT CTA GCT ACC TGG GTG GGT AAT AAT TTG CAA GAT CCA GCA TCC AGA GAT CTA GTA GTC AAT TAT ACT AAC ATG GGT TTA AAG ATC AGG CAA CTA TTG TGG TTT CAT ATA TCT TGC CTT ACT TTT GGA AGA GAG ACT GTA CTT GAA " 'TAT TTG GTC Pack-This paper scale is used in the B country «quasi (CNS) T4 specifications ( 210x297 male *) 81. 4. 10,000 sheets (11) V. Description of the invention () SEQ ID NO: 17 SEQ category: nucleotide and corresponding protein sequence length: 60 bp stranding: single strand form: linear molecule category: Gene DNA Original source: HBV SI ay Direct source: Chemical synthesis AAT CC? CTG GGA TTC TTT ccc GAT CAC CAG TTG GAT CCA GCC TTC AGA C-CA AAC ACC GCA Asn Pro Leu Gly? He Phe Pro Asp His GinLeu Asp Pro Ala? He Arg Ala Asn Thr Ala V, ..: 〆 (please read the precautions and fill in this page first) Binding-Order-Line-KX industrial and consumer cooperation du printed version of the Central Standards Bureau of the Ministry of Economic Affairs Paper-scale Chinese National Standard (CNS) T4 specifications (210X297 mm) for free use 81. 4. 10,000 ^ (1!) V. Description of invention Λ (i Β 6 SEQ ID Ν0: 18 SEQ category: nucleotide and Corresponding protein sequence length: 63bp 鐽 strand property: single strand form: linear molecule category: genomic DNA original source: HBV SI ay direct experiment source: chemical synthesis (please read the precautions before filling this page) CC7 TCC ACC AAT CAG TCA GGA Λ Guang — .-LVjvy CAG-CCZ ACC CCG dagger · θ CCA »· ρ Ff« AOS. AAC Pro Ala Ser Thr Asn Arg Gin Ser C-ly Arg Gin Pro Thr Pro He Ser Pro Pro Leu Asn * 81. 4. The 10,000 sheets (Η) of this paper are printed in Chinese National Standard (CNS) Τ4 specifications (210 > < 297 public address). Printed by the Ministry of Economic Affairs, quasi-bureau of quasi-bureau ΚΧ 工 Consumer Cooperatives. Description of the invention () SEQ ID NO: 19 SEQ category: nucleotide and corresponding Protein sequence length: 63bp Bond strand: Single strand form: Linear molecule category: Genotype DNA Original source: HBV SI ay Direct source of verification: Chemical synthesis (please read back to the precautions # fill in this page) GAT CCA GCC TTC AGA GCA? ΛΟ ACC C-CA ΛΑΤ CCA GAT TGG GAC TTC CCC AAC AAG GAC Λ / " r * AS? Pro Ala Phe Arg Ala Asn Thr Ala Asn Pro Asp Trp Asp Phe Asn Pro Asn Lys Asp Tiir 81. 4. 10,000 sheets (Η) of this paper are used in China National Standards (CNS) T4 specification (2 丨 0X297); ») Printed by the employees of the Central Bureau of Standards of the Ministry of Economic Affairs & Cooperatives 5. Description of invention () Λ 6 It 6 SEQ ID N0: 20 SEQ category: Nucleotide and corresponding protein sequence length: 108bp Leng strand: single strand morphology: linear molecule category: Gene Huan DNA Original source: HBV SI ay Direct experiment source: Chemical synthesis CCG CAC Guangsan 'CTT TTG r · m «AC-C CCT CAG Guangyi CAG ΑΤΑ CTA ΟΛΑ ACT TTG CCA GCA AAT rr · ^ v〇v *» CCT C-CC TCC ACC ^ > rn CC-C CAG TCA AGG * CAG CCT Pro His Gly Gly Leu Leu Gly. Trp Ser Pro Gin Ala Gin Gly lie Leu Glu Thr Leu Pro Ala Asn Pro Pro Pro Ala Ser Thr Asn Arg Gin Ser Gly Arg Gin Pro Λ " (please read the notes on the back # fill in this page) Employees of the Central Bureau of Standards of the Ministry of Economic Affairs have cooperated with Du to print this paper. "8 Standards (〇 阳) 肀 4 Specifications (210 father 297 public;« :) 81. 夂 10,000 sheets (H) V. Description of invention () SEQ ID NO: 21 SEQ category: nucleotide sequence length: 63bP strand strand: single strand morphology: linear molecule category: genomic DNA source: HBV SI ay direct experiment source: chemical synthesis Λ 6η 6 (please (First read the back-end notes # 塡 write this page)
CCT GCC ACC AA? t"* ^ CAG TCA GGA AC-C- CAG CCT ACT CCC ATC TCT1 CCA CCT CTA C-AC - X 裝- 經濟部屮央標準局貝工消赀合作社印製 本紙張尺度逍用中國S家標準(CNS)T4規格(210X297公*) 81. 4. !0,000張(H) 209^43 經濟部中央榣準局貝工消费合作社印製 五、發明説明() SEQ ID NO : SEQ類別: 序列長度: 鐽股性: 形態: 分子類別: 原始來源: 直接實驗來源: 22 核苷酸與對應的蛋白質 108bp 單股 線形 基因體DNA HBV SI V^· 化學合成 (請先WI讀背而之注意事項#填寫本頁)CCT GCC ACC AA? T " * ^ CAG TCA GGA AC-C- CAG CCT ACT CCC ATC TCT1 CCA CCT CTA C-AC-X Pack-Printed by the Ministry of Economic Affairs, Bureau of Standards, Beigong Consumer Cooperative Cooperative, paper size China S Home Standards (CNS) T4 specification (210X297 g *) 81. 4.! 0,000 sheets (H) 209 ^ 43 Printed by the Ministry of Economic Affairs Central Bureau of Precision Industry Beigong Consumer Cooperative V. Description of invention () SEQ ID NO: SEQ Category: Sequence Length: Molecular Weight: Morphology: Molecular Category: Original Source: Direct Experimental Source: 22 nucleotides and corresponding protein 108 bp single strand linear genomic DNA HBV SI V ^ · Chemical Synthesis (please read WI first Matters needing attention # fill in this page)
cc.\ CAC C*GT CAG 广 ATA TTG CCT TCC ACCcc. \ CAC C * GT CAG Guang ATA TTG CCT TCC ACC
ATT C-GG TGG ACC ACA GTC- TCA AAT r* Lurvj CAG TCAATT C-GG TGG ACC ACA GTC- TCA AAT r * Lurvj CAG TCA
Pro His Gly Gly Ala Gin Gly lie Pro Pro Pro Ala Arg Gin ProPro His Gly Gly Ala Gin Gly lie Pro Pro Pro Ala Arg Gin Pro
He Leu Gly Trp Leu Thr Thr Val Ser Thr Asn Arg AGC CAG GCT ACA ATT 产 V» · CCT GGA •\ r-r* Λ\Σ\3 CAG r Ser Pro Gin Ser Thr lie Gin Ser GlyHe Leu Gly Trp Leu Thr Thr Val Ser Thr Asn Arg AGC CAG GCT ACA ATT production V »· CCT GGA • \ r-r * Λ \ Σ \ 3 CAG r Ser Pro Gin Ser Thr lie Gin Ser Gly
裝· 線· 本紙張尺度逍用中《«家標準^胳)<?4規格(2丨0父29‘/公;«:) 81. 4. 10,000» (II) Λ fi η 6 五、發明説明() SEQ ID NO : SEQ類別: 序列長度: 鏈股性: 形態: 分子類別: 原始來源: 直接實驗來源Installation · Line · This paper standard is used in "« Home Standard ^ "<? 4 specifications (2 丨 0 father 29 '/ male;« :) 81. 4. 10,000 »(II) Λ fi η 6 V. Description of the invention () SEQ ID NO: SEQ category: Sequence length: Chain strand: Morphology: Molecular category: Original source: Direct experimental source
CAG TGG AATCAG TGG AAT
23 核苷酸 60bp 單股 、 線形 基因體DNA HBV SI ay 化學合成 TCC AGA ACC TT.C CAC CAA ACT CTG (請先間讀背而之注意事項#堝寫本頁) 裝· 經濟部中央櫺準局貞工消t合作杜印製23 nucleotides 60bp single strand, linear genomic DNA HBV SI ay chemical synthesis TCC AGA ACC TT.C CAC CAA ACT CTG (please read back and note beforehand #pot write this page) Installation · Central Ministry of Economic Affairs Cooperate with Duzheng for printing
CAA GAT CCC AGA GTG AGA GGC C^G.***\TAT - X SEQ ID NO : 24 SEQ類別: 核苷酸 序列長度: 60bp 鍵股性: 單股 形態: 線形 分子類別: 基因體DNA 原始來源: HBV SI ay 直接實驗來源: 化學合成 GAT CCC AGA GTG GGT GGC TCC AGT AGA GGC CTG TAT TTC CCT TCA GGA ACA GTA AAC - X 線. 本紙張尺度边用中《Η家標準(CNS)T4規格(210X297公;«:) 81. 4. 10,000張(11) 2〇924〇 Λ 6 Η 6 五、發明説明() SEQ ID NO : 25 SEQ類別: 核苷酸 序列長度: 558bp 鏈股性: 單股 形態: 線形 分子類別: 基因讎DNA 原始來源: HBV核心adw 直接資驗來源: PCR-擴大 (請先閲讀背而之注意事項#填寫本頁) 經濟部中央檑準局貝工消t合作社印製CAA GAT CCC AGA GTG AGA GGC C ^ G. *** \ TAT-X SEQ ID NO: 24 SEQ category: nucleotide sequence length: 60bp bond strand: single strand morphology: linear molecule category: genomic DNA original source : HBV SI ay Direct experiment source: Chemical synthesis GAT CCC AGA GTG GGT GGC TCC AGT AGA GGC CTG TAT TTC CCT TCA GGA ACA GTA AAC-X-ray. This paper is used in the "H home standard (CNS) T4 specification (210X297 Public; «:) 81. 4. 10,000 (11) 2〇924〇Λ 6 Η 6 V. Description of the invention () SEQ ID NO: 25 SEQ category: nucleotide sequence length: 558bp strand strand: single strand form : Linear molecular category: Gene DNA Original source: HBV core adw Direct source of verification: PCR-Amplification (please read the back and the precautions # fill out this page) Printed by Beigongxiaot Cooperative of the Central Bureau of Economics of the Ministry of Economic Affairs
ATG GAC ATT GAC CCT TAT AAA GAA TTT GGA GCT ACT GTG GAG TTA CTC TCG TTT TTG CCT TCT GAC TTC TTT CCT TCC GTA CGA GAT CTC CTA GAC ACC GCC TCA GCT CTG TAT CGA GAA GCC TTA GAG TCT CCT: GAG CAT TGC TCA CCT CAC CAT ACT GCA CTC AGG CAA GCC ATT CTC TGC TGG GGG GAA TTG ATG ACT CTA GCT ACC TGG GTG GGT AAT AAT TTG CAA GAT CCA GCA TCC AGA GAT CTA GTA GTC AAT TAT GTT AAT ACT AAC ATG GGT TTA AAG ATC AGG CAA CTA TTG TGG TTT CAT ATA TCT TGC CTT ACT TTT GGA AGA GAG ACT GTA CTT GAA TAT TTG GTC TCT TTC GGA GTG TGG ATT CGC ACT CCT CCA GCC TAT AGA CCA CCA AAT GCC CCT ATG TTA TCA ACA CTT CCG GAA ACT ACT GTT GTT AGA CGA CGG GAC CGA GGC AGG TCC CCT AGA AGA AGA ACT CCC TCG CCT CGC AGA CGT AGA TCT CAA TCG CCG CGT CGC AGA AGA TCT CAA TCT CGG . GAA TCT CAA TGT TAG 裝· 線. 本紙》尺度遑用中《國家«準(CNS) T4規格(210X297公;Jt) 81. 4. 10,000張(Η) 〇〇924〇 Λ 6 __Π6 五、發明説明() SEQ ID NO : 26 SEQ類別: 核苷酸 序列長度: 678bp 鍵股性: 單股 形態: 線形ATG GAC ATT GAC CCT TAT AAA GAA TTT GGA GCT ACT GTG GAG TTA CTC TCG TTT TTG CCT TCT GAC TTC TTT CCT TCC GTA CGA GAT CTC CTA GAC ACC GCC TCA GCT CTG TAT CGA GAA GCC TTA GAG TCT CCT: GAG CAT TGC CCT CAC CAT ACT GCA CTC AGG CAA GCC ATT CTC TGC TGG GGG GAA TTG ATG ACT CTA GCT ACC TGG GTG GGT AAT AAT TTG CAA GAT CCA GCA TCC AGA GAT CTA GTA GTC AAT TAT GTT AAT ACT AAC ATG GGT TTA AAG ATCGG CTA TTG TGG TTT CAT ATA TCT TGC CTT ACT TTT GGA AGA GAG ACT GTA CTT GAA TAT TTG GTC TCT TTC GGA GTG TGG ATT CGC ACT CCT CCA GCC TAT AGA CCA CCA AAT GCC CCT ATG TTA TCA ACA CTT CCG GAA ACT ACT GTT AGA CGA CGG GAC CGA GGC AGG TCC CCT AGA AGA AGA ACT CCC TCG CCT CGC AGA CGT AGA TCT CAA TCG CCG CGT CGC AGA AGA TCT CAA TCT CGG. GAA TCT CAA TGT TAG installation and line. This paper is used in the "national" «Standard (CNS) T4 specifications (210X297; Jt) 81. 4. 10,000 sheets (Η) 〇〇924〇Λ 6 __Π6 V. Description of the invention () SEQ ID NO: 26 SEQ category: nucleotide sequence length: 678bp Key strands: single strand : Linear
分子類別: 基因鼸DNA 原姶來源: HBV S adw/aywMolecule category: Gene Molecule DNA Original source: HBV S adw / ayw
直接實驗來源:HBV DNA (請先閲讀背而之注意事項#填窍本頁) 經濟部屮央楳準局员工消1V-合作社印製Direct experiment source: HBV DNA (please read the back-to-back notes # fill in this page) Employee Consumers 1V-Co-op Print
ΑΤΑ GAG AAC ATC ACA TCA GGA TTC CTA GGA CCC CTT CTC GTG TTA CAG GCG GGG TTT TTC TTG TTG ACA AGA ATC CTC ACA ATA CCG CAG AGT CTA GAC TCG TGG TGG ACT TCT CTC ΑΑΤ TTT CTA GGG GGA ACT ACC GTG TGT CTT GGC CAA AAT TCG CAG tCc TCA ACC TCC AAT CAC TCA CCA ACC TCT TGT CCT CCA ACT TGT CCT GGT TAT CGC TGG ATG TGT CTG CGG CGT TTT ATC ATC TTC CTC TTC ATC CTG CTG CTA TGC CTC ATC TTC TTG TTG GTT CTT CTG GAC TAT CAA GGT ATG TTG CCC GTT TGT CCT CTA ATT CCA GGA TCC TCA ACA ACC AGC ACG GGA CCA TGC CGG ACC TGC ATG ACT ACT GCT CAA GGA ACC TCT ATG TAT CCC TCC TGT TGC TGT ACC AAA CCT TCG GAC •GGA AAT TGC ACC TGT ATT CCC ATC CCA TCA TCC TGG GCT TTC GGA AAA TTC CTA TGG GAG TGG GCC TCA GCC CGT TTC TCC TGG CTC AGT TTA CTA GTG CCA TTT GTT CAG TGG TTC GTA GGG CTT TCC CCC ACT GTT TGG CTT TCA GTT ATA TGG ATG ATG TGG TAT TGG * GGG CCA AGT CTG TAC AGC ATC TTG AGT CCC TTT TTA CCG CTG TTA CCA ATT TTC TTT TGT CTT TGG GTA TAC ATT 裝· 訂..j ·線· 本紙張尺度边用中《Β家«準(CNS)T4規格(210X297公;¢) 81. 4. 10,000張(H) -ο,'^ 920 66 ΛΠ 五、發明説明()ΑΤΑ GAG AAC ATC ACA TCA GGA TTC CTA GGA CCC CTT CTC GTG TTA CAG GCG GGG TTT TTC TTG TTG ACA AGA ATC CTC ACA ATA CCG CAG AGT CTA GAC TCG TGG TGG ACT TCT CTC ΑΑΤ TTT CTA GGG GGA ACT ACC GT CAA AAT TCG CAG tCc TCA ACC TCC AAT CAC TCA CCA ACC TCT TGT CCT CCA ACT TGT CCT GGT TAT CGC TGG ATG TGT CTG CGG CGT TTT ATC ATC TTC CTC TTC ATC CTG CTG CTA TGC CTC ATC TTC TTG TTG GTT CTT CTG CAA GGT ATG TTG CCC GTT TGT CCT CTA ATT CCA GGA TCC TCA ACA ACC AGC ACG GGA CCA TGC CGG ACC TGC ATG ACT ACT GCT CAA GGA ACC TCT ATG TAT CCC TCC TGT TGC TGT ACC AAA CCT TCG GAC • GGA AAT TGC ACC ATT CCC ATC CCA TCA TCC TGG GCT TTC GGA AAA TTC CTA TGG GAG TGG GCC TCA GCC CGT TTC TCC TGG CTC AGT TTA CTA GTG CCA TTT GTT CAG TGG TTC GTA GGG CTT TCC CCC ACT GTT TGG CTT TCA GTT TATA TGG ATG ATGTG TAT TGG * GGG CCA AGT CTG TAC AGC ATC TTG AGT CCC TTT TTA CCG CTG TTA CCA ATT TTC TTT TGT CTT TGG GTA TAC ATT Binding · Ordering · j · Line · This paper scale is used in the "B home" standard (CNS ) T4 regulation Grid (210X297; ¢) 81. 4. 10,000 sheets (H)-ο, '^ 920 66 ΛΠ V. Description of invention ()
SEQ ID NO : 27 SEQ類別: 核苷酸 序列長度: 585bp 鏈股性: 單股 形態: 線形 分子類別: 基因膿DNA 原始來源: HBV S adw/ayw 直接實驗來源: HBV DNA (請先閲讀背而之注意事項#填寫本頁) 經濟部中央楳準/¾员工消费合作杜印製SEQ ID NO: 27 SEQ category: nucleotide sequence length: 585bp strand strand: single strand morphology: linear molecule category: gene pus DNA original source: HBV S adw / ayw direct experiment source: HBV DNA (please read the back first Zhi Note #fill this page) Central Ministry of Economic Affairs / ¾ Employee Consumption Cooperation Du Print
CTA C-AC TCG TC-G TGG ACT TCT CTC AAT pt< pn m • 上 CTA GGG GGA TCT CCC GTG m/*·· »71 CTT 广 CAA ΑΛΤ TCG CAG TCC CCA ACC TCC ΛΑΤ CAC TCA CCA ACC TCC TGT 广 w w — 〇 v—Λ ATT ?GT CCT 产产 1 TAT cc-c m ^ i. \Z\2 ATG m ^ CTG CGG ΤΤΊ* ATC ATA TTC CTC TTC ATC CTG CTG CTA TGC CTC ATC TTC TTA TTG GTT CTT CTG GAT TAT CAA GGT ATG TTG CCC .GTT TGT CCT CTA ATT CCA GGA TCA ACA ACA ACC AGT ACG GGA CCA TGC AAA ACC TGC ACG AC? CCT GCT CAA C-GC AAC TCT ATG TTT CCC TCA TGT TGC TGT ACA AAA CCT ACG GAT GGA AAT TGC ACC TGT ATT CCC ATC CCA TCG TCC TGG GCT TTC GCA AAA TAC CTA TGG GAG TGG GCC TCA GTC CGT TTC TCT TGG CTC AGT TTA CTA GTG CCA TTT GTT CAG TGG TTC GTA GGG CTT TCC ccc ACT GTT TGG CTT TCA GCT ATA TGG ATG ATG TGG TAT TGG GGG CCA AGT CTG TAC AGC ATC GTG AGT CCC TTT ATA CCG CTG TTA CCA ATT ' TTC TTT TGT CTC TGG GTA TAG ATT ,> 裝. 本紙張尺度逍用中a B家«毕(CHS) Ή規格(210X297公*) 81. 4. 10,000張(Η) 〇〇9^b 經濟部中央標準局KX工消费合作社印製 五、發明説明() SEQ ID NO : SEQ類別: 序列長度: 鏈股性: 形態: 分子類別: 原始來源: 直接實驗來源 28 核苷酸 106bp 單股 線形 基因鼸DNA HBV S1 化學合成 3 ·-GAT-CTT-TAA-CAT-GGA-GAA-CAA-TCC-TCT-G C-G-A77-C7T-TCC-CGA-TCA-CCA-3TT-GGA-TCC-A GC-CTT-CAG-AGC-AAA-CAC-CSC-AAA-TCC-AGA-T TS-GGA-CTT-CAA-TCC-CAG-(7)-3' SEQ ID NO : SEQ類別: 序列長度: 鏈股性: 形態: 分子類別: 原始來源: 直接實驗來源: 29 核苷酸 115bp 單股 線形 基因鼸DNA HBV S 化學合成 5'-AAT-TCT-A6A-CTC-GA6-TCT-EAA-CA7-AGA-G AA-CAT—CAC—AT C-AGG-ATT —CCT-AGG—ACC—CCT —T CT-CGT—GTT—ACA~GGC~5*3G-GTT—TTT—CTT-GTTt^ AC-AAG-AAT-CCT-CAC-AATthCC-GCA-GAG- (OVV· 本紙》尺度逍用中《國家樣準(CNS)T4規格(210X297公:«:) 81. 4, 10,000張(II) (請先閲讀背而之注意事項再填寫本頁) 五、發明説明( SEQ ID NO : SEQ類別: 序列長度: 鏈股性: 形態: 分子類別: 原姶來源: 30 核苷酸 108bp 單股 線形 基因醱DNA HBV核心 Λ 6 B 6 直接實驗來源:化學合成 3,一GAT-CTT-TTA-AAG-SGA^T匚C-TCT-(2CT-.:5SS-.3 gG-ί二.A 丁一 5SA_TGA-C:TC_ 丁 AG-CTA-CCT-r.SG-TGG-G CA_A.TA_ATT_TGG—AAG-ATC_C:AG_CAT_C:TA_33{B—A C匚一TTG—TAS-TAA—ATC-TAG—AC- : A) -3,〆〆… (請先閲讀背而之注意事項#填寫本頁) 經濟部中央標準局员工消费合作社印製 SEQ ID NO : SEQ類別: 序列長度: 鏈股性: 形態: 分子類別: 原姶來源: 直接實驗來源CTA C-AC TCG TC-G TGG ACT TCT CTC AAT pt < pn m • Up CTA GGG GGA TCT CCC GTG m / * ·· 71 CTT Guang CAA ΑΛΤ TCG CAG TCC CCA ACC TCC ΛΑΤ CAC TCA CCA ACC TCC TGT Guang ww — 〇v—Λ ATT? GT CCT production 1 TAT cc-c m ^ i. \ Z \ 2 ATG m ^ CTG CGG ΤΤΊ * ATC ATA TTC CTC TTC ATC CTG CTG CTA TGC CTC ATC TTC TTA TTG GTT CTT CTG GAT TAT CAA GGT ATG TTG CCC .GTT TGT CCT CTA ATT CCA GGA TCA ACA ACA ACC AGT ACG GGA CCA TGC AAA ACC TGC ACG AC? CCT GCT CAA C-GC AAC TCT ATG TTT CCC TCA TGT TGC TGT ACA AAA CCT ACG GAT GGA AAT TGC ACC TGT ATT CCC ATC CCA TCG TCC TGG GCT TTC GCA AAA TAC CTA TGG GAG TGG GCC TCA GTC CGT TTC TCT TGG CTC AGT TTA CTA GTG CCA TTT GTT CAG TGG TTC GTA GGG CTT TCC ccc ACT GTT TGG CTT ATA TGG ATG ATG TGG TAT TGG GGG CCA AGT CTG TAC AGC ATC GTG AGT CCC TTT ATA CCG CTG TTA CCA ATT 'TTC TTT TGT CTC TGG GTA TAG ATT, > Pack. This paper scale is used in a B home «Bi ( CHS) Ή specifications (210X297 g *) 81. 4. 10,000 sheets (Η) 〇〇9 ^ b Ministry of Economic Affairs Central Standards Bureau KX Engineering Printed by Fei Cooperative Society 5. Description of the invention () SEQ ID NO: SEQ category: Sequence length: Chain strand: Morphology: Molecular category: Original source: Direct experiment source 28 nucleotides 106bp Single strand linear gene mule DNA HBV S1 Chemical synthesis 3 · -GAT-CTT-TAA-CAT-GGA-GAA-CAA-TCC-TCT-G CG-A77-C7T-TCC-CGA-TCA-CCA-3TT-GGA-TCC-A GC-CTT-CAG-AGC -AAA-CAC-CSC-AAA-TCC-AGA-T TS-GGA-CTT-CAA-TCC-CAG- (7) -3 'SEQ ID NO: SEQ category: Sequence length: Chain strand: Morphology: Molecular category : Original source: Direct experiment source: 29 nucleotide 115bp single stranded linear gene mule DNA HBV S Chemical synthesis 5'-AAT-TCT-A6A-CTC-GA6-TCT-EAA-CA7-AGA-G AA-CAT-CAC —AT C-AGG-ATT —CCT-AGG—ACC—CCT —T CT-CGT—GTT—ACA ~ GGC ~ 5 * 3G-GTT—TTT—CTT-GTTt ^ AC-AAG-AAT-CCT-CAC-AATthCC -GCA-GAG- (OVV · This paper) Standard "National Sample Standard (CNS) T4 Specification (210X297):« :) 81. 4, 10,000 sheets (II) (Please read the precautions before filling in (This page) V. Description of the invention (SEQ ID NO: SEQ category: Sequence length: Chain strands: Form: points Subcategory: Original source: 30 nucleotides 108 bp single-stranded linear gene DNA HBV core Λ 6 B 6 Direct experimental source: chemical synthesis 3, a GAT-CTT-TTA-AAG-SGA ^ T 匚 C-TCT- ( 2CT- .: 5SS-.3 gG-ί 二 .A Ding Yi 5SA_TGA-C: TC_ Ding AG-CTA-CCT-r.SG-TGG-G CA_A.TA_ATT_TGG—AAG-ATC_C: AG_CAT_C: TA_33 {B-AC匚 一 TTG—TAS-TAA—ATC-TAG—AC-: A) -3, 〆〆… (please read the back-up notes first # fill in this page) SEQ ID NO is printed by the employee consumer cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs : SEQ category: Sequence length: Stranding: Morphology: Molecular category: Original source: Direct experimental source
31 核苷酸 106bp 單股 線形 基因體DNA HBV核心 化學合成 5 · —丁""C"TC_C36——丁TC一GT(3—{3GG — CAT_,Z'GA—二 AT-丁 GA—CC.C-TTA—丁 A.A—AGA — ATT — TSG—AGC:— 丁 AC — T m « ·— —· · ·-* ~. Η —^ I —HU. ί 一L·- ; —.·Γ 丨! 一 ♦ · ί 一二U.u一 I ί。一 I 一U/ ;2: 本紙張尺度逍用中困Η家楳準(CNS)甲4規格(210X297公;Ϊ) 81. 4.】0,000張⑻31 nucleotide 106bp single stranded linear genomic DNA HBV core chemical synthesis 5-Ding " " C " TC_C36-Ding TC-GT (3-{3GG-CAT_, Z'GA-two AT-Ding GA- CC.C-TTA—Ding AA—AGA — ATT — TSG—AGC: — Ding AC — T m «· — — · · ·-* ~. Η — ^ I —HU. Ί 一 L ·-; —. · Γ 丨! One ◆ · Two Uu-I I. One I-U /; 2: This paper scale is easy to use and difficult to use 掳 楳 樳 (CNS) A 4 specifications (210X297 public; Ϊ) 81. 4.] 0,000 sheets⑻
It 6 五、發明説明() 經濟部中央標準局Μ工消伢合作杜印製 SEQ ID NO : 32 SEQ類別: 核苷酸 序列長度: 89bp 鏈股性: 單股 形態: 線形 分子類別: 基因鼸DNA 原始來源: HBV核心 直接實驗來源: 化學合成 5 ' —SAT-C丁C一CTA—GAC-ACC-GCC—TCA—SCT-GTS — T AT-C5A-GAA-,3CC — 了丁 A_GAG—丁 !1了_*二[:丁 一3AG-CA 丁一 T _5C^TCA-C 匚了 一CAC-匚 AT-ACT-0CA-·二 TC-AGS-CAA—3 -(G)-3 * SEQ ID N0 : 33 .) SEQ類別: 核苷酸 序列長度: 25bp 鏈股性: 單股 形態: 線形 分子類別: 基因鼸DNA 原始來源: HBV核心 直接實驗來源: 化學合成 j ' -TT-3—SAT-CCT-CCA-ACC-TST—Ξ·2Ζ-Τ~·- 本紙張尺度遑用中Bffl家標準(CNS)T4規格(210X297公;¢) 81. 4. 10,000¾ (II) (請先閲讀背而之注意事項#填寫本頁) 五、發明説明() SEQ ID NO : SE(i類別: 序列長度: 鍵股性: 形態: 分子類別: 原始來源: 直接實驗來源: 34 核苷酸 25bp 單股 線形 基因體DNA HBV核心 化學合成 (請先閲讀背而之注意事項再填寫本頁) 裝· 訂_ 線- 經济部中央標準局貝工消伢合作杜印製 本紙張尺度逍用中國Η家«準(CNS) «Ρ 4規格(210X297公;《:) 81. 4. 10,000張(|.i) η 6 五、發明説明() SEQ ID NO : 35 SEQ類別: 核苷酸 序列長度: 588bP 鐽股性: 單股 形態: 線形 分子類別: 基因鎌DNA 原始來源: HBV核心 直接實驗來源: PCR-鑛大 (請先閲讀背而之注意事項#填寫本页) 經濟部中央標準局β工消费合作社印製It 6 V. Description of the invention () Co-printed by the Ministry of Economic Affairs, Central Bureau of Standards, Migong Consumers SEQ ID NO: 32 SEQ Category: Nucleotide Sequence Length: 89bp Strand Strand: Single Strand Form: Linear Molecule Class: Gene Mole Original DNA source: HBV core direct experimental source: Chemical synthesis 5 '—SAT-C D C-CTA—GAC-ACC-GCC—TCA—SCT-GTS—T AT-C5A-GAA-, 3CC—Ding A_GAG—D ! 1 了 _ * 二 [: 丁 一 3AG-CA 丁 一 T _5C ^ TCA-C 匚 一 CAC- 匚 AT-ACT-0CA- · 二 二 TC-AGS-CAA—3-(G) -3 * SEQ ID N0: 33.) SEQ category: nucleotide sequence length: 25bp strand strand: single strand morphology: linear molecule category: gene mule DNA original source: HBV core direct experimental source: chemical synthesis j'-TT-3—SAT -CCT-CCA-ACC-TST—Ξ · 2Z-Τ ~ ·-This paper size is used in the Bffl family standard (CNS) T4 specification (210X297; ¢) 81. 4. 10,000¾ (II) (Please read first Contrary note #fill in this page) 5. Description of the invention () SEQ ID NO: SE (i category: sequence length: bond strand: shape: molecular category: original source: straight Access to the experiment source: 34 nucleotides 25bp single strand linear genomic DNA HBV core chemical synthesis (please read the precautions before filling this page) Pack · Order _ Line-Central Ministry of Economic Affairs Bureau of Industrial Engineering Cooperative The size of the paper used for printing is easy to use. The Chinese ďzhun (CNS) «P 4 specifications (210X297 ;; :) 81. 4. 10,000 sheets (| .i) η 6 V. Description of invention () SEQ ID NO: 35 SEQ category: Nucleotide sequence length: 588bP Leng strand: single strand form: linear molecular category: gene sickle DNA original source: HBV core direct experiment source: PCR-Mining University (please read the back to the precautions # fill in this Page) Printed by the β-Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs
TCC AAC CTG TGC CTT GGG TGG CTT TGG GGC ATG GAC ATT GAC CCT TAT AAA GAA TTT GGA GCT ACT GTG GAG TTA CTC TCG TTT TTG CCT TCT GAC TTC TTT CCT TCC GTA CGA GAT CTC CTA GAC ACC GCC TCA GCT CTG ΤΑΓ CGA GAA GCC TTA GAG TCT CCT GAG CAT TGC TCA CCT CAC CAT ACT GCA CTC AGG CAA GCC ATT CTC TGC TGG GGG GAA TTG ATG ACT CTA GCT ACC TGG GTG GGT AAT AAT TTG CAA GAT CCA GCA TCC AGA GAT CTA GTA GTC AAT TAT GTT AAT ACT AAC ATG GGT TTA AAG ATC AGG CAA CTA TTG TGG TTT CAT ATA TCT TGC CTT ACT TTT GGA AGA GAG ACT GTA CTT GAA TAT TTG GTC TCT TTC GGA GTG TGG ATT CGC ACT CCT CCA GCC TAT AGA CCA CCA AAT GCC CCT ATG TTA TCA ACA CTT CCG GAA ACT ACT GTT GTT AGA CGA CGG GAC CGA GGC AGG TCC CCT AGA AGA AGA ACT CCC TCG CCT CGC AGA CGT AGA TCT CAA TCG CCG CGT CGC AGA AGA TCT CAA TCT CGG GAA TCT CAA TGT TAG 本紙张尺度逡用中a國家標毕(CNS)〒4規格(2丨0X297公:- 70 - 81. 4. 10,000» (Η)TCC AAC CTG TGC CTT GGG TGG CTT TGG GGC ATG GAC ATT GAC CCT TAT AAA GAA TTT GGA GCT ACT GTG GAG TTA CTC TCG TTT TTG CCT TCT GAC TTC TTT CCT TCC GTA CGA GAT CTC CTA GAC ACC GCC TCA GCT CTG ATA GCC TTA GAG TCT CCT GAG CAT TGC TCA CCT CAC CAT ACT GCA CTC AGG CAA GCC ATT CTC TGC TGG GGG GAA TTG ATG ACT CTA GCT ACC TGG GTG GGT AAT AAT TTG CAA GAT CCA GCA TCC AGA GAT CTA GTA GTC AAT TAT ACT AAC ATG GGT TTA AAG ATC AGG CAA CTA TTG TGG TTT CAT ATA TCT TGC CTT ACT TTT GGA AGA GAG ACT GTA CTT GAA TAT TTG GTC TCT TTC GGA GTG TGG ATT CGC ACT CCT CCA GCC TAT AGA CCA CCA AAT GCC CCT ATC TCA ACA CTT CCG GAA ACT ACT GTT GTT AGA CGA CGG GAC CGA GGC AGG TCC CCT AGA AGA AGA ACT CCC TCG CCT CGC AGA CGT AGA TCT CAA TCG CCG CGT CGC AGA AGA TCT CAA TCT CGG GAA TCT CAA TGT TAG Used in the national standard (CNS) 〒4 specifications (2 丨 0X297 male:-70-81. 4. 10,000 »(Η)
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