TWI334438B - Optimized expression of hpv 31 l1 in yeast - Google Patents

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1334438 九、發明說明： 【發明所屬之技術領域】 本發明通常係關於人類乳突狀瘤病毒（HP V)之治療。更詳 言之，本發明係關於編碼HP V3 1 L1蛋白質之合成聚核苷 酸、及包含該等聚核苷酸之重組載體.及宿主。本發明亦係 關於HPV3 1似病毒粒子（VLP)及其在用於預防及治療HPV 之疫苗及醫藥組合物中的用途。 【先前技術】 存在超過80種之人類乳突狀瘤病毒（HPV)，其中許多種已 與自良性增生性瘤至惡性癌瘤之廣泛多種生物表型（關於 評述參見McMurray等人，Int. J. Exp. Pathol. 82(1): 15-33 (2001))相關聯。1^¥6及1^¥11為與良性瘤、非惡性尖銳濕 疣及/或生殖器黏膜或呼吸黏膜之低度發育異常最一般性 關聯的類型。HPV16及HPV18為與子宮頸、***、***及 肛道之原位及侵犯性癌最頻繁關聯的高危險性類型。超過 90%之宮頸癌與HPV16、HPV18或不太流行之致癌類型 HPV31、HPV33、HPV45、HPV52、及 HPV58之感染相關聯 (Schiffman 等人，J. Natl· Cancer Inst. 85(12): 958-64 (1993))。在90-100%之宮頸癌中偵測到HPV DNA之觀察結 果提供HPV導致宮頸癌之有力疫學證據（參見Bosch等人，J. Clin. Pathol. 55: 244-265(2002))。 乳突狀瘤病毒為小（50-60 nm)的無包被二十面體DNA病 毒，其編碼高達8個早期及2個晚期基因。病毒基因組之開 放讀架（ORF)受指定為E1至E7、及L1與L2，其中”E”表示早 95258.doc 1334438 期且”L”表示晚期。L1&L2對病毒外殼蛋白質編碼，而£基 因與諸如病毒複製及細胞轉化之功能相關聯。 L1蛋白質為主要外殼蛋白質且具有55_6〇之分子 量。L2蛋白質為次要外殼蛋白質。免疫學資料顯示大:分 L2蛋白質在L1蛋白質之内部^ ^與！^蛋白質兩者皆高度保 存於不同乳突狀瘤病毒中。 U蛋白質或^與“蛋白質之組合於酵母菌、昆蟲細胞、 哺乳動物細胞或細菌中之表現導致似病毒粒子（VLp)之自 我組合（seif-assembiy)(關於述評，參見SchiUer and R〇den， in Papillomavirus Reviews: Current Research on Papillomaviruses; Lacey, ed. Leeds, UK: Leeds Medical Information，第i 〇 i _丨2頁（丨996))。VLp形態學上類似於真實 病毒體且能夠在投藥於動物或人類中誘發高滴度之中和抗 體。因為VLP不含有潛在致癌病毒基因組，所以它們為Hpv 疫苗研發中使用活病毒（關於評述，參見SchiUer and1334438 IX. Description of the Invention: [Technical Field to Which the Invention Is Ascribed] The present invention generally relates to the treatment of human papillomavirus (HP V). More specifically, the present invention relates to synthetic polynucleotides encoding HP V3 1 L1 proteins, and recombinant vectors and hosts comprising the same. The invention also relates to HPV31 virus-like particles (VLPs) and their use in vaccines and pharmaceutical compositions for the prevention and treatment of HPV. [Prior Art] There are more than 80 human papillomaviruses (HPV), many of which have been associated with a wide variety of biological phenotypes from benign proliferative neoplasias to malignant carcinomas (for review, see McMurray et al., Int. J). Exp. Pathol. 82(1): 15-33 (2001)). 1^¥6 and 1^¥11 are the most common types associated with low-grade dysplasia of benign tumors, non-malignant condyloma acuminata and/or genital mucosa or respiratory mucosa. HPV16 and HPV18 are the most frequently associated high-risk types associated with in situ and aggressive cancer of the cervix, vagina, vulva and anal. More than 90% of cervical cancers are associated with infection with HPV16, HPV18 or less prevalent cancer types HPV31, HPV33, HPV45, HPV52, and HPV58 (Schiffman et al, J. Natl. Cancer Inst. 85(12): 958- 64 (1993)). The observation of HPV DNA detected in 90-100% of cervical cancers provides strong epidemiological evidence that HPV causes cervical cancer (see Bosch et al., J. Clin. Pathol. 55: 244-265 (2002)). The papilloma virus is a small (50-60 nm) uncoated icosahedral DNA virus that encodes up to 8 early and 2 late genes. The open reading frame (ORF) of the viral genome is designated E1 to E7, and L1 and L2, where "E" indicates early 95258.doc 1334438 and "L" indicates late. L1 & L2 encodes viral coat proteins, while £ genes are associated with functions such as viral replication and cell transformation. The L1 protein is the major coat protein and has a molecular weight of 55-6 〇. The L2 protein is a minor coat protein. Immunology data shows that the L2 protein is inside the L1 protein ^ ^ and! Both proteins are highly preserved in different papillomaviruses. The expression of a U protein or a combination of "proteins" in yeast, insect cells, mammalian cells or bacteria results in a self-assembly (seif-assembiy) of virion-like particles (VLp) (for a review, see SchiUer and R〇den, In Papillomavirus Reviews: Current Research on Papillomaviruses; Lacey, ed. Leeds, UK: Leeds Medical Information, page i 〇i _丨 2 (丨 996). VLp is morphologically similar to a real virion and can be administered to animals High-titer neutralizing antibodies are induced in humans. Because VLPs do not contain the potential oncogenic viral genome, they use live viruses for Hpv vaccine development (for review, see SchiUer and

Hidesheim，J. Clin. Virol. 19: 67-74(2000))顯現安全之替代 方法。為此原因，已將L1及L2基因識別為研發針對感 染及疾病之預防性及治療性疫苗的免疫學目標。 HPV疫苗研發及商品化已受到與在經成功轉化之宿主體 内獲得高表現水平之外殼蛋白質相關聯之困難（限制純化 蛋白質之製造）所阻礙。因此，不管識別編碼諸如HP V3 1 L1 蛋白質之HP V L1蛋白質之野生型核苷酸序列（G〇ldsb〇r〇ugh 荨人，Virology 171(1): 306-311(1989)) ’ 仍將非常希望研發 粗製HPV蛋白質之容易更新源，該hpv蛋白質利用在期望 95258.doc 1334438 宿主細胞中經最適化表現之編碼HP V3 1 L1核苷酸序列。此 外，製備大量用於疫苗研發中之天然蛋白質的免疫對照特 性之HPV31 LI VLP將是有用的。 【發明内容】 本發明係關於引出或增強對由已與宮頸癌相關聯之. HPV3 1 L1基因所表現的蛋白質產物之免疫性的組合物及方 法。詳言之，本發明提供編碼HPV31 L1蛋白質之聚核苷酸， 其中該等聚核苷酸沒有受酵母菌辨識之内部轉錄終止訊 號。亦提供編碼HPV31 L1之合成聚核苷酸，其中該等聚核 苷酸為在酵母菌細胞中之高水平表現而經密碼子最適化。 本發明進一步提供HPV3 1似病毒粒子（VLP)且揭示該等 VLP在預防及/或治療HP V疾病或與HP V關聯之癌症的致免 疫組合物及疫苗中的用途。 本發明係關於編碼HPV31 L1蛋白質之合成DNA分子。於 本發明之一態樣中，將合成分子之核苷酸序列改變以消除 受酵母菌辨識之轉錄終止訊號。於另一態樣中，設計該等 合成分子之密碼子以便使用受酵母菌細胞首選之密碼子。 該等合成分子可用作可自我組合至VLP之HPV31 L1蛋白質 源。該等VLP可使用於基於VLP之疫苗中。 本發明之一特定實施例包含一合成核酸分子，該核酸分 子編碼如8£卩1〇]^0:4所設定之1^¥311^1蛋白質，該核酸 分子包含一如SEQ ID Ν0··2或SEQ ID Ν0··3中所設定之核苷 酸序列。 如上所陳述，本文提供編碼HPV31 L1基因之合成聚核苷 95258.doc 1334438 酸，該等核苷酸沒有受酵母菌辨識之内部轉錄終止訊號。 本發明亦提供所描述之編碼HPV31L1之合成聚核苷酸，其 經進一步改變以便含有受酵母菌細胞首選之密碼子。 本發明亦提供重組載體及重組宿主細胞（既包括原核細 胞又包括真核細胞），其含有貫穿本說明書所揭示之核酸分 〇 本發明係關於在一重組宿主細胞中表現Hpv3 i L1蛋白 質之方法，其包含：（a)將一包含編碼HpV3i li蛋白質之核 酸的載體引入至一酵母菌宿主細胞中；其中該核酸分子‘ 有受酵母菌辨識之内部轉錄終止訊號及；（1?)在允許該 HPV31 L1蛋白質表現之條件下培養該酵母菌宿主細胞。 本發明係'進一步關於在一重組宿主細胞中表現h p v 3 2 L1蛋白質之方法’其包含：⑷將—包含編碼蛋白 質之核酸的载體引人-酵母g宿主細胞中；其中該核酸分 子經密碼子最適化在輯母菌宿主細胞t之最適化表現 及；⑻在允許該HPV31 L1蛋白f表現之條件下培養酵母菌 宿主細胞。 於較佳實施例中，該核酸包含如SEQ ID N〇:hlsEQ m N〇:3中所設定之核苷酸序列。 本發明亦係關於HPV3 1似病毒粒子（VLp)、製備HpV3 1 VLP之方法、及使用HPV3 1 VLP之方法。 於本發明之一較佳實施例中’在酵母菌中製備Hpv3i VLP。於一進一步較佳之實施例中，該酵母菌係由下列各 物組成之群中選出：_酵母加如⑽州wisiae)、 95258.doc 1334438 多形漢遜酵母（Hansenula polymorpha)、巴氏畢赤酵母 一 (Pichia pastoris)、脆壁克魯維酵母（Kluyvermyces fragilis)、 乳酸克魯維酵母（Kluveromyces lactis)、及栗酒裂殖酵母菌 (Schizosaccharomyces pombe) ° ' 本發明之另一.態樣為HPV31 VLP，其包含由HPV31 L1基. . 因製備之HPV31 L1蛋白質，該HPV31 L1基因沒有受酵母菌 辨識之内部轉錄終止訊號。 本發明之又一態樣為HP V3 1 VLP，其包含由經密碼子最 _ 適化之HPV31 L1基因製備的HPV31 L1蛋白質。於本發明之 此態樣之一較佳實施例中，經密碼子最適化之HPV3 1 L1基 因大體由一如SEQ ID NO:2或SEQ ID N0:3中所設定之核普 酸序列組成。 - 本發明亦提供在動物體内誘發免疫響應之方法，其包括 · 將HP V3 1似病毒粒子投予該動物。於一較佳實施例中，由 經密碼子最適化之基因製備HPV3 1 VLP。於一進一步較佳 之貫施例中’由沒有受酵母菌辨識之内部轉錄終止訊號之 · 基因製備該等HPV3 1 VLP。 本發明之另一態樣為預防或治療與Hpv關聯之宮頸癌的 方法，其包括向嗔乳動物投予一包含HPV31 VLP之疫苗。 於本發明之此態樣的一較佳實施例中，在酵母菌中製備該 等HPV3 1 VLP。 _ 本發明亦係關於一包含HP V3〖似病毒粒子（VLp)之疫苗。 於本發明之此態樣的替代實施例中，該疫苗進一步包含 於一較佳實施例中 該至少 至少一種額外HPV類型之VLP。 95258.doc •10· 1334438 一種額外HPV類型係由下列各物組成之群中選出：HPV6、 HPV1 卜 HPV16、HPV18、HPV3 3、HPV35、HPV39、HPV45、 HPV5 卜 HPV52、HPV5 5、HPV56、HPV5 8、HPV59、及 HPV68。 本發明亦係關於包含HP V3 1似病毒粒子之醫藥組合物。 此外，本發明係關於包含HPV3 1 VLP及至少一種額外HPV. 類型之VLP的醫藥組合物。於一較佳實施例中，該至少一 種額外HPV類型係由下列各物組成之群中選出：HPV6、 HPVn、HPV16 ' HPV18、HPV33、HPV35、HPV39、HPV45、 HPV51、HPV52、HPV5 5、HPV5 6、HPV5 8、HPV59、及 HPV68。 貫穿本說明書及所附申請專利範圍中所使用之單數形式 "一”（”a”、"an”）及”該"包括複數引用，除非上下文另外明確 要求。 採用貫穿本說明書及所附申請專利範圍中所使用之下列 定義及縮寫： 術語”啓動子"係指一 DNA股上RNA聚合酶所結合之識別 位點。該啓動子與RNA聚合酶形成起始複合物以啓動及推 動轉錄活性。該複合物可藉由命名為”増強子"或"上游活化 序列'’之活化序列或命名為”沈默子"之抑制序列來改質。 術語”載體"係指某些裝置，由其可將DNA片段引入宿主 生物體或宿主組織中。存在多種載體，包括質粒、病毒（包 括腺病毒）、抗菌素及柯斯質粒（cosmids)。 名稱”3 1 L1野生型序列"係指本文中如SEQ ID NO: 1所揭 示之HPV31 L1序列。儘管先前已描述過HPV31 L1野生型序 列，發現在由臨床分離株中所得DNA間之微小序列變化並 95258.doc 1334438 非罕見。因此，—代表性HPV31 L1野生型序列自先前表現 出含有HPV 31 DNA之臨床樣本（見實例1}中分離。將3i Ll 野生型序列用m考序歹以與本文所揭示之經密碼子最適 化的HPV 31 L1序列相比較（參見圖1)。 名稱3 1 L1 4为重建'係指一本文所揭示之構造（seq瓜 N0:2)，其中該HPV31 L1核苷酸序列經部分重建以含有在 酵母菌中最優表現之受酵母菌首選的密碼子。31 L1部分重 建包含改變HPV 3 1 L1野生型核苷酸序列之中部（核苷酸 697-1249)。亦以受酵母菌首選之密碼子重建完整11]?¥ 31 Li 序列，該重建在本文中稱為"31 L1全部重建，,（SEq ID N〇:3)。 術5吾有效里，¾明引入充足疫苗組合物以製備適當水平 之多肽，以使得發生免疫響應。熟習此項技術者認識到此 水平可改變。 一·'保守胺基酸取代”係指將一胺基酸殘基由另一化學上 類似之胺基酸殘基置換。該等保守取代之實例為：以一疏 水殘基（異亮氨酸、亮氨酸、纈氨酸、或蛋氨酸）取代另一殘 基、以一極性殘基取代具有相同電荷之另一極性殘基（例如 精氨酸取代離胺酸、谷氨酸取代天冬氨酸）。 術語''哺乳動物”係指任何哺乳動物，包括人類。 "VLP"或”VLP"意謂似病毒粒子。 '合成的意5胃HP V 3 1 L1已經改良以使其含有一核苦酸序 列’該序列與存在於自然產生之野生型HPV3 1 L1基因中之 核苷酸序列不同。如上所陳述，本文提供包含一核苦酸序 95258.doc 12 1334438 列之合成分子，該序列經改變以消除受酵母菌辨識之内部 轉錄終止訊號。本文亦提供包含受酵母菌細胞首選表現之 密碼子的合成分子。本文所提供之合成分子編碼與該野生 型HP V3 1 L1基因相同之胺基酸序列。 【實施方式】 大部分宮頸癌與特殊致癌類型之人類乳突狀瘤病毒 (HPV)感染相關聯。本發明係關於引起或增強對於致癌HPV 類型之基因所表現之蛋白質產物的免疫性之組合物及方 法。詳言之，本發明提供編碼HPV3 1 L1之聚核苷酸及HP V3 1 似病毒粒子（VLP)且揭示該等聚核苷酸及VLP在預防及/或 治療與HPV相關聯之癌症的致免疫組合物及疫苗中之用 途。 野生型HPV3 1 L 1核苷酸序列已有報道（Goldsborough等 人，Virology 171(1): 306-311(1989) ; Genbank Accession # J04353)。本發明提供編碼HPV31 L1蛋白質之合成DNA分 子。本發明之合成分子包含一核苷酸序列，其中某些該等 核苷酸已被改變以便消除受酵母菌辨識之轉錄終止訊號。 於替代實施例中，將合成分子之密碼子進行設計以便使用 受酵母菌細胞首選高水平表現之密碼子。該等合成分子可 用作可自我組合至VLP之HPV31 L1蛋白質源。該等VLP可 用於基於VLP之疫苗中以提供經由中和抗體及細胞調節免 疫性對抗乳突狀瘤病毒感染之有效免疫預防。該等基於 VLP之疫苗亦可用於治療已確定之HPV感染。 HPV VLP於酵母菌細胞中之表現提供節省成本且容易適 95258.doc -13- 丄幻4438 應在發酵器中大規模生長之優勢。然而，包括Hpv3iU2 許多HPV L1蛋白質（參見實例4)於酵母菌細胞中以低水平 表現。根據本發明已確定HPV31 L1之低水平表現歸因於 mRNA轉錄體之十刀斷’該切斷係起因於受酵母菌辨識之轉錄 終止訊號之存在》藉由改變HPV31L1DNA以消除任何類似 於酵母ϋ轉錄終止位點之潛在序列，有可能促進導致增強 之HP V3 1 L1蛋白質表現的全長mRNA轉錄。 因此，於本發明之某些實施例中，已對HPV3 1 L· 1 DNA 進行改變以消除任何類似於酵母菌轉錄終止訊號之潛在序 列。此等改變允許與截短之轉錄體相反之全長Hp V3丨轉錄 體之表現（參見實例4)其改良表現量。 如上所述，本發明之合成DNA包含來自野生型HpV3丨L工 序列為消除受酵母菌辨識之轉錄終止點之改變。熟習此項 技術者將s忍識到可建構編碼HPV3 1 L1蛋白質之額外DNA 刀子’但§亥專分子不含有酵母菌轉錄終止位點。發現酵母 菌轉錄終止序列之技術在此項技術中為人所熟知。酵母菌 mRNA之轉錄終止及3’末端形成需要存在三個訊號：一效 率元素’諸如TATATA或有關序列，其提高位於下游之元素 的定位效率；（2)定位元素，其確定聚（A)位點之位置及（3) 聚腺苷醯作用位點（通常為Py(A)n)。 科學文獻充滿編碼酵母菌轉錄終止訊號之序列的描述。 例如參見：Guo 及 Sherman，Trends Biochem. Sci. 21: 477-481(1986); Guo及 Sherman，Mol. Cell. Biol. 16(6): 2772-2776(1996); Zaret等人，Cell 28:563-573(1982); Henikoff等 95258.doc -14- 1334438 人，Cell 33:607-614(1983); Thalenfeld等人，J. Biol. Chem. 258(23): 14065-14068(1983); Zaret等人，J. Mol. Biol. 176: 107-135(1984); Heidmann等人，Mol. Cell Biol 14:4633-4642( 1984)及 Russo, Yeast 11:447-453(1985)。因此，熟習此 項技術者將沒有困難地確定消除何序列以建構一產生根據 - 本發明之全長mRNA轉錄體的合成HPV31 L1基因。此外， 此項技術中已充分建立了評估該合成序列中是否存在一酵 母菌轉錄終止序列之檢定及程序，所以普通熟習此項技術 鲁 者將能夠確定一建構之HPV3 1 L1序列是否包含需要消除之 終止序列。 如上所述，本發明係關於一種編碼Hpv型3丨L丨蛋白質之 核酸分子，該核酸分子沒有受酵母菌辨識之内部轉錄終止 - 讯號。於本發明之一例示性實施例中，該等合成核酸分子 . 包含一如SEQ ID NO:2或SEQ ID ΝΟ··3所設定之核苷酸序 列。 於本發明之替代實施例中，HpV31 L1基因序列在酵母菌 籲 細胞環境中經"最適化，，為高水平表現。本發明所涵蓋之經 密碼子最適化之HPV31 L1基因包括編碼沒有受酵母菌辨識 之内部轉錄終止訊號之HPV3 1 L1的合成分子，該等合成分 子進一步包含至少一個為在酵母菌細胞中之高水平表現而 經也碼子最適化之密碼子。 四個可能之核苷酸鹼基之”三聯體”密碼子可存在超過6 〇 · 自不同形式。因為此等密碼子提供僅2〇種不同胺基酸之訊 心（及轉錄啓動及終止），故某些胺基酸可由多於—個密碼子 95258.doc -15- 1334438 進行編碼，此現象稱為密碼子重複性。由於未完全瞭解之 原因，替代之密碼子不均句存在於不同類型細胞之内生性 DNA中。確貫看起來存在特定類型細胞中對於特定密瑪子 之可變自然等級或"優先選擇例如，胺基酸亮胺酸由包 括 CTA、CTC、CTG、CTT、丁TA、及 ttg之六種 DNA 密碼 子之任何一者來指定。對微生物之基因組密碼子頻率的徹 底分析已揭示出大腸桿菌之内生性〇1^人最一般地含有ctg 亮胺酸指定密碼子，而酵母菌及黏菌iDNA最一般地包括 TTA亮胺酸指定密碼子。鑑於此等級，據信一般大腸桿菌 宿主得到富亮胺酸多肽之高水平表現的可能性將在一定程 度上取決於密碼子之使用頻率。例如，很可能一富有 密碼子之基因會在大腸桿菌中表現不佳，而一富有CTG之 基因將可能在此宿主中高度表現。類似地，在酵母菌宿主 細胞中表現富有亮胺酸之多肽的較佳密碼子可能為丁丁A。 在重組DNA技術中密碼子優先選擇現象之含義是顯而易 見的，且該現象將用以解釋許多先前為在經成功轉化之宿 主有機體内達到高表現水平之外生性基因所經歷的失敗— 一更欠”較佳”之密碼子可重複存在於所嵌入之基因中且用 於表現之宿主細胞機構可不進行有效操作。此現象說明已 經設計以包括-計劃宿主細胞之較佳密瑪子的合成基因為 實施重組DNA技術提供一最適化形式之外來遺傳材料。因 此，本發明之一態樣為一在酵母菌細胞中經·密碼子最適化 表現的HPV31 L1基因。在本發明之—較佳實施例中，吾人 已發現使用編碼相同蛋白質序列之替代密碼子可移除由酵 95258.doc • Ϊ6 - 1334438 母菌細胞表現HPV31 L1蛋白質上之約束。 根據本發明，將HPV31 L1基因片斷轉變為具有同樣經翻 譯之序列但具有如由Sharp及c〇we(Syn〇nym〇us口卿 in Saccharomyces cerevisiae. Yeast 7: 657-678(1991)，其以引用 之方式倂入本文中）所述之替代密碼子用法的序列。該方法 通常由識別與經高度表現之酵母菌基因通常不相關聯之野 生型序列中的密碼子及將它們以在酵母菌中經最適化高度 表現密碼子置換而組成。接著檢查該新基因序列中由此等 欲碼子置換產生之不合需要之序列（例如，”ΑΤττα"序列、 内含子剪接辨識位點之無意創造、不必要之限制酶位點， 等等）。藉由以編碼相同胺基酸之不同密褐子取代存在之密 碼子來消除不合需要之序列。接著測試合成基因片斷之改 良表現。 上述方法用於創造HPV31 L1之合成基因片斷，導致一包 含經高水平表現最適化之基因。雖然上述程序提供對設計 用於HPV疫苗中之經密碼子最適化的基因方法概要，熟習 此項技術者應瞭解可藉由在該程序中之微小變化或該序列 中之微小變化來達到類似之疫苗功效或增強之基因表現。 因此’本發明係關於一包含編碼HP V3 1 L1蛋白質之核；g： 酸序列、或HPV31 L1蛋白質之生物學活性片段或突變形式 的合成聚核音酸’該聚核普酸序列包含在酵母菌宿主中經 最適化表現之密碼子。該等HP V31 L1蛋白質之突變形式包 括（但不限於）：保守胺基酸取代、胺基-末端切斷、叛基_ 末端切斷、刪除、或添加。任何該生物學活性片段及/或突 95258.doc -17- 1334438 變體將編碼蛋白質或蛋白質片段兩者中之任一者，該蛋白 質或蛋白質片段至少充分模擬如SEQ ID N0:4中所設定之 HPV3 1 L1蛋白質的免疫學特性。本發明之合成聚核苷酸編 碼表現功能性HPV31 L1蛋白質之mRNA分子以便可用於研 發治療性或預防性HPV疫苗。Hidesheim, J. Clin. Virol. 19: 67-74 (2000)) An alternative to the emergence of safety. For this reason, the L1 and L2 genes have been identified as immunological targets for the development of prophylactic and therapeutic vaccines against infections and diseases. The development and commercialization of HPV vaccines has been hampered by the difficulties associated with obtaining high levels of coat protein in successfully transformed hosts, limiting the manufacture of purified proteins. Thus, regardless of the recognition of a wild-type nucleotide sequence encoding an HP V L1 protein such as the HP V3 1 L1 protein (G〇ldsb〇r〇ugh 荨人, Virology 171(1): 306-311 (1989)) will still It is highly desirable to develop an easily updated source of crude HPV protein that utilizes the HP V3 1 L1 nucleotide sequence encoded by the optimal expression in the desired 95258.doc 1334438 host cell. In addition, it would be useful to prepare a large number of HPV31 LI VLPs for the immunological control properties of natural proteins used in vaccine development. SUMMARY OF THE INVENTION The present invention relates to compositions and methods for eliciting or enhancing immunity to protein products exhibited by the HPV3 1 L1 gene that has been associated with cervical cancer. In particular, the invention provides polynucleotides encoding HPV31 L1 proteins, wherein the polynucleotides are free of internal transcriptional termination signals recognized by the yeast. Synthetic polynucleotides encoding HPV31 L1 are also provided, wherein the polynucleotides are codon-optimized for high levels of expression in yeast cells. The invention further provides HPV31 virus-like particles (VLPs) and discloses the use of such VLPs in immunogenic compositions and vaccines for the prevention and/or treatment of HPV diseases or cancers associated with HPV. The present invention relates to synthetic DNA molecules encoding the HPV31 L1 protein. In one aspect of the invention, the nucleotide sequence of the synthetic molecule is altered to eliminate transcription termination signals recognized by the yeast. In another aspect, the codons of the synthetic molecules are designed to use codons preferred by the yeast cells. These synthetic molecules can be used as a source of HPV31 L1 protein that can self-assemble to VLPs. These VLPs can be used in VLP-based vaccines. A particular embodiment of the invention comprises a synthetic nucleic acid molecule encoding a protein of 1 ^ ¥ 311 ^ 1 as set forth in SEQ ID NO: 2 or the nucleotide sequence set in SEQ ID Ν0··3. As set forth above, herein is provided a synthetic polynucleoside 95258.doc 1334438 acid encoding the HPV31 L1 gene, which has no internal transcription termination signal recognized by the yeast. The invention also provides a synthetic polynucleotide encoding HPV31L1 as described which is further altered to contain a codon which is preferred by the yeast cell. The invention also provides recombinant vectors and recombinant host cells (including both prokaryotic and eukaryotic cells) comprising nucleic acid bifurcations as disclosed in the present specification. The present invention relates to a method for expressing Hpv3 i L1 protein in a recombinant host cell. And comprising: (a) introducing a vector comprising a nucleic acid encoding a HpV3i li protein into a yeast host cell; wherein the nucleic acid molecule has an internal transcription termination signal recognized by the yeast; and (1?) The yeast host cell is cultured under conditions in which the HPV31 L1 protein is expressed. The present invention is a method for further expressing a hpv 3 2 L1 protein in a recombinant host cell, which comprises: (4) introducing a vector comprising a nucleic acid encoding a protein into a yeast g host cell; wherein the nucleic acid molecule is subjected to a password Optimizing the optimal expression of the host cell t in the mother strain; and (8) cultivating the yeast host cell under the condition that the expression of the HPV31 L1 protein f is allowed. In a preferred embodiment, the nucleic acid comprises the nucleotide sequence set forth in SEQ ID N:hlsEQ m N〇:3. The present invention also relates to HPV31 virus-like particles (VLp), a method of preparing HpV3 1 VLP, and a method of using HPV3 1 VLP. In a preferred embodiment of the invention, Hpv3i VLPs are prepared in yeast. In a further preferred embodiment, the yeast strain is selected from the group consisting of: yeast plus (10) state wisiae, 95258.doc 1334438 Hansenula polymorpha, Papillon Pichia pastoris, Kluyvermyces fragilis, Kluveromyces lactis, and Schizosaccharomyces pombe ° 'Another aspect of the present invention is HPV31 VLP, which contains the HPV31 L1 protein produced by HPV31 L1. The HPV31 L1 gene has no internal transcription termination signal recognized by yeast. A further aspect of the invention is the HP V3 1 VLP comprising the HPV31 L1 protein prepared from the codon-optimized HPV31 L1 gene. In a preferred embodiment of this aspect of the invention, the codon-optimized HPV3 1 L1 gene consists essentially of a nucleotide sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 3. - The invention also provides a method of eliciting an immune response in an animal comprising: administering an HP V3.1 virion to the animal. In a preferred embodiment, the HPV3 1 VLP is prepared from a codon-optimized gene. In a further preferred embodiment, the HPV3 1 VLPs are prepared from genes that are not recognized by yeast for internal transcription termination signals. Another aspect of the invention is a method of preventing or treating cervical cancer associated with Hpv comprising administering to a suckling animal a vaccine comprising an HPV31 VLP. In a preferred embodiment of this aspect of the invention, the HPV3 1 VLPs are prepared in yeast. The invention also relates to a vaccine comprising HP V3 virus-like particles (VLp). In an alternative embodiment of this aspect of the invention, the vaccine further comprises the at least one additional HPV type of VLP in a preferred embodiment. 95258.doc •10· 1334438 An additional HPV type is selected from the group consisting of: HPV6, HPV1, HPV16, HPV18, HPV3 3, HPV35, HPV39, HPV45, HPV5, HPV52, HPV5 5, HPV56, HPV5 8 , HPV59, and HPV68. The invention also relates to pharmaceutical compositions comprising HP V31 virus-like particles. Furthermore, the invention relates to pharmaceutical compositions comprising a HPV3 1 VLP and at least one additional HPV. In a preferred embodiment, the at least one additional HPV type is selected from the group consisting of: HPV6, HPVn, HPV16 'HPV18, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV5 5, HPV5 6 , HPV5 8, HPV59, and HPV68. The singular forms "a", "the", "the" and "the" are used throughout the specification and the appended claims. The following definitions and abbreviations used in the scope of the patent application: The term "promoter" refers to a recognition site to which RNA polymerase binds to a DNA strand. The promoter forms an initiation complex with RNA polymerase to initiate and drive transcriptional activity. The complex can be modified by an activation sequence designated "増子子" or "upstream activation sequence" or an inhibitory sequence designated "silencer". The term "vector" refers to a device by which a DNA fragment can be introduced into a host organism or host tissue. A variety of vectors are present, including plasmids, viruses (including adenoviruses), antibiotics, and cosmids. "3 1 L1 wild type sequence" refers to the HPV31 L1 sequence disclosed herein as SEQ ID NO: 1. Although the HPV31 L1 wild-type sequence has been previously described, a small sequence change between the DNA obtained from clinical isolates was found and 95258.doc 1334438 is not uncommon. Thus, the representative HPV31 L1 wild-type sequence was isolated from a clinical sample previously shown to contain HPV 31 DNA (see Example 1). The 3i Ll wild-type sequence was sequenced with m to optimize the codons disclosed herein. Comparison of the HPV 31 L1 sequences (see Figure 1). Name 3 1 L1 4 is a reconstructed 'system refers to a construct disclosed herein (seq melon N0:2), wherein the HPV31 L1 nucleotide sequence is partially reconstructed Contains the preferred codon for yeasts that are optimally expressed in yeast. 31 L1 partial reconstitution contains the middle part of the HPV 3 1 L1 wild-type nucleotide sequence (nucleotides 697-1949). Also preferred by yeast The codon reconstruction is complete 11]? ¥ 31 Li sequence, this reconstruction is referred to herein as "31 L1 all reconstruction, (SEq ID N〇: 3). In the 5 effective, 3⁄4 明 introduced sufficient vaccine composition To prepare an appropriate level of polypeptide to cause an immune response to occur. Those skilled in the art recognize that this level can vary. A 'conservative amino acid substitution' refers to the addition of an amino acid residue to another chemically similar Amino acid residue substitution. Examples of such conservative substitutions are: A hydrophobic residue (isoleucine, leucine, valine, or methionine) is substituted for another residue, and a polar residue is substituted for another polar residue having the same charge (eg, arginine substituted lysine) , glutamic acid replaces aspartic acid.) The term ''mammal'' refers to any mammal, including humans. "VLP" or "VLP" means virion-like particles. 'Synthetic Italian 5 stomach HP V 3 1 L1 has been modified to contain a nucleotide sequence which differs from the nucleotide sequence present in the naturally occurring wild-type HPV3 1 L1 gene. As stated above, the present invention provides a nuclear ascorbic acid sequence 95258.doc 12 1334438 A synthetic molecule that has been altered to eliminate internal transcriptional termination signals recognized by yeast. Also provided herein are synthetic molecules comprising codons preferred by yeast cells. The synthetic molecules provided herein are encoded in the wild. The same amino acid sequence of the HP V3 1 L1 gene. [Embodiment] Most cervical cancers are associated with a specific carcinogenic type of human papillomavirus (HPV) infection. Compositions and methods for enhancing immunity to protein products expressed by genes of oncogenic HPV types. In particular, the invention provides polynucleotides encoding HPV3 1 L1 and HP V3.1 virus-like particles (VLPs) and reveals Use of isonucleotides and VLPs in immunogenic compositions and vaccines for the prevention and/or treatment of cancer associated with HPV. Wild-type HPV3 1 L 1 nucleotide sequence has been reported (Goldsborough et al., Virology 171) (1): 306-311 (1989); Genbank Accession # J04353). The present invention provides synthetic DNA molecules encoding the HPV31 L1 protein. The synthetic molecules of the invention comprise a nucleotide sequence in which some of the nucleotides have been altered to eliminate transcription termination signals recognized by the yeast. In an alternate embodiment, the codons of the synthetic molecule are designed to use codons that are preferred for high levels of expression by the yeast cells. These synthetic molecules can be used as a source of HPV31 L1 protein that can self-assemble to VLPs. Such VLPs can be used in VLP-based vaccines to provide effective immunoprophylaxis against neutralizing antibodies and cell-regulated immunity against papillomavirus infection. These VLP-based vaccines can also be used to treat established HPV infections. The performance of HPV VLPs in yeast cells provides cost savings and is easy to adapt to the advantages of large-scale growth in fermenters. However, many HPV L1 proteins including Hpv3iU2 (see Example 4) were expressed at low levels in yeast cells. It has been determined in accordance with the present invention that the low level of performance of HPV31 L1 is attributed to the cleavage of the mRNA transcript, which results from the presence of a transcriptional termination signal recognized by the yeast, by altering HPV31L1 DNA to eliminate any yeast-like sputum. The potential sequence of the transcription termination site has the potential to promote full-length mRNA transcription leading to enhanced HP V3 1 L1 protein expression. Thus, in certain embodiments of the invention, HPV3 1 L·1 DNA has been altered to eliminate any potential sequence similar to the yeast transcription termination signal. These changes allow for the performance of the full-length Hp V3 丨 transcript (see Example 4) as opposed to the truncated transcript. As described above, the synthetic DNA of the present invention comprises a change from the wild-type HpV3丨L sequence to eliminate the transcription termination point recognized by the yeast. Those skilled in the art will be acquainted with the ability to construct additional DNA knives encoding the HPV3 1 L1 protein', but § hai special molecules do not contain yeast transcription termination sites. Techniques for the discovery of yeast transcription termination sequences are well known in the art. The transcription termination and 3' end formation of yeast mRNA requires three signals: an efficiency element such as TATATA or related sequences, which enhances the localization efficiency of elements located downstream; and (2) localization elements, which determine the poly(A) position. The position of the point and (3) the site of polyadenylation (usually Py(A)n). The scientific literature is full of descriptions of sequences encoding yeast transcription termination signals. See, for example, Guo and Sherman, Trends Biochem. Sci. 21: 477-481 (1986); Guo and Sherman, Mol. Cell. Biol. 16(6): 2772-2776 (1996); Zaret et al., Cell 28: 563-573 (1982); Henikoff et al. 95258.doc -14- 1334438, Cell 33:607-614 (1983); Thalenfeld et al, J. Biol. Chem. 258(23): 14065-14068 (1983); Zaret et al, J. Mol. Biol. 176: 107-135 (1984); Heidmann et al, Mol. Cell Biol 14: 4633-4642 (1984) and Russo, Yeast 11: 447-453 (1985). Thus, those skilled in the art will have no difficulty in determining which sequence to eliminate to construct a synthetic HPV31 L1 gene that produces a full-length mRNA transcript according to the present invention. In addition, assays and procedures for assessing the presence or absence of a yeast transcription termination sequence in the synthetic sequence have been well established in the art, so that it is common for those skilled in the art to be able to determine whether a constructed HPV3 1 L1 sequence needs to be eliminated. Termination sequence. As described above, the present invention relates to a nucleic acid molecule encoding a Hpv-type 3丨L丨 protein which has no internal transcription termination-signal recognized by yeast. In an exemplary embodiment of the invention, the synthetic nucleic acid molecule comprises a nucleotide sequence set forth in SEQ ID NO: 2 or SEQ ID ΝΟ. In an alternative embodiment of the invention, the HpV31 L1 gene sequence is "optimized" in a yeast cell environment to exhibit high levels of performance. The codon-optimized HPV31 L1 gene encompassed by the present invention includes a synthetic molecule encoding HPV3 1 L1 which is not recognized by the yeast for internal transcription termination signals, and the synthetic molecules further comprise at least one of which is high in the yeast cells. A cohort that is horizontally expressed and optimized by the code. The "triplet" codons of the four possible nucleotide bases may exist in more than 6 〇 · from different forms. Because these codons provide a signal (and transcription initiation and termination) of only two different amino acids, some amino acids can be encoded by more than one codon 95258.doc -15- 1334438. Called codon repeatability. For reasons not fully understood, alternative codons are present in endogenous DNA of different cell types. Surely there appears to be a variable natural level for a particular type of cell in a particular type of cell or "preferred choice, for example, amino acid leucine is comprised of six species including CTA, CTC, CTG, CTT, DTA, and ttg Any one of the DNA codons is specified. Thorough analysis of the genomic codon frequency of microorganisms has revealed that the endogenous 大肠杆菌1 of human E. coli contains the most commonly designated codon for ctg leucine, while yeast and mucoid iDNA most commonly include TTA leucine designation. a. In view of this level, it is believed that the likelihood that a typical E. coli host will obtain a high level of expression of a leucine-rich polypeptide will depend to some extent on the frequency of use of the codon. For example, it is likely that a codon-rich gene will perform poorly in E. coli, and a CTG-rich gene will likely be highly expressed in this host. Similarly, a preferred codon for a leucine-rich polypeptide in a yeast host cell may be Tinidine A. The implications of codon preference in recombinant DNA technology are obvious, and this phenomenon will be used to explain many of the failures previously experienced in achieving high levels of exogenous genes in successfully transformed host organisms - The "preferred" codon can be repetitively present in the embedded gene and the host cell machinery used for expression can be deactivated. This phenomenon demonstrates that synthetic genes that have been designed to include the preferred Mimas of the host cell are genetic material that provides an optimized form for performing recombinant DNA techniques. Therefore, one aspect of the present invention is an HPV31 L1 gene which is expressed by codons in yeast cells. In a preferred embodiment of the invention, we have found that the use of alternative codons encoding the same protein sequence removes the constraint on the HPV31 L1 protein expressed by the yeast 95258.doc • Ϊ6 - 1334438 mother cells. According to the present invention, the HPV31 L1 gene fragment is transformed to have the same translated sequence but has as described by Sharp and c〇we (Syn〇nym〇us kouqing in Saccharomyces cerevisiae. Yeast 7: 657-678 (1991) The sequence of alternative codon usage described herein is incorporated by reference. This method typically consists of identifying codons in wild-type sequences that are not normally associated with highly expressed yeast genes and replacing them with codons that are optimally expressed in yeast. Next, the undesired sequence generated by the substitution of the same code in the new gene sequence is examined (for example, "ΑΤττα" sequence, unintentional creation of intron splicing recognition sites, unnecessary restriction enzyme sites, etc.) Eliminating undesirable sequences by replacing the codons present with different dense browns encoding the same amino acid. Next, test the improved expression of the synthetic gene fragment. The above method is used to create a synthetic gene fragment of HPV31 L1, resulting in an inclusion Genes that are optimized at a high level. While the above procedure provides a summary of the genetic methods that are optimized for codon design in HPV vaccines, those skilled in the art should be aware of the small changes that can be made in the procedure or Small changes in the sequence to achieve similar vaccine efficacy or enhanced gene expression. Thus the present invention relates to a nucleus comprising an HP V3 1 L1 protein; a g: acid sequence, or a biologically active fragment or mutation of the HPV31 L1 protein. A form of synthetic polynucleic acid' the polynucleotide sequence comprises a codon that is optimally expressed in a yeast host. Mutated forms of the HP V31 L1 protein include, but are not limited to, conservative amino acid substitutions, amino-terminal cleavage, retino-end cleavage, deletion, or addition. Any such biologically active fragment and/or spur 95258 .doc -17- 1334438 A variant will encode either a protein or a protein fragment that at least substantially mimics the immunological properties of the HPV3 1 L1 protein as set forth in SEQ ID NO: 4. The synthetic polynucleotide of the invention encodes an mRNA molecule that expresses a functional HPV31 L1 protein so that it can be used to develop a therapeutic or prophylactic HPV vaccine.

本發明之一態樣為經密碼子最適化之核酸分子，其編碼 如SEQ ID N0:4中所設定之HPV31 L1蛋白質，該核酸分子 包含如SEQ ID NO:2中所設定之核苷酸序列。 本發明之另一態樣為經密碼子最適化之核酸分子，其編 碼如SEQ ID NO:4中所設定之HPV31 L1蛋白質，該核酸分 子包含如SEQ ID N0:3中所設定之核苷酸序列。 本發明亦係關於重組載體及既包括原核又包括真核之重 組宿主細胞，其含有貫穿本發明書所揭示之核酸分子。One aspect of the invention is a codon-optimized nucleic acid molecule encoding the HPV31 L1 protein set forth in SEQ ID NO: 4, the nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO: . Another aspect of the invention is a codon-optimized nucleic acid molecule encoding the HPV31 L1 protein set forth in SEQ ID NO: 4, the nucleic acid molecule comprising the nucleotide set forth in SEQ ID NO: sequence. The invention also relates to recombinant vectors and recombinant host cells comprising both prokaryotic and eukaryotic vectors, which comprise a nucleic acid molecule disclosed throughout the present disclosure.

經由本文所描述之方法建構的合成HPV3 1 DNA或其片段 可藉由分子複製入一含有適宜啓動子及其它適當轉錄調整 元素之表現載體而重組表現，且轉移入原核或真核宿主細 胞以製備重組HPV31 L1。該等操作技術於此項技術中有所 描述（Sambrook 等人，Molecular Cloning: A Laboratory Manual ; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, (1989) i Current Protocols in Molecular Biology, AusubelThe synthetic HPV3 1 DNA or a fragment thereof constructed by the methods described herein can be recombinantly expressed by molecular replication into a expression vector containing a suitable promoter and other appropriate transcriptional regulatory elements, and transferred into a prokaryotic or eukaryotic host cell for preparation. Recombinant HPV31 L1. Such techniques are described in this art (Sambrook et al, Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, (1989) i Current Protocols in Molecular Biology, Ausubel

Interscience, New York(1988) ； Yeast Genetics: A Laboratory Course Manual, Rose等人，Cold Spring Harbor Laboratory, Cold Spring Harbor, New York，（1990)，其以引用之方式全 95258.doc -18- 1334438 部併入本文中）。 因此’本發明係進-步關於—種在—重組宿主細胞中表 現HPV31 L1蛋白質之方法，1勹人 ''包含：（a)將包含編碼HPV31 U蛋白質之核酸的載體引入至_酵母菌宿主細胞中；其中 該核酸分子在該酵母g宿主細胞中經密碼子最適化最優表 現及⑽在允許該HPV31L1蛋白質之表現的條件下培養酵 母菌宿主細胞。 本發明亦係關於-種在-重组宿主細胞中表現HPV31 L1蛋白質之方法’其包含：（a)將包含編碼Hpv3iLi蛋白質 之核酸的載體引人至酵母菌宿主細胞中；#中該核酸分子 沒有受酵母菌辨識之内部轉錄終止訊號及㈨在允許該 HPV31 L1蛋白質之表現的條件下培養酵母菌宿主細胞。 本發明係進-步關於在—重組宿主細胞中表現爾η ㈣白質之方法’其包含：⑷將包含如seqidn〇^seq 1〇趾3中所設定之核酸的载體引入至酵母菌宿主細胞 中；及⑻在允許該HPV31L1蛋白質之表現的條件下培養酵 母宿主細胞。 可將本發明之合成基因組合至包含經設計以提供猶31 L1蛋白質在宿主細胞中之有效表現的序列之表料笑中。 該卡荚較佳含有該合成基因，諸如啓動子及終止序列之相 關轉錄及翻譯控制序列有效地連接於其上。在一較佳實施 例中，該啓動子為s. cerevisiae GAL1啓動子，儘管熟習此 項技術者將認識到可使用任何大量其它已知之酵母菌啓動 子，諸如GAL10、GAL7、ADm、TDH3或pGK啓動子:或 95258.doc 1334438 可使用其它真核基因啓動子。儘管亦可使用其它已知之轉 錄終止劑，一較佳轉錄終止劑為S. cerevisiae ADH1終止 子。尤其較佳為GAL 1啓動子- ADH1終止子之組合。 本發明之另一態樣為HPV3 1似病毒粒子（VLP)、製備 HPV31 VLP之方法、及使用HPV31 VLP之方法。當人類及. 動物乳突狀瘤病毒之主要外殼蛋白質L1在酵母菌、昆蟲細 胞、哺乳動物細胞或細菌中表現時VLP可自我組合（對於評 述，參見 Schiller 及 Roden，KPapillomavirusReviews*: Current Research on Papillomaviruses i Lacey, ed. Leeds, UK: Leeds Medical Information，第 101-12 頁（1996))。亦可藉由 表現L1與L2外殼蛋白質之組合來製備形態學上不明顯之 HPV VLP。VLP包含在T=7之二十面構造中之72個L1五聚物 (Baker等人，Biophys. J. 60(6): 1445-56(1991))。 VLP形態學上類似於真實病毒體且能夠將經投藥至動物 體内誘發高滴度之中和抗體。以VLP使兔（Breitburd等人’ J. Virol. 69(6): 3959-63(1995))及狗（Suzich等人，Proc. Natl. Acad. Sci. USA 92(25): 11553-57( 1995))免疫化顯示出既誘 發中和抗體又保護免受實驗乳突狀瘤病毒之感染。然而’ 因為該等VLP不含有潛在致癌之病毒基因組且可由單一基 因自我組合，它們在HP V疫苗研發中呈現對於使用活病毒 之的安全替代（對於評述，參見Schiller及Hidesheim，J. Clin· Virol. 19: 67-74(2000)) 〇 因此，本發明係關於包含HPV3 1之重組L1蛋白質或重組 L1+L2蛋白質的似病毒離子。 95258.doc •20- 於本發明之一較佳實施例中，在酵母菌中製備HPV3 1 VLP。於一進一步較佳之實施例中，該酵母菌係由下列各 物組成之群中選出：釀酒酵母、多形漢遜酵母、巴氏畢赤 酵母、脆壁克魯維酵母、乳酸克魯維酵母、及栗酒裂殖酵 母菌。 本發明之另一態樣為HP V3 1 VLP，其包含由沒有受酵母 菌辨識之内部轉錄終止訊號的HPV31 L1基因製備之HPV31 L1蛋白質。 本發明之又一態樣為HPV3 1 VLP，其包含由經密碼子最 適化之HPV31 L1基因製備的HPV31 L1蛋白質。於本發明之 此態樣之一較佳實施例中，該經密碼子最適化之HP V3 1 L 1 基因大體由一如SEQ ID NO:2或SEQ ID N0:3中所設定之核 苷酸序列所組成。 本發明之又一態樣為製備HPV31 VLP之方法，其包含： (a)以編碼HPV31 L1蛋白質或HPV31 L1+L2蛋白質之重組 DNA分子來轉化酵母菌；（b)在容許表現重組DNA分子之條 件下培養該經轉化之酵母菌以製備重組HPV3 1蛋白質；及 (c)分離該重組HPV31蛋白質以製備HPV31 VLP。 於本發明之此態樣的一較佳實施例中，以一沒有受酵母 菌辨識之内部轉錄終止訊號的HPV31 L1基因來轉化該酵母 菌。於另一較佳實施例中，以一經密碼子最適化之HPV3 1 L1 基因來轉化該酵母菌以製備HPV3 1 VLP。於一尤其較佳之 實施例中，經密碼子最適化之HPV31 L1基因大體由如SEQ ID NO:2或SEQ ID N0:3中所設定之核苷酸序列所組成。 95258.doc •21 - 1334438 本發明亦提供在動物體内誘發免疫響應之方法，其包含 將HPV3 1似病毒粒子投予該動物。於一較佳實施例中，係 藉由沒有受酵母菌辨識之内部轉錄終止序列的基因來製備 該等HPV3 1 VLP。於一進一步較佳之實施例中，由經密碼 子最適化之基因來製備該等HPV3 1 VLP。 本發明之又一態樣為預防或治療與Η P V關聯之宮頸癌之 方法，其包含向哺乳動物投予一包含HPV3 1 VLP之疫苗。 在本發明之此態樣的一較佳實施例中，在酵母菌中製備該 等HPV31 VLP。 本發明亦係關於一種包含HPV3 1似病毒粒子（VLP)之疫 苗。 於本發明之此態樣的替代實施例中，該疫苗進一步包含 至少一種額外HPV類型之VLP。於一較佳實施例中，該至少 一種額外HPV類型係由下列各物組成之群中選出：HPV6、 HPV1 卜 HPV16、HPV18、HPV33、HPV3 5、HPV39、HPV4 5、 HPV5 卜 HPV52、HPV5 5、HPV56、HPV5 8、HPV59、及 HPV68。 於本發明之此態樣的一較佳實施例中，該疫苗進一步包 含HPV16 VLP。 於本發明之此態樣的另一較佳實施例中’該疫苗進一步 包含 HPV16 VLP 及 HPV18 VLP。 於本發明之此態樣的又一較佳實施例中，該疫苗進一步 包含 HPV6 VLP、HPV11 VLP、HPV16 VLP 及 HPV18 VLP °Interscience, New York (1988); Yeast Genetics: A Laboratory Course Manual, Rose et al, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1990), by way of reference, all 95258.doc -18- 1334438 Incorporated herein). Thus, the present invention relates to a method for expressing HPV31 L1 protein in a recombinant host cell, and the method comprises: (a) introducing a vector comprising a nucleic acid encoding the HPV31 U protein into a yeast host In the cell; wherein the nucleic acid molecule is optimally expressed by codon optimization in the yeast g host cell and (10) the yeast host cell is cultured under conditions permitting expression of the HPV31L1 protein. The invention also relates to a method for expressing an HPV31 L1 protein in a recombinant host cell comprising: (a) introducing a vector comprising a nucleic acid encoding a Hpv3iLi protein into a yeast host cell; The internal transcription termination signal recognized by the yeast and (9) the yeast host cell is cultured under conditions permitting the expression of the HPV31 L1 protein. The present invention relates to a method for expressing η (tetra) white matter in a recombinant host cell, which comprises: (4) introducing a vector comprising a nucleic acid as set forth in seqidn〇^seq 1 toe 3 into a yeast host cell And (8) cultivating the yeast host cell under conditions permitting expression of the HPV31L1 protein. The synthetic gene of the invention can be combined into a superscript comprising a sequence designed to provide efficient expression of the J31 L1 protein in a host cell. Preferably, the card pod contains the synthetic gene, such as the promoter and the relevant transcriptional and translational control sequences of the termination sequence operably linked thereto. In a preferred embodiment, the promoter is the s. cerevisiae GAL1 promoter, although those skilled in the art will recognize that any of a number of other known yeast promoters, such as GAL10, GAL7, ADm, TDH3 or pGK, can be used. Promoter: or 95258.doc 1334438 Other eukaryotic gene promoters can be used. A preferred transcription terminator is the S. cerevisiae ADH1 terminator, although other known transcription terminators can also be used. Particularly preferred is a combination of the GAL 1 promoter - ADH1 terminator. Another aspect of the invention is HPV31 virus-like particles (VLP), methods for preparing HPV31 VLPs, and methods for using HPV31 VLPs. VLPs can self-assemble when human and human papillomavirus major coat protein L1 is expressed in yeast, insect cells, mammalian cells or bacteria (for review, see Schiller and Roden, KPapillomavirus Reviews*: Current Research on Papillomaviruses i Lacey, ed. Leeds, UK: Leeds Medical Information, pp. 101-12 (1996)). Morphologically indistinct HPV VLPs can also be prepared by expressing a combination of L1 and L2 coat proteins. The VLP contains 72 L1 pentamers in an icosahedral configuration of T = 7 (Baker et al., Biophys. J. 60(6): 1445-56 (1991)). VLPs are morphologically similar to real virions and are capable of inducing high titer neutralizing antibodies when administered to an animal. Rabbits were made with VLP (Breitburd et al. 'J. Virol. 69(6): 3959-63 (1995)) and dogs (Suzich et al, Proc. Natl. Acad. Sci. USA 92(25): 11553-57 ( 1995)) Immuneization has been shown to both induce neutralizing antibodies and protect against infection by experimental papillomavirus. However, because these VLPs do not contain potentially carcinogenic viral genomes and can be self-assembled by a single gene, they present a safe alternative to the use of live viruses in HP V vaccine development (for review, see Schiller and Hidesheim, J. Clin Virol) 19: 67-74 (2000)) Accordingly, the present invention relates to a virus-like ion comprising a recombinant L1 protein of HPV31 or a recombinant L1+L2 protein. 95258.doc • 20- In a preferred embodiment of the invention, HPV3 1 VLPs are prepared in yeast. In a further preferred embodiment, the yeast strain is selected from the group consisting of: Saccharomyces cerevisiae, Hansenula polymorpha, Pichia pastoris, Kluyveromyces cerevisiae, Kluyveromyces lactis And chestnut wine fission yeast. Another aspect of the invention is the HP V3 1 VLP comprising the HPV31 L1 protein prepared from the HPV31 L1 gene which is not recognized by the yeast for internal transcription termination signals. A further aspect of the invention is the HPV3 1 VLP comprising the HPV31 L1 protein prepared from the codon-optimized HPV31 L1 gene. In a preferred embodiment of this aspect of the invention, the codon-optimized HP V3 1 L 1 gene is substantially represented by a nucleotide set forth in SEQ ID NO: 2 or SEQ ID NO: 3. The sequence consists of. A further aspect of the invention is a method of making an HPV31 VLP comprising: (a) transforming a yeast with a recombinant DNA molecule encoding HPV31 L1 protein or HPV31 L1+L2 protein; (b) permitting expression of a recombinant DNA molecule The transformed yeast is cultured to prepare a recombinant HPV31 protein; and (c) the recombinant HPV31 protein is isolated to prepare an HPV31 VLP. In a preferred embodiment of this aspect of the invention, the yeast is transformed with an HPV31 L1 gene that is not recognized by the yeast for internal transcription termination signals. In another preferred embodiment, the yeast is transformed with a codon-optimized HPV3 1 L1 gene to produce an HPV3.1 VLP. In a particularly preferred embodiment, the codon-optimized HPV31 L1 gene consists essentially of the nucleotide sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3. 95258.doc • 21 - 1334438 The invention also provides a method of eliciting an immune response in an animal comprising administering HPV31 virus-like particles to the animal. In a preferred embodiment, the HPV3 1 VLPs are prepared by a gene that is not endogenously recognized by the yeast. In a further preferred embodiment, the HPV3 1 VLPs are prepared from codon-optimized genes. A further aspect of the invention is a method of preventing or treating cervical cancer associated with Η P V comprising administering to a mammal a vaccine comprising an HPV 31 VLP. In a preferred embodiment of this aspect of the invention, the HPV31 VLPs are prepared in yeast. The invention also relates to a vaccine comprising HPV31 virus-like particles (VLP). In an alternate embodiment of this aspect of the invention, the vaccine further comprises at least one additional HPV type of VLP. In a preferred embodiment, the at least one additional HPV type is selected from the group consisting of: HPV6, HPV1, HPV16, HPV18, HPV33, HPV3 5, HPV39, HPV4 5, HPV5, HPV52, HPV5 5, HPV56, HPV5 8, HPV59, and HPV68. In a preferred embodiment of this aspect of the invention, the vaccine further comprises an HPV 16 VLP. In another preferred embodiment of this aspect of the invention, the vaccine further comprises an HPV16 VLP and an HPV18 VLP. In still another preferred embodiment of this aspect of the invention, the vaccine further comprises HPV6 VLP, HPV11 VLP, HPV16 VLP and HPV18 VLP °

本發明亦係關於包含HP V 3 1似病毒粒子之醫藥組合物。 此外，本發明係關於包含HPV3 1 VLP及至少一種額外HPV 95258.doc -22- 1334438 類型之VLP的醫藥組合物。於一較佳實施例中，該至少一 種額外HPV類型係由下列各物組成之群中選出：Ηρν6、 HPV1 卜 HPV1 6、HPV1 8、HPV33、HPV35、HPV39、HPV45、 HPV5 卜 HPV52、HPV55、HPV56、HPV58、HPV59mPV68。 可以由常規測試所界定之適當劑量來單獨使用本發明之 疫苗組合物以獲得HPV31感染之最優抑制同時將任何潛在 之毒性最小化。此外，可希望其它藥劑之共投藥或順序投 藥。 · 引入至疫苗受體之似病毒粒子量將取決於所表達之基因 產物的致免疫性。大體上，直接向肌肉組織投予約心8至 100 μ§、且較佳約20 kg至60吨之免疫學或預防學上有效量 之VLP:亦預期皮下注射，皮内注射，經由皮膚廢印，及 . 其它投藥之模式，諸如腹膜内、靜脈内、或吸入傳輸。'亦 . 預期到可提供加強接種。諸如靜脈内、肌肉、皮下或其它 與諸如明戴或Merck明蓉佐劑之佐劑一起投藥的方式之非 腸道奴藥同時或隨後非腸道引入本發明之疫苗亦為有利。鲁 為描述及揭示可能與本發明結合使用之方法與材料的目 的將本文所提及之所有公開案以引用方式倂入本文中。不 應理解本文巾之任何料承認本㈣沒有㈣先於 前之發明的該揭示内容。 无 參考伴隨之圖式描述了本發明之較佳實施例，應瞭解本· 發明不限於彼等精確之實施例，且熟習此項技術者可在i — :進行多種變化及修改而不背離如所附巾請專利範圍以 * 疋義之本發明的範疇或精神。 95258.doc •23- 1334438 下列實例例示但不限制本發明。 實例1 測定代表性HPV31 L1序列 HPV 31 L1野生型序列已在先前加以描述（Goldsborough 等人，Virology 171(1): 306-311(1989) ; Genbank Accession # J04353)。然而，發現在自臨床分離所得到之DNA之間的 微小序列變化並非罕見。為分離代表性HPV31 L1野生型序 列，自三個先前顯示含有HPV3 1 DNA之臨床樣本中分離 DNA。使用Taq DNA聚合酶及下列引子在一聚合酶鏈反應 (PCR)中放大HPV 31 L1序列：HPV 3 1 LI F 5' - CGT CGA CGT AAA CGT GTA TCA TAT TTT TTT ACA G - 3，(SEQ ID NO:5)及 HPV 31 LIB 5' - CAG ACA CAT GTA TTA CAT ACA CAA C - 3'(SEQ ID NO:6)。使經放大之產物在瓊脂糖凝膠 上發生電泳且藉由溴化乙錠著色而顯現。切除〜1500 bp L1 帶且使用QIA快速PCR純化套組（Qiagen，Hilden, Germany) 來純化DNA。接著將DNA連接至ΤΑ選殖載體pCR-II (Invitrogen Corp.，Carlsbad, CA)、經大腸桿菌轉化、且置 放於具有安比西林（ampicillin)外加用於藍/白菌落選擇之 IPTG及X-gal之LB瓊脂上。將該等板倒置且在37°C下培育16 小時。將白色菌落在具有安比西林之LB介質中進行培養， 在3 7°C下振盪16小時，且執行小規模純化（miniprep)以提取 質粒DNA。 為證明質體中存在L1基因，由瓊脂糖凝膠電泳及溴化乙 鍵著色來進行及觀察限制性酶切。於含有自三個臨床分離 95258.doc -24- 1334438 之每一者選殖之L1的質體上執行DNA測序。將DNA及經翻 譯之胺基酸序列相互對比且與Genbank HPV 31 L1序列對 比。該等三個臨床分離之序列分析揭示出沒有與Genbank 序列相同之序列。將pCR-II-HPV 31L1/81純系選擇為代表 性31 L1序列且在本文中稱為”31 L1野生型序列"（SEQ ID. NO: 1，參見圖1)。選為31L1野生型之序列在核苷酸1266處 含有一沈默取代及在核苷酸1295處自C至G之變化，其將經 編碼之胺基酸自蘇氨酸改變為絲氨酸。3 1 L1經部分及全部 重建之基因（SEQ ID NO分別為2及3)亦在該位置編碼絲氨 酸（參見圖1)。於所有狀況中，該等胺基酸序列是相同的。 於重建之構造中變化核苷酸以使用受酵母菌首選之密碼子 序列編碼胺基酸且消除潛在之轉錄終止訊號（參見實例2)。 使用 LS-101 5, - CTC AGA TCT CAC AAA ACA AAA TGT CTC TGT GGC GGC CTA GC - 3'(SEQ ID NO:7)及LS-102 5'-GAC AGA TCT TAC TTT TTA GTT TTT TTA CGT TTT GCT GG - 3'(SEQ ID NO:8)引子放大31 LI野生型序列以添加 Bglll延伸。使用Vent™ DNA聚合酶來執行PCR。藉由填脂 糖凝膠之溴化乙錠著色使該PCR產物顯現。切除〜1500 bp 帶且使用QIAEX II凝膠提取DNA套組（Qiagen)來純化 DNA。接著以Bglll於37°C下消化該PCR產物2小時且使用 QIA快速PCR純化套組將其純化。將經Bglll消化之3 1 L1 PCR產物連接於經BamHI消化之pGAL 11 0且轉化DH5大腸 桿菌。由PCR篩選HPV31 L1嵌入正確方位之菌落。由DNA 測序證實了序列及方位。將經選擇之純系命名為 95258.doc -25- 1334438The invention also relates to pharmaceutical compositions comprising HP V 31 -like virions. Furthermore, the invention relates to a pharmaceutical composition comprising a HPV3 1 VLP and at least one additional VLP of the type HPV 95258.doc -22-1334438. In a preferred embodiment, the at least one additional HPV type is selected from the group consisting of: Ηρν6, HPV1, HPV1 6, HPV1 8, HPV33, HPV35, HPV39, HPV45, HPV5, HPV52, HPV55, HPV56 , HPV58, HPV59mPV68. The vaccine compositions of the present invention can be used alone at appropriate dosages as defined by routine testing to achieve optimal inhibition of HPV31 infection while minimizing any potential toxicity. In addition, co-administration or sequential administration of other agents may be desirable. • The amount of virion-like particles introduced into the vaccine receptor will depend on the immunogenicity of the expressed gene product. In general, an immunologically or prophylactically effective amount of VLP of about 8 to 100 μ§, and preferably about 20 to 60 tons, is administered directly to the muscle tissue: subcutaneous injection, intradermal injection, and skin imprinting are also contemplated. , and other modes of administration, such as intraperitoneal, intravenous, or inhalation. 'Yes. Enhanced vaccination is expected. It is also advantageous to introduce the vaccine of the present invention simultaneously or subsequently parenterally, such as intravenously, intramuscularly, subcutaneously, or other means of administration with an adjuvant such as Mingdai or Merck Mingrong adjuvant. All of the publications mentioned herein are hereby incorporated by reference in their entirety for all purposes for the purposes of the disclosure. It should not be understood that any of the materials herein will recognize that this disclosure does not have (4) prior inventions. The present invention is not limited to the precise embodiments thereof, and those skilled in the art can make various changes and modifications without departing from the invention. The scope of the invention is to be limited to the scope or spirit of the invention. 95258.doc • 23- 1334438 The following examples illustrate but do not limit the invention. Example 1 Determination of a representative HPV31 L1 sequence The HPV 31 L1 wild type sequence has been previously described (Goldsborough et al, Virology 171(1): 306-311 (1989); Genbank Accession #J04353). However, it has not been found that small sequence changes between DNA obtained from clinical isolates are not uncommon. To isolate a representative HPV31 L1 wild type sequence, DNA was isolated from three clinical samples previously shown to contain HPV3.1 DNA. Amplification of HPV 31 L1 sequence in a polymerase chain reaction (PCR) using Taq DNA polymerase and the following primers: HPV 3 1 LI F 5' - CGT CGA CGT AAA CGT GTA TCA TAT TTT TTT ACA G - 3, (SEQ ID NO: 5) and HPV 31 LIB 5' - CAG ACA CAT GTA TTA CAT ACA CAA C - 3' (SEQ ID NO: 6). The amplified product was electrophoresed on an agarose gel and visualized by ethidium bromide coloration. The ~1500 bp L1 band was excised and the DNA was purified using a QIA rapid PCR purification kit (Qiagen, Hilden, Germany). The DNA was then ligated into the sputum selection vector pCR-II (Invitrogen Corp., Carlsbad, CA), transformed with E. coli, and placed in IPTG and X- for ampicillin plus blue/white colony selection. Gal on LB agar. The plates were inverted and incubated for 16 hours at 37 °C. White colonies were cultured in LB medium with ampicillin, shaken at 37 ° C for 16 hours, and miniprep was performed to extract plasmid DNA. In order to prove the presence of the L1 gene in the plastid, restriction enzyme digestion was carried out by agarose gel electrophoresis and bromide broma coloring. DNA sequencing was performed on plastids containing L1 cloned from each of the three clinical isolates 95258.doc -24 - 1334438. The DNA and the translated amino acid sequences were compared to each other and compared to the Genbank HPV 31 L1 sequence. Sequence analysis of these three clinical isolates revealed no sequence identical to the Genbank sequence. The pCR-II-HPV 31L1/81 pure line was selected as a representative 31 L1 sequence and is referred to herein as "31 L1 wild type sequence " (SEQ ID. NO: 1, see Figure 1). Selected as 31L1 wild type The sequence contains a silent substitution at nucleotide 1266 and a change from C to G at nucleotide 1295, which changes the encoded amino acid from threonine to serine. 3 1 L1 is partially and completely reconstituted The genes (SEQ ID NOs 2 and 3, respectively) also encode serine at this position (see Figure 1). In all cases, the amino acid sequences are identical. The nucleotides are altered in the reconstructed construct to be used. The yeast preferred codon sequence encodes an amino acid and eliminates potential transcriptional termination signals (see Example 2). Using LS-101 5, - CTC AGA TCT CAC AAA ACA AAA TGT CTC TGT GGC GGC CTA GC - 3' (SEQ ID NO: 7) and LS-102 5'-GAC AGA TCT TAC TTT TTA GTT TTT TTA CGT TTT GCT GG - 3' (SEQ ID NO: 8) primer amplification 31 LI wild type sequence to add Bglll extension. Using VentTM DNA polymerase to perform PCR. The PCR product was visualized by ethidium bromide staining of a fat-filled gel. The ~1500 bp band was removed. The DNA was purified using QIAEX II gel extraction DNA kit (Qiagen). The PCR product was then digested with Bglll at 37 °C for 2 hours and purified using QIA rapid PCR purification kit. Bglll digested 3 1 L1 The PCR product was ligated into BamHI-digested pGAL 1 0 and transformed into DH5 E. E. coli was screened by HPV31 L1 into the correct orientation of the colony. The sequence and orientation were confirmed by DNA sequencing. The selected pure line was named 95258.doc -25- 1334438

PgalllO-HPV 31L1 #2。 接著製備了大規模純化（maxiprep)DNA且使釀酒酵母變 為感受態（competent)且將其進行轉化。將酵母菌轉化沈積 於Leu·山梨醇平皿上之Leu-山梨醇軟性瓊脂（t〇p_agar)中且 在30C下反向培育3_5日。挑選及劃線在Leu•山梨醇板上進 打分離之菌落。為誘發L1轉錄及蛋白質表現，隨後於5 ml 5 X Leu Ade山梨醇中以ι.6%葡萄糖及4%半乳糖在3〇。〇下在 渦旋管培養中生長經分離之菌落。 實例2 酵母菌密碼子最適化 酵母菌較佳之推瑪子已作描述（Sharp及Cowe，Yeast 7: 657-678(1991))。最初，利用受酵母菌首選之密碼子重建了 代表核音酸697-1249之HPV 31 L1之當中部分。重建所採用 之策略為設計長重疊跨越待重建區域之意識及抗意識低聚 物’以受酵母菌首選之密碼子序列取代核苷酸同時維持相 同之胺基酸序列。此等低聚物用於在PCR反應中代替模板 DNA。額外之放大引子經設計及使用於以pfu dna聚合酶 (Stratagene，La Jolla, CA)自模板低聚物放大經重建之序 列。放大之最優條件為特定於區段的；然而，大多數使用 類似下文之程序：1分鐘之94°C初始變性步驟、隨後15-25 輪30 sec之95°C變性、30 sec之55°C退火、72°C 3.5分鐘延 伸、隨後10分鐘之72°C最終延伸及4°C固定。 由違脂糖凝膠電泳檢查PCR產物。切除適當尺寸之帶且 凝膠純化DNA。接著將該等放大之片段作為模板使用以組 95258.doc -26· 1334438 合552核苷酸重建之HPV 31中部L1片段。接著使用PCR放大 野生型核苷酸1-725(5,末端）及1221-1515(3,末端）。執行使用 5'末端、3'末端、及經重建之中部的最終pCR來生成全長31 L1部分重建，本文稱為”3 1 l 1部分重建”。 亦以受酵母菌首選之密碼子重建完整31L1序列。此構造 在本文中稱為”31 L1全部重建使用9種長重疊之低聚物自 1-753生成受酵母菌首選之密碼子核苷酸序列且使用4種長 重疊之低聚物自1207-1515生成受酵母菌首選之密碼子核 苷酸序列。在放大及凝膠純化之後，將此等片段隨上文所 述之經中部重建之區段（核苷酸697_1249)—同使用於一 PCR反應中以產生全長3丨L1全部重建序列。以BamHI延伸 來生成此段序列。經凝膠純化重建之31 L1 DNA以BamHI 消化、連接於經BamHI消化之pGAL110表現載體且轉化至 大腸桿菌DH5細胞中。由PCR篩選Hpv31 L1嵌入正確方位 之菌落。由DNA測序來證實序列及方位。 製備了質粒DNA。使釀酒酵母細胞變為感受態且將其進 行轉化。將該酵母菌沈積於Leu-山梨醇平皿上之Leu•山梨醇 軟性瓊脂中且反向培育3-5曰。將菌落劃線以用於在Leu-山 梨醇平皿上進行分離。隨後於5 ml 5 X Leu. Ade•山梨醇中 以1.6%葡萄糖及4%半乳糖在30〇C下在渦旋管培養中生長 分離之菌落以誘發L1轉錄及蛋白質表現。48_72小時之後， 將等於OD600=10之培養體積製成小球、移除上清液且將該 等小球冷凍及貯藏於-7〇°C。 實例3 95258.doc -27- 1334438PgalllO-HPV 31L1 #2. Large scale purified (maxiprep) DNA was then prepared and Saccharomyces cerevisiae was made competent and transformed. The yeast was transformed and deposited on Leu-sorbitol soft agar (t〇p_agar) on a Leu·sorbitol plate and cultured in reverse for 3 to 5 days at 30C. The colonies that were separated on the Leu•sorbitol plate were selected and crossed. To induce L1 transcription and protein expression, it was then in iv.6% glucose and 4% galactose in 3 ml of 5 X Leu Ade sorbitol. The isolated colonies are grown in the vortex tube culture under the armpit. Example 2 Optimization of Yeast Codons The preferred pusher of yeast has been described (Sharp and Cowe, Yeast 7: 657-678 (1991)). Initially, the middle part of HPV 31 L1 representing the nuclear acid 691-2949 was reconstructed using the yeast preferred codon. The strategy used for reconstruction is to design a consciousness and anti-conscious oligomer that overlaps over the area to be reconstructed to replace nucleotides with the preferred codon sequence of yeast while maintaining the same amino acid sequence. These oligomers are used to replace template DNA in a PCR reaction. Additional amplification primers were designed and used to amplify the reconstructed sequence from the template oligomer by pfu dna polymerase (Stratagene, La Jolla, CA). The optimal conditions for amplification are segment-specific; however, most use a procedure similar to the following: a 94°C initial denaturation step of 1 minute followed by a 95°C denaturation of 15-25 sec 30 sec, 55° 30 sec. C annealing, extension at 72 ° C for 3.5 minutes, subsequent extension of 72 ° C for 10 minutes and fixation at 4 ° C. The PCR product was examined by lipase gel electrophoresis. The band of the appropriate size is removed and the DNA is gel purified. The amplified fragment was then used as a template for the HPV 31 central L1 fragment reconstituted with the group 95258.doc -26·1334438 and 552 nucleotides. Next, the wild type nucleotides 1-725 (5, terminal) and 1221-1515 (3, terminal) were amplified using PCR. The full-length 31 L1 partial reconstruction is generated using the final pCR at the 5' end, the 3' end, and the reconstructed middle portion, referred to herein as "3 1 l 1 partial reconstruction." The complete 31L1 sequence was also reconstructed with the yeast preferred codon. This construct is referred to herein as "31 L1 all reconstitution using 9 long overlapping oligomers from 1-753 to generate yeast-preferred codon nucleotide sequences and using 4 long overlapping oligomers from 1207- 1515 generates a codon nucleotide sequence preferred by the yeast. After amplification and gel purification, the fragments are used in a centrally reconstructed segment (nucleotides 697_1249) as described above. The reaction was carried out to generate a full-length 3丨L1 reconstituted sequence. This sequence was generated by BamHI extension. The 31 L1 DNA reconstituted by gel purification was digested with BamHI, ligated into BamHI-digested pGAL110 expression vector and transformed into E. coli DH5 cells. The Hpv31 L1 was screened by PCR to insert the colony in the correct orientation. The sequence and orientation were confirmed by DNA sequencing. Plasmid DNA was prepared. The Saccharomyces cerevisiae cells were made competent and transformed. The yeast was deposited in Leu-Yaman The Leu•sorbitol soft agar on the alcohol plate was incubated in the reverse direction for 3-5 曰. The colonies were streaked for separation on Leu-sorbitol plates, followed by 5 ml 5 X Leu. Ade•sorbitol With 1.6% grapes And 4% galactose was grown in vortex tube culture at 30 ° C to induce L1 transcription and protein expression. After 48-72 hours, the culture volume equal to OD600=10 was made into pellets, and the supernatant was removed. And the pellets were frozen and stored at -7 ° C. Example 3 95258.doc -27- 1334438

製備RNA 將經轉化之酵母菌細胞小球（其經半乳糖誘發作用誘發 以表現HPV 31 L1)在冰上解凍且懸浮於1 ml經DEPC-處理 之冷水中。藉由離心將細胞製成小球且移除所得上清液。 接著將該細胞小球再次懸浮於400 μΐ TES(pH 7.0之10 mM. Tris，10 mM EDTA及0.5% SDS)中。添加等體積之AE缓衝液 飽和之苯酚（50 mM NaOAc及10 mM EDTA)。將管渦旋10秒 且在65°C下加熱50分鐘且每隔10分鐘進行混合。接著將該 管置放於冰上5分鐘，隨後在4°C下離心5分鐘。將上清液收 集且轉移至一無菌管中。添加額外之400 μΐ苯酚、將該管渦 旋、置放於冰上5分鐘且進行離心。將上清液轉移至一無菌 管中且添加入4〇0 μΐ氯仿、將其混合且進行離心。將上清液 再次收集且轉移至一無菌管中且除了添加1 ml 1 00% EtOH 外還添加pH 5.2之40 μΐ 3 Μ乙酸鈉。將該管置放於乾冰上1 小時，其後將該管高速離心以將RNA製成小球。以70% EtOH洗滌RNA—次且使其風乾。接著將RNA懸浮於100 μΐ 經DEPC處理之水中且在65 °C下加熱5分鐘至溶解。假定當 八260/280為1.7-2.0日寺八260讀數為1=40 pg/ml執行分光光度 法以測定樣本中之RNA濃度。 實例4 北方墨點分析 表現3 1 L1野生型之酵母菌的初始分析說明HP V 3 1 L1蛋 白質之表現量顯著小於預期。為確定發生低表現是否歸因 於一轉錄水平對比翻譯水平之問題，執行了 HP V 3 1 L1轉錄 95258.doc -28- 1334438 體之北方墨點分析。由凝膠製成北方墨點，其中以相同凝 膠上來自表現HPV31 L1之酵母菌的RNA運行來自表現Hpv 16 L1之酵母菌的rnA以比較轉錄體尺寸。 熔煉（cast) 了 1.2%瓊脂糖甲醛凝膠。將十微克RNA與變性 緩衝液組合（最終濃度：6%甲醛、45%f醯胺與〇9xM〇ps) 且在55C下加熱15分鐘。添加1/1〇體積之裝載凝膠缓衝液 且將樣本裝載於凝膠上。在65伏下於i χ M〇ps緩衝液中執 行電泳〜5小時。將凝膠在無菌水中洗滌丨5分鐘隨後在丨〇 X SSC中洗滌5分鐘兩次。藉由毛細管作用在1〇 χ SS(：中經16 小時將RNA轉移至一 Hyb〇nd-N+尼龍薄膜（Amersham -Bi〇SCiences’ Piscataway，NJ)。接著藉由使用 Amersham交聯 劑裝置以700能量單位進行交聯來將RNA固定於尼龍薄 膜。固定之後，使尼龍薄膜風乾。在55。〇下將該薄膜置放 於30 ml Zetaprobe緩衝液中兩小時，其後添加經32p_標記之 探針且在53-65°C下培育16小時。接著將該薄膜於5 χ SS(： 中在室溫下洗滌20分鐘3次、隨後於〇 4 χ ssc中在室溫下洗 滌20分鐘兩次且在60°C下洗滌1〇分鐘一次。由使用Hpv 31 L1序列特殊同義及反義引子之PCR來生成探針DNA。藉由 以聚核苷酸激酶（PNK)與γ-32Ρ ATP在37°C下處理1小時來 標記放大之DNA。該墨點包裹於保鮮膜（saran wrap)中且曝 露於χ-射線膠片16小時。沖洗膠片時偵測在自動射線照相 上呈黑帶之經雜交探針之RNA。 上述之北方墨點分析揭示大多數全31 L1野生型 轉錄體顯著小於全長（參見圖4)。然而，將3丨L丨部分重建設 95258.doc -29- 1334438 計為不僅在該基因之中部嵌入受酵母菌首選之密碼子，而 且消除任何類似酵母菌轉錄終止位點之潛在序列。北方墨 點分析法清晰地顯示了經重建時，該31 L1基因轉錄體之長 度已顯著增加至與全長HPV 16 L1轉錄體（未圖示）之長度對 應之尺寸。因此，未成熟轉錄終止可能已占顯著部分之來 自31 L1野生型構造的低表現量。 實例5 HPV 31 L1蛋白質表現 等於OD600 = 1 〇之經半乳糖誘發的培養基之冷凍酵母菌 細胞小球在冰上經解凍且懸浮於3〇〇 μ1 pc緩衝液（1〇〇Preparation of RNA The transformed yeast cell pellets (which were induced by galactose induction to express HPV 31 L1) were thawed on ice and suspended in 1 ml of DEPC-treated cold water. The cells were pelleted by centrifugation and the resulting supernatant was removed. The cell pellet was then resuspended in 400 μΐ TES (10 mM. Tris, 10 mM EDTA and 0.5% SDS, pH 7.0). Add equal volumes of AE buffer saturated phenol (50 mM NaOAc and 10 mM EDTA). The tube was vortexed for 10 seconds and heated at 65 ° C for 50 minutes and mixed every 10 minutes. The tube was then placed on ice for 5 minutes and then centrifuged at 4 °C for 5 minutes. The supernatant was collected and transferred to a sterile tube. An additional 400 μM of phenol was added, the tube was vortexed, placed on ice for 5 minutes and centrifuged. The supernatant was transferred to a sterile tube and added to 4 〇 ΐ chloroform, mixed and centrifuged. The supernatant was again collected and transferred to a sterile tube and 40 μΐ 3 Μ sodium acetate, pH 5.2, was added in addition to 1 ml of 100% EtOH. The tube was placed on dry ice for 1 hour, after which the tube was centrifuged at high speed to make RNA into pellets. RNA was washed with 70% EtOH and allowed to air dry. The RNA was then suspended in 100 μM DEPC-treated water and heated at 65 °C for 5 minutes until dissolved. It is assumed that when eight 260/280 is 1.7-2.0, Temple 8 260 reads 1 = 40 pg/ml to perform spectrophotometry to determine the RNA concentration in the sample. Example 4 Northern blot analysis Initial analysis of the yeast expressing 3 1 L1 wild type indicated that the performance of HP V 3 1 L1 protein was significantly less than expected. To determine whether low performance was attributed to a level of transcription versus translation, a northern blot analysis of HP V 3 1 L1 transcription 95258.doc -28-1334438 was performed. A northern ink dot was prepared from the gel in which rnA from the yeast expressing Hpv 16 L1 was run with RNA from the yeast expressing HPV31 L1 on the same gel to compare the transcript size. A 1.2% agarose formaldehyde gel was cast. Ten micrograms of RNA was combined with denaturing buffer (final concentration: 6% formaldehyde, 45% f guanamine and 〇9 x M 〇 ps) and heated at 55 C for 15 minutes. Add 1/1 inch volume of loading gel buffer and load the sample onto the gel. Electrophoresis was carried out in i χ M〇ps buffer at 65 volts for ~5 hours. The gel was washed in sterile water for 5 minutes and then washed twice in 丨〇X SSC for 5 minutes. The RNA was transferred to a Hyb〇nd-N+ nylon film (Amersham-Bi〇SCiences' Piscataway, NJ) by capillary action in 1 〇χ SS (: 16 hours. Then by using the Amersham crosslinker device to 700 The energy unit was cross-linked to fix the RNA to the nylon membrane. After fixation, the nylon membrane was air-dried. The membrane was placed in 30 ml Zetaprobe buffer for two hours at 55., followed by the addition of 32p-labeled probe. The needle was incubated for 16 hours at 53-65 ° C. The film was then washed 3 times in 5 χ SS (: at room temperature for 20 minutes, then washed twice in 〇 4 χ ssc for 20 minutes at room temperature And washing once every 1 minute at 60 ° C. The probe DNA was generated by PCR using the Hpv 31 L1 sequence specific synonymous and antisense primers. By using polynucleotide kinase (PNK) and γ-32Ρ ATP at 37 The amplified DNA was labeled by treatment at ° C for 1 hour. The ink dots were wrapped in saran wrap and exposed to xenon-ray film for 16 hours. When the film was processed, it was detected as a black band hybridization on autoradiography. Probe RNA. The above northern blot analysis reveals most of the full 31 L1 wild type The transcript was significantly smaller than the full length (see Figure 4). However, the construction of the 3丨L丨 part 95258.doc -29-1334438 was counted as not only embedding the yeast preferred codon in the middle of the gene, but also eliminating any similar yeast. The potential sequence of the transcription termination site of the bacterium. The Northern blot analysis clearly shows that the length of the 31 L1 transcript has been significantly increased to correspond to the length of the full-length HPV 16 L1 transcript (not shown) upon reconstitution. Size. Therefore, immature transcription termination may have accounted for a significant portion of the low performance from the 31 L1 wild-type construct. Example 5 HPV 31 L1 protein performance equals OD600 = 1 〇 galactose-induced medium of frozen yeast cells is small The ball was thawed on ice and suspended in 3 μl of pc buffer (1〇〇

Na2HP〇4及 〇·5 M NaCl，pH 7.0)與 2 mM PMSF 中。添加酸洗 之0.5 mm玻璃珠，〜〇.5g/管。在4它下渦旋該等管15分鐘。 添加7.5 μΐ 20%之TritonXIOO且在4°C下重複渦旋5分鐘。將 該等官置放於冰上15分鐘，接著在4°C下離心15分鐘。將上 清液轉移至一無菌微型離心管中且貯藏於_7〇它。 實例6 西方墨點分析 自20至40個為之的全部由西方墨點法分析了每一 ΗΡν 3 j L1構造之來自二十至四十個經分離之酵母菌菌落的全部酵 母菌蛋白質提取物之以在半乳糖誘導之後證實HP v 3 1 l i 蛋白質之表現。 將十微克全部酵母菌蛋白質提取物與SDS-PAGE裝栽緩 衝液相組合且在95°C下加熱1〇分鐘。將蛋白質裝載於8% SDS-PAGE凝膠上且在Tris-甘胺酸缓衝液中進行電泳。蛋白 95258.doc -30· 1334438 質刀離之後，將蛋白質自凝膠經西方轉化至硝化纖維且將 墨點封阻於在TTBS(Tris緩衝生理食鹽水與Tween 2〇)中之 10%無脂乳粉中16小時。將墨點於TTBS中洗滌3次。施用山 羊抗-trpE-HPV 16 L1血清（一種與扭^ 31 u交互反應之多 - 純系血清）在TTBS中1:1〇00之稀釋液於室溫下丄小時。在 · TTBS中洗蘇該墨點3次且抗山羊-HRp共軛之抗體以在 TTBS中1 : 2500之稀釋液丨小時。墨點再次洗滌3次且使用 ECLtm偵測劑（Amersham Bi〇sciences，^咖隱^ 間。接· 著執行自動射線照相術。在自動射線照相上由偵測劑使抗 灰清所辨識之蛋白質顯現為黑帶。 於所有狀況中，在自動射線照相上偵測Hpv31 L1蛋白質 為對應於大約55 kD(資料未圖示）之獨特帶。將Hpv丨6 L} - 蛋白質作為凝膠上之陽性質控而包括在内。 實例7 放射免疫檢定法（RIA) >由多種方法來生長表現HPV31L1之酵母菌細胞’包括渦 # 旋s培養、振盪培養瓶及發酵器。溶解酵母菌且製備蛋白 質提取物以測定每毫克全部蛋白質令所製備之Hpv 3丨 似病毒粒子（VLP)量。為證明HPV 31 LI VLP表現，由捕獲 放射免疫檢定法（RIA)分析了每種全部酵母菌蛋白質提取 物之一部分。 0 使用偵測單純系抗體H31.A6、意即HPV型31-特異型及 . VLP構型-特異型來執行RIA。H31 A6對31 u具有特 異性因為發現其結合無損傷2Hpv 31 li vLp且不辨識變 95258.doc -31 · 1334438 性之HPV 31 VLP。隨後可藉由一以1125放射性同位素標記 之山羊抗小鼠抗體來偵測該mAb。因此，每分鐘記數（cpm) 值對應於相對水平之HPV31 LI VLP表現。 以在PBS中以1:1000稀釋隔夜之山羊抗_trpE_HPV3i L1 多純系也清塗覆聚苯乙烯珠β接著以5體積之無菌蒸餾水洗. 務該等小珠且將其風乾》接著將該抗原、即來自分離之酵 母菌菌落的全部酵母菌蛋白質提取物藉由將其在PBs中以 1% BSA、0.1% Tween-20及〇.1%疊氮化鈉稀釋來裝載於該 等小珠上且以渦旋培育1小時。洗滌之後，將該等小珠在一 20-井聚苯乙烯板中每井分配一個且以丨：5〇,〇(j〇稀釋之 H31.A6 mAb在室溫下培育17_24小時。小珠洗蘇且在 23000-27000 cpm每1〇 μΐ之活性範圍添加經1125標記之山 羊抗小鼠IgG。2小時之後.，洗滌該等小珠且以cpm/ml記錄 放射性記數。自總cpm/ml中減去來自空白井之背景記數給 出扣除背景值之RIA。 執行兩個實驗.於實驗1中將來自31 L1野生型與31 L1部 分重建之蛋白質提取物作對比且於實驗2中將來自31匕丨部 为重建與31 L1全部重建之蛋白質提取物作對比（參見圖 5)。結果顯示31 L1部分重建VLP表現大於31 L1野生型69 倍。該31 L1全部重建具有超過該31 L1部分重建丨7倍之增 強表現。因此，藉由引入受酵母菌首選之密碼子序列及消 除潛在轉錄終止訊號使該等31 L1表現水平增加了 > 7倍。 實例8 透射電子顯微鏡 95258.doc -32· 1334438 為證明HPV 31 L1蛋白質事實上自我組合形成五員 (pentamericO-L i殼粒該等殼粒依次自我組合至似病毒粒子 中’-部分經純化之31 L1全部重建蛋白質提取物經：透射 電子顯微鏡（TEM)。將酵母菌在小規模發酵下生長且製成小 球。該等小球經受純化處理。由免疫墨點分析該等小球及 經淨化之酵母菌提取物以證明經由該純化程序之l丨蛋白質 表現及保持。接著經淨化之酵母菌經45%_蔗醣緩解液 (sucrose cushion)而經受離心且將所得小球懸浮於緩衝液 中用於TEM分析（參見圖6)。結果顯示在此粗製樣本中之該 等球狀粒子直徑範圍在3〇至60 nm之間且某些粒子展現殼 粒之規則排列。 【圖式簡單說明】 圖1為顯示經部分（SEQ ID NO:2)及全部重建（SEq ID NO··3) 3 1 L1基因改變之核苷酸的序列比對（參見實例2)。參 考序列為31 L1野生型序列（SEq ID ΝΟ:1、參見實例丨）。與 該參考序列同樣之部分及全部重建3 1 L1序列中之核苷酸 以點表示。經改變之核苷酸表示於其相應位置。核苷酸數 目含於括號内。 圖2顯示了 31 L1全部重建核苷（SEQ ID ΝΟ··3)及胺基酸序 列（SEQ ID ΝΟ:4)。核苷酸數目在左側表示。 圖3總結列於左側之三個HP V 3 1 L1序列構造之間的變 化。第四行表示所表示之構造與3 1 L 1野生型序列之間的百 分比核苷酸同一性且第五行表示胺基酸同一性。最後一行 表示經改變為受酵母菌首選之密碼子序列的核苷酸數量及 95258.doc -33- 1334438 進行改變之區域。 圖4顯示在高嚴謹性下具體對HP V3 1 L1進行探測之北方 墨點法（參見實例4)。左側箭頭表示HPV3 1 L1之全長及截短 轉錄體之位置。標s己為3 1 wt"之色帶（lane)來自含31 L1野 生型序列之酵母菌的相同RNA製劑。標記為，，16"之色帶含 有來自HPV16之RNA，其由於高嚴謹性條件而未受HpV31 L1探針辨識。標記為，’Neg”之色帶為不含有L1編碼序列之酵 母菌提取物》標記為”3 1 R”之色帶來自表現3丨L丨部分重建 序列之兩個經分開分離之菌落的Rna。 圖5顯示一部分來自兩個捕獲放射免疫檢定法（ria)實驗 以每分鐘記數（cpm)/毫克全部蛋白質表示之資料（參見實例 7)。在RIA中得到之cpm為HPV 31 L1 VLP之相對指標。該 等RIA資料證明来自受酵母菌首選之經密碼子重建之基因 序列的酵母菌蛋白質提取物中增強之31 L1 VLp表現。 圖6顯示如透射電子顯微鏡所顯現本文所述之3ι [丨 的代表性樣本（見實例8)。橫棒代表1〇〇 nm。 95258.doc •34· 序列表 <110> Merck & Co., Inc·Na2HP〇4 and 〇·5 M NaCl, pH 7.0) and 2 mM PMSF. Add pickled 0.5 mm glass beads, ~〇5g/tube. The tubes were vortexed under 4 for 15 minutes. 7.5 μΐ 20% Triton XIOO was added and vortexed for 5 minutes at 4 °C. The officers were placed on ice for 15 minutes and then centrifuged at 4 °C for 15 minutes. The supernatant was transferred to a sterile microcentrifuge tube and stored at _7 〇. Example 6 Western blot analysis All of the yeast protein extracts from twenty to forty isolated yeast colonies of each ΗΡν 3 j L1 construct were analyzed by Western blotting methods from 20 to 40 This confirmed the performance of the HP v 3 1 li protein after galactose induction. Ten micrograms of all yeast protein extracts were combined with SDS-PAGE loading buffer and heated at 95 °C for 1 minute. The protein was loaded onto an 8% SDS-PAGE gel and electrophoresed in Tris-glycine buffer. Protein 95258.doc -30· 1334438 After the knife is separated, the protein is converted from the gel to the nitrocellulose by the gel and the ink dots are blocked in 10% fat-free in TTBS (Tris buffered saline and Tween 2). 16 hours in the milk powder. The dots were washed 3 times in TTBS. The goat anti-trpE-HPV 16 L1 serum (a type of interaction with the twisted ^ 31 u - pure serum) was applied to a dilution of 1:1 00 in TTBS at room temperature for one hour. The ink spot was washed 3 times in the TTBS and the anti-goat-HRp conjugated antibody was incubated for 1 hour in the TTBS. The ink dots were washed 3 times again and the ECLtm detection agent (Amersham Bi〇sciences, ^ 隐 ^ 。 。 执行 执行 执行 执行 执行 执行 执行 执行 执行 执行 执行 执行 执行 执行 执行 执行 执行 执行 执行 执行 执行 执行 执行 执行 执行 执行 执行 执行 执行 执行 执行 执行 执行 执行 执行 执行 执行 执行Appears as a black band. In all cases, Hpv31 L1 protein was detected on autoradiography as a unique band corresponding to approximately 55 kD (data not shown). Hpv丨6 L} - protein was used as the yang on the gel Example 7 Radioimmunoassay (RIA) > Growth of yeast cells expressing HPV31L1 by a variety of methods including vortex s culture, shaking flasks and fermenters. Dissolving yeast and preparing proteins The extract was assayed for the amount of Hpv 3 病毒-like virus particles (VLP) prepared per mg of total protein. To demonstrate HPV 31 LI VLP performance, each of the yeast protein extracts was analyzed by capture radioimmunoassay (RIA). Part of it. 0 RIA was performed using the detection of the simple antibody H31.A6, meaning HPV type 31-specific and VLP configuration-specific. H31 A6 is specific for 31 u because its binding is found to be non-invasive 2Hpv 3 1 li vLp does not recognize the HPV 31 VLP of 95258.doc -31 · 1334438. The mAb can then be detected by a 1125 radioisotope-labeled goat anti-mouse antibody. Therefore, the count per minute (cpm The values correspond to the relative levels of HPV31 LI VLP performance. The goat anti- _trpE_HPV3i L1 multi-pure multi-pure coated polystyrene beads were diluted 1:1000 in PBS and then washed with 5 volumes of sterile distilled water. Waiting for the beads and air drying them, then the antigen, ie all yeast protein extracts from the isolated yeast colonies, by stacking them in PBs with 1% BSA, 0.1% Tween-20 and 〇.1% Sodium nitride was diluted to be loaded on the beads and incubated for 1 hour with vortexing. After washing, the beads were dispensed one well per well in a 20-well polystyrene plate and 丨: 5 〇, 〇 (j〇 diluted H31.A6 mAb was incubated for 17-24 hours at room temperature. Beads were washed and 1125-labeled goat anti-mouse IgG was added at an activity range of 23,000-27000 cpm per 1 μM. After 2 hours. The beads were washed and the radioactivity count was recorded in cpm/ml. Subtracted from the total cpm/ml from the blank The background count gives the RIA minus the background value. Two experiments were performed. In the experiment 1, the 31 L1 wild type was compared with the 31 L1 partially reconstituted protein extract and in Experiment 2 it was from the 31 part. Reconstruction was compared to the 31 L1 fully reconstituted protein extract (see Figure 5). The results showed that the 31 L1 partially reconstructed VLP showed 69 times more than the 31 L1 wild type. The 31 L1 full reconstruction has an 7-fold increase in performance over the 31 L1 partial reconstruction. Therefore, these 31 L1 expression levels were increased by > 7 times by introducing a codon sequence preferred by yeast and eliminating potential transcription termination signals. Example 8 Transmission Electron Microscopy 95258.doc -32· 1334438 To demonstrate that the HPV 31 L1 protein actually self-assembles to form five members (pentamericO-L i shell particles, which are self-assembled into virions in turn - part purified) 31 L1 all reconstructed protein extracts by transmission electron microscopy (TEM). Yeasts were grown under small-scale fermentation and made into pellets. The pellets were subjected to purification treatment. The pellets were analyzed by immunoblots. Purified yeast extract to demonstrate and maintain the protein via the purification procedure. The purified yeast is then subjected to centrifugation via a 45% sucrose cushion and the resulting pellet is suspended in a buffer. Used for TEM analysis (see Figure 6). The results show that the spherical particles in this crude sample range in diameter from 3〇 to 60 nm and some particles exhibit a regular arrangement of shell particles. Figure 1 is a sequence alignment showing nucleotides altered by partial (SEQ ID NO: 2) and all reconstituted (SEq ID NO··3) 3 1 L1 genes (see Example 2). The reference sequence is 31 L1 wild. Type sequence (SEq ID ΝΟ: 1, see example 丨). The same part and all of the same as the reference sequence. The nucleotides in the 3 1 L1 sequence are represented by dots. The changed nucleotides are represented at their corresponding positions. In parentheses, Figure 2 shows 31 L1 all reconstituted nucleosides (SEQ ID ΝΟ··3) and amino acid sequence (SEQ ID ΝΟ: 4). The number of nucleotides is shown on the left. Figure 3 is summarized on the left side. A change between the three HP V 3 1 L1 sequence constructs. The fourth row indicates the percent nucleotide identity between the constructed construct and the 3 1 L 1 wild type sequence and the fifth row indicates the amino acid identity. One row indicates the number of nucleotides changed to the codon sequence preferred by yeast and the region changed by 95258.doc -33- 1334438. Figure 4 shows the northern ink specifically for HP V3 1 L1 under high stringency Point method (see Example 4). The left arrow indicates the full length of HPV3 1 L1 and the position of the truncated transcript. The label is 3 1 wt" The band is from the same yeast containing 31 L1 wild type sequence RNA preparation. Labeled as, 16" ribbon containing HPV16 RNA, which is not recognized by the HpV31 L1 probe due to high stringency conditions. The band labeled 'Neg' is a yeast extract that does not contain the L1 coding sequence. The band labeled "3 1 R" is derived from the performance. 3丨L丨 Partially reconstructed sequence of two separately isolated colonies of Rna. Figure 5 shows a portion of data from two capture radioimmunoassay (ria) experiments expressed in minutes per minute (cpm) / mg of total protein ( See example 7). The cpm obtained in the RIA is a relative indicator of the HPV 31 L1 VLP. These RIA data demonstrate enhanced 31 L1 VLp expression in yeast protein extracts from the codon-reconstructed gene sequences preferred by yeast. Figure 6 shows a representative sample of 3ι [丨 (see Example 8) as described herein as seen by transmission electron microscopy. The horizontal bar represents 1 〇〇 nm. 95258.doc •34· Sequence Listing <110> Merck & Co., Inc·

Jansen, Kathrin U.Jansen, Kathrin U.

Schultz, Loren D.Schultz, Loren D.

Neeper, Michael P.Neeper, Michael P.

Markus, Henry Z. <120>酵母菌中HPV 31 LI之最適化表現Markus, Henry Z. <120> Optimal performance of HPV 31 LI in yeast

<130> 21188-PCT <140> 093124870 <141> 2004-08-18 <150> 60/457,172 <151〉 2003-03-24 <160> 8 <170> FastSEQ for Windows Version 4.0 > > > > 0 12 3 111 i—I 2 2 2 2 < < < < 1515<130> 21188-PCT <140> 093124870 <141> 2004-08-18 <150> 60/457,172 <151> 2003-03-24 <160> 8 <170> FastSEQ for Windows Version 4.0 >>>> 0 12 3 111 i—I 2 2 2 2 <<<< 1515

DNA HPV31 L1野生型 <400> 1 atgtctctgt ggcggcctag cgaggctact gtctacttac cacctgtccc agtgtctaaa 60 gttgtaagca cggatgaata tgtaacacga accaacatat attatcacgc aggcagtgct 120 aggctgctta cagtaggcca tccatattat tccataccta aatctgacaa tcctaaaaaa 180 atagttgtac caaaggtgtc aggattacaa tatagggtat ttagggttcg tttaccagat 240 ccaaacaaat ttggatttcc tgatacatct ttttataatc ctgaaactca acgcttagtt 300 tgggcctgtg ttggtttaga ggtaggtcgc gggcagccat taggtgtagg tattagtggt 360 catccattat taaataaatt tgatgacact gaaaactcta atagatatgc cggtggtcct 420 ggcactgata atagggaatg tatatcaatg gattataaac aaacacaact gtgtttactt 480 ggttgcaaac cacctattgg agagcattgg ggtaaaggta gtccttgtag taacaatgct 540 attacccctg gtgattgtcc tccattagaa ttaaaaaatt cagttataca agatggggat 600 atggttgata caggctttgg agctatggat tttactgctt tacaagacac taaaagtaat 660 gttcctttgg acatttgtaa ttctatttgt aaatatccag attatcttaa aatggttgct 720 gagccatatg gcgatacatt atttttttat ttacgtaggg aacaaatgtt tgtaaggcat 780 ttttttaata gatcaggcac ggttggtgaa tcggtcccta ctgacttata tattaaaggc 840 tccggttcaa cagctacttt agctaacagt acatactttc ctacacctag cggctccatg 900 gttacttcag atgcacaaat ttttaataaa ccatattgga tgcaacgtgc tcagggacac 960 aataatggta tttgttgggg caatcagtta tttgttactg tggtagatac cacacgtagt 1020 accaatatgt ctgtttgtgc tgcaattgca aacagtgata ctacatttaa aagtagtaat 1080 tttaaagagt atttaagaca tggtgaggaa tttgatttac aatttatatt tcagttatgc 1140 aaaataacat tatctgcaga cataatgaca tatattcaca gtatgaatcc tgctattttg 1200 gaagattgga attttggatt gaccacacct ccctcaggtt ctttggagga tacctatagg 1260 tttgtaacct cacaggccat tacatgtcaa aaaagtgccc cccaaaagcc caaggaagat 1320 ccatttaaag attatgtatt ttgggaggtt aatttaaaag aaaagttttc tgcagattta 1380 gatcagtttc cactgggtcg caaattttta ttacaggcag gatatagggc acgtcctaaa 1440 tttaaagcag gtaaacgtag tgcaccctca gcatctacca ctacaccagc aaaacgtaaa 1500 aaaactaaaa agtaa 1515 95258 1334438 <210> 2 <211> 1515 <212> DNA <213>人工序列 <220> <223> 31部分重建 <400> 2 atgtctctgt gttgtaagca aggctgctta atagttgtac ccaaacaaat tgggcctgtg catccattat ggcactgata ggttgcaaac attacccctg atggttgata gttcctttgg gagccatacg ttcttcaaca tccggttcca gtcacctccg aacaacggta accaacatgt ttcaaggagt aagatcaccc gaggactgga tttgtaacct ccatttaaag gatcagtttc tttaaagcag aaaactaaaa ggcggcctag cggatgaata cagtaggcca caaaggtgtc ttggatttcc ttggtttaga taaataaatt atagggaatg cacctattgg gtgattgtcc caggctttgg acatttgtaa gcgacacctt gatccggcac ccgctaccct acgctcagat tctgttgggg ctgtctgtgc acctgagaca tgtctgctga acttcggtct cacaggccat attatgtatt cactgggtcg gtaaacgtag agtaa cgaggctact tgtaacacga tccatattat aggattacaa tgatacatct ggtaggtcgc tgatgacact tatatcaatg agagcattgg tccattagaa agctatggat ttctatttgt gttcttctat cgtaggtgaa ggctaactcc cttcaacaag taaccagctg tgcaatcgct tggtgaggaa catcatgacc gaccactcca tacatgtcaa ttgggaggtt caaattttta tgcaccctca gtctacttac accaacatat tccataccta tatagggtat ttttataatc gggcagccat gaaaactcta gattataaac ggtaaaggta ttaaaaaatt tttactgctt aaatatccag ttgcgtagag tctgtcccaa acctacttcc ccatactgga ttcgtgactg aactctgaca ttcgatctgc tacatccaca ccttccggtt aaaagtgccc aatttaaaag ttacaggcag gcatctacca cacctgtccc attatcacgc aatctgacaa ttagggttcg ctgaaactca taggtgtagg atagatatgc aaacacaact gtccttgtag cagttataca tacaagacac attatcttaa aacagatgtt ccgacctgta caactccatc tgcagcgtgc tggtcgatac ctaccttcaa aattcatctt gtatgaaccc ctttggagga cccaaaagcc aaaagttttc gatatagggc ctacaccagc agtgtctaaa aggcagtgct tcctaaaaaa tttaccagat acgcttagtt tattagtggt cggtggtcct gtgtttactt taacaatgct agatggggat taaaagtaat aatggttgct cgtaaggcac catcaagggc tggctccatg acagggtcac cacgcgttct gtcctctaac ccagttgtgc tgccatcctg tacctatagg caaggaagat tgcagattta acgtcctaaa aaaacgtaaa 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1515 <210> 3 <211> 1515 <212> DNA <213>人工序列 <220> <223> 31全部重建 <400〉 3 atgtctttgt ggagaccatc tgaagctacc gtctacttgc caccagtccc agtctctaag 60 gtcgtctcta ccgacgaata cgtcaccaga accaacatct actaccacgc tggttctgct 120 agattgttga ccgtcggtca cccatactac tctatcccaa agtctgacaa cccaaagaag 180 atcgtcgtcc caaaggtctc tggtttgcaa tacagagtct tcagagtcag attgccagac 240 ccaaacaagt tcggtttccc agacacctct ttctacaacc cagaaaccca aagattggtc 300 tgggcttgtg tcggtttgga agtcggtaga ggtcaaccat tgggtgtcgg tatctctggt 360 cacccattgt tgaacaagtt cgacgacacc gaaaactcta acagatacgc tggtggtcca 420 ggtaccgaca acagagaatg tatctctatg gactacaagc aaacccaatt gtgtttgttg 480 ggttgtaagc caccaatcgg tgaacactgg ggtaagggtt ctccatgttc taacaacgct 540 -2- 95258 1334438 atcaccccag gtgactgtcc accattggaa ttgaagaact ctgtcatcca agacggtgac 600 atggtcgaca ccggtttcgg tgctatggac ttcaccgctt tgcaagacac caagtctaac 660 gtcccattgg acatctgtaa ctctatctgt aagtacccag actacttgaa gatggtcgct 720 gaaccatacg gcgacacctt gttcttctac ttgcgtagag aacagatgtt cgtaaggcac 780 ttcttcaaca gatccggcac cgtaggtgaa tctgtcccaa ccgacctgta catcaagggc 840 tccggttcca ccgctaccct ggctaactcc acctacttcc caactccatc tggctccatg 900 gtcacctccg acgctcagat cttcaacaag ccatactgga tgcagcgtgc acagggtcac 960 aacaacggta tctgttgggg taaccagctg ttcgtgactg tggtcgatac cacgcgttct 1020 accaacatgt ctgtctgtgc tgcaatcgct aactctgaca ctaccttcaa gtcctctaac 1080 ttcaaggagt acctgagaca tggtgaggaa ttcgatctgc aattcatctt ccagttgtgc 1140 aagatcaccc tgtctgctga catcatgacc tacatccaca gtatgaaccc tgccatcctg 1200 gaggactgga acttcggtct gaccactcca ccttccggtt ctttggaaga cacctacaga 1260 ttcgtcacct ctcaagctat cacctgtcaa aagtctgctc cacaaaagcc aaaggaagac 1320 ccattcaagg actacgtctt ctgggaagtc aacttgaagg aaaagttctc tgctgacttg 1380 gaccaattcc cattgggtag aaagttcttg ttgcaagctg gttacagagc tagaccaaag 1440 ttcaaggctg gtaagagatc tgctccatct gcttctacca ccaccccagc taagagaaag 1500 aagaccaaga agtaa 1515 <210> 4 <211> 504 <212> PRT <213>人工序列 <220> <223> HPV 31 LI <400> 4DNA HPV31 L1 wild-type < 400 > 1 atgtctctgt ggcggcctag cgaggctact gtctacttac cacctgtccc agtgtctaaa 60 gttgtaagca cggatgaata tgtaacacga accaacatat attatcacgc aggcagtgct 120 aggctgctta cagtaggcca tccatattat tccataccta aatctgacaa tcctaaaaaa 180 atagttgtac caaaggtgtc aggattacaa tatagggtat ttagggttcg tttaccagat 240 ccaaacaaat ttggatttcc tgatacatct ttttataatc ctgaaactca acgcttagtt 300 tgggcctgtg ttggtttaga ggtaggtcgc gggcagccat taggtgtagg tattagtggt 360 catccattat taaataaatt tgatgacact gaaaactcta atagatatgc cggtggtcct 420 ggcactgata atagggaatg tatatcaatg gattataaac aaacacaact gtgtttactt 480 ggttgcaaac cacctattgg agagcattgg ggtaaaggta gtccttgtag taacaatgct 540 attacccctg gtgattgtcc tccattagaa ttaaaaaatt cagttataca agatggggat 600 atggttgata caggctttgg agctatggat tttactgctt tacaagacac taaaagtaat 660 gttcctttgg acatttgtaa ttctatttgt aaatatccag attatcttaa aatggttgct 720 gagccatatg gcgatacatt atttttttat ttacgtaggg aacaaatgtt tgtaaggcat 780 ttttttaata gatcaggcac ggttggtgaa tcggtcccta ctgacttata tattaaaggc 840 tccggttcaa cagctacttt agctaacagt acatactttc ctacacctag cggctccatg 900 gttacttcag atgcacaaat ttttaataaa ccatattgga tgcaacgtgc tcagggacac 960 aataatggta tttgttgggg caatcagtta tttgttactg tggtagatac cacacgtagt 1020 accaatatgt ctgtttgtgc tgcaattgca aacagtgata ctacatttaa aagtagtaat 1080 tttaaagagt atttaagaca tggtgaggaa tttgatttac aatttatatt tcagttatgc 1140 aaaataacat tatctgcaga cataatgaca tatattcaca gtatgaatcc tgctattttg 1200 gaagattgga attttggatt gaccacacct ccctcaggtt ctttggagga tacctatagg 1260 tttgtaacct cacaggccat tacatgtcaa aaaagtgccc cccaaaagcc caaggaagat 1320 ccatttaaag attatgtatt ttgggaggtt aatttaaaag aaaagttttc tgcagattta 1380 gatcagtttc cactgggtcg caaattttta ttacaggcag gatatagggc acgtcctaaa 1440 tttaaagcag gtaaacgtag tgcaccctca gcatctacca ctacaccagc aaaacgtaaa 1500 aaaactaaaa agtaa 1515 95258 1334438 < 210 > 2 < 211 > 1515 < 212 > DNA <213>Artificialsequence<220><223> 31 partial reconstruction <400> 2 atgtctctgt gttgtaagca aggctgctta atagttgtac cc aaacaaat tgggcctgtg catccattat ggcactgata ggttgcaaac attacccctg atggttgata gttcctttgg gagccatacg ttcttcaaca tccggttcca gtcacctccg aacaacggta accaacatgt ttcaaggagt aagatcaccc gaggactgga tttgtaacct ccatttaaag gatcagtttc tttaaagcag aaaactaaaa ggcggcctag cggatgaata cagtaggcca caaaggtgtc ttggatttcc ttggtttaga taaataaatt atagggaatg cacctattgg gtgattgtcc caggctttgg acatttgtaa gcgacacctt gatccggcac ccgctaccct acgctcagat tctgttgggg ctgtctgtgc acctgagaca tgtctgctga acttcggtct cacaggccat attatgtatt cactgggtcg gtaaacgtag agtaa cgaggctact tgtaacacga tccatattat aggattacaa tgatacatct ggtaggtcgc tgatgacact tatatcaatg agagcattgg tccattagaa agctatggat ttctatttgt gttcttctat cgtaggtgaa ggctaactcc cttcaacaag taaccagctg tgcaatcgct tggtgaggaa catcatgacc gaccactcca tacatgtcaa ttgggaggtt caaattttta tgcaccctca gtctacttac accaacatat tccataccta tatagggtat ttttataatc gggcagccat gaaaactcta gattataaac ggtaaaggta ttaaaaaatt tttactgctt aaatatccag ttgcgtagag tctgtcccaa acctacttcc ccatactgga ttcgtgactg aactctgaca ttcgat ctgc tacatccaca ccttccggtt aaaagtgccc aatttaaaag ttacaggcag gcatctacca cacctgtccc attatcacgc aatctgacaa ttagggttcg ctgaaactca taggtgtagg atagatatgc aaacacaact gtccttgtag cagttataca tacaagacac attatcttaa aacagatgtt ccgacctgta caactccatc tgcagcgtgc tggtcgatac ctaccttcaa aattcatctt gtatgaaccc ctttggagga cccaaaagcc aaaagttttc gatatagggc ctacaccagc agtgtctaaa aggcagtgct tcctaaaaaa tttaccagat acgcttagtt tattagtggt cggtggtcct gtgtttactt taacaatgct agatggggat taaaagtaat aatggttgct cgtaaggcac catcaagggc tggctccatg acagggtcac cacgcgttct gtcctctaac Ccagttgtgc tgccatcctg tacctatagg caaggaagat tgcagattta acgtcctaaa aaaacgtaaa 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1515 <210> 3 <211> 1515 <212> DNA <213>Artificialsequence<220><223> 31 all reconstruction <400> 3 atgtctttgt ggagaccatc tgaagctacc gtctacttgc caccagtccc agtctctaag 60 gtcgtctcta ccgacgaata cgtcaccaga accaacatct actaccacgc tggttctgct 120 agat tgttga ccgtcggtca cccatactac tctatcccaa agtctgacaa cccaaagaag 180 atcgtcgtcc caaaggtctc tggtttgcaa tacagagtct tcagagtcag attgccagac 240 ccaaacaagt tcggtttccc agacacctct ttctacaacc cagaaaccca aagattggtc 300 tgggcttgtg tcggtttgga agtcggtaga ggtcaaccat tgggtgtcgg tatctctggt 360 cacccattgt tgaacaagtt cgacgacacc gaaaactcta acagatacgc tggtggtcca 420 ggtaccgaca acagagaatg tatctctatg gactacaagc aaacccaatt gtgtttgttg 480 ggttgtaagc caccaatcgg tgaacactgg ggtaagggtt ctccatgttc taacaacgct 540 - 2- 95258 1334438 atcaccccag gtgactgtcc accattggaa ttgaagaact ctgtcatcca agacggtgac 600 atggtcgaca ccggtttcgg tgctatggac ttcaccgctt tgcaagacac caagtctaac 660 gtcccattgg acatctgtaa ctctatctgt aagtacccag actacttgaa gatggtcgct 720 gaaccatacg gcgacacctt gttcttctac ttgcgtagag aacagatgtt cgtaaggcac 780 ttcttcaaca gatccggcac cgtaggtgaa tctgtcccaa ccgacctgta catcaagggc 840 tccggttcca ccgctaccct ggctaactcc acctacttcc caactccatc tggctccatg 900 gtcacctccg acgctcagat cttcaacaag ccatactgga Tgcagcgtgc acagggtcac 960 aacaac ggta tctgttgggg taaccagctg ttcgtgactg tggtcgatac cacgcgttct 1020 accaacatgt ctgtctgtgc tgcaatcgct aactctgaca ctaccttcaa gtcctctaac 1080 ttcaaggagt acctgagaca tggtgaggaa ttcgatctgc aattcatctt ccagttgtgc 1140 aagatcaccc tgtctgctga catcatgacc tacatccaca gtatgaaccc tgccatcctg 1200 gaggactgga acttcggtct gaccactcca ccttccggtt ctttggaaga cacctacaga 1260 ttcgtcacct ctcaagctat cacctgtcaa aagtctgctc cacaaaagcc aaaggaagac 1320 ccattcaagg actacgtctt ctgggaagtc aacttgaagg aaaagttctc tgctgacttg 1380 gaccaattcc Cattgggtag aaagttcttg ttgcaagctg gttacagagc tagaccaaag 1440 ttcaaggctg gtaagagatc tgctccatct gcttctacca ccaccccagc taagagaaag 1500 aagaccaaga agtaa 1515 <210> 4 <211> 504 <212> PRT <213> artificial sequence <220><223> HPV 31 LI <400> 4

Met Ser Leu Trp Arg Pro Ser Glu Ala Thr Val Tyr Leu Pro Pro Val 15 10 15Met Ser Leu Trp Arg Pro Ser Glu Ala Thr Val Tyr Leu Pro Pro Val 15 10 15

Pro Val Ser Lys Val Val Ser Thr Asp Glu Tyr Val Thr Arg Thr Asn 20 25 30 lie Tyr Tyr His Ala Gly Ser Ala Arg Leu Leu Thr Val Gly His Pro 35 40 45Pro Val Ser Lys Val Val Ser Thr Asp Glu Tyr Val Thr Arg Thr Asn 20 25 30 lie Tyr Tyr His Ala Gly Ser Ala Arg Leu Leu Thr Val Gly His Pro 35 40 45

Tyr Tyr Ser lie Pro Lys Ser Asp Asn Pro Lys Lys lie Val Val Pro 50 55 60Tyr Tyr Ser lie Pro Lys Ser Asp Asn Pro Lys Lys lie Val Val Pro 50 55 60

Lys Val Ser Gly Leu Gin Tyr Arg Val Phe Arg Val Arg Leu Pro Asp 65 70 75 80Lys Val Ser Gly Leu Gin Tyr Arg Val Phe Arg Val Arg Leu Pro Asp 65 70 75 80

Pro Asn Lys Phe Gly Phe Pro Asp Thr Ser Phe Tyr Asn Pro Glu Thr 85 90 95Pro Asn Lys Phe Gly Phe Pro Asp Thr Ser Phe Tyr Asn Pro Glu Thr 85 90 95

Gin Arg Leu Val Trp Ala Cys Val Gly Leu Glu Val Gly Arg Gly Gin 100 105 110Gin Arg Leu Val Trp Ala Cys Val Gly Leu Glu Val Gly Arg Gly Gin 100 105 110

Pro Leu Gly Val Gly lie Ser Gly His Pro Leu Leu Asn Lys Phe Asp 115 120 125Pro Leu Gly Val Gly lie Ser Gly His Pro Leu Leu Asn Lys Phe Asp 115 120 125

Asp Thr Glu Asn Ser Asn Arg Tyr Ala Gly Gly Pro Gly Thr Asp Asn 130 135 140Asp Thr Glu Asn Ser Asn Arg Tyr Ala Gly Gly Pro Gly Thr Asp Asn 130 135 140

Arg Glu Cys lie Ser Met Asp Tyr Lys Gin Thr Gin Leu Cys Leu Leu 145 150 155 160Arg Glu Cys lie Ser Met Asp Tyr Lys Gin Thr Gin Leu Cys Leu Leu 145 150 155 160

Gly Cys Lys Pro Pro lie Gly Glu His Trp Gly Lys Gly Ser Pro Cys 165 170 175Gly Cys Lys Pro Pro lie Gly Glu His Trp Gly Lys Gly Ser Pro Cys 165 170 175

Ser Asn Asn Ala lie Thr Pro Gly Asp Cys Pro Pro Leu Glu Leu Lys 180 185 190Ser Asn Asn Ala lie Thr Pro Gly Asp Cys Pro Pro Leu Glu Leu Lys 180 185 190

Asn Ser Val He Gin Asp Gly Asp Met Val Asp Thr Gly Phe Gly Ala 195 200 205Asn Ser Val He Gin Asp Gly Asp Met Val Asp Thr Gly Phe Gly Ala 195 200 205

Met Asp Phe Thr Ala Leu Gin Asp Thr Lys Ser Asn Val Pro Leu Asp 210 215 220 95258 lie Cys Asn Ser lie Cys Lys Tyr Pro Asp Tyr Leu Lys Met Val Ala 225 230 235 240Met Asp Phe Thr Ala Leu Gin Asp Thr Lys Ser Asn Val Pro Leu Asp 210 215 220 95258 lie Cys Asn Ser lie Cys Lys Tyr Pro Asp Tyr Leu Lys Met Val Ala 225 230 235 240

Glu Pro Tyr Gly Asp Thr Leu Phe Phe Tyr Leu Arg Arg Glu Gin Met 245 250 255Glu Pro Tyr Gly Asp Thr Leu Phe Phe Tyr Leu Arg Arg Glu Gin Met 245 250 255

Phe Val Arg His Phe Phe Asn Arg Ser Gly Thr Val Gly Glu Ser Val 260 265 270Phe Val Arg His Phe Phe Asn Arg Ser Gly Thr Val Gly Glu Ser Val 260 265 270

Pro Thr Asp Leu Tyr lie Lys Gly Ser Gly Ser Thr Ala Thr Leu Ala 275 280 285Pro Thr Asp Leu Tyr lie Lys Gly Ser Gly Ser Thr Ala Thr Leu Ala 275 280 285

Asn Ser Thr Tyr Phe Pro Thr Pro Ser Gly Ser Met Val Thr Ser Asp 290 295 300Asn Ser Thr Tyr Phe Pro Thr Pro Ser Gly Ser Met Val Thr Ser Asp 290 295 300

Ala Gin lie Phe Asn Lys Pro Tyr Trp Met Gin Arg Ala Gin Gly His 305 310 315 320Ala Gin lie Phe Asn Lys Pro Tyr Trp Met Gin Arg Ala Gin Gly His 305 310 315 320

Asn Asn Gly lie Cys Trp Gly Asn Gin Leu Phe Val Thr Val Val Asp 325 330 335Asn Asn Gly lie Cys Trp Gly Asn Gin Leu Phe Val Thr Val Val Asp 325 330 335

Thr Thr Arg Ser Thr Asn Met Ser Val Cys Ala Ala lie Ala Asn Ser 340 345 350Thr Thr Arg Ser Thr Asn Met Ser Val Cys Ala Ala lie Ala Asn Ser 340 345 350

Asp Thr Thr Phe Lys Ser Ser Asn Phe Lys Glu Tyr Leu Arg His Gly 355 360 365Asp Thr Thr Phe Lys Ser Ser Asn Phe Lys Glu Tyr Leu Arg His Gly 355 360 365

Glu Glu Phe Asp Leu Gin Phe lie Phe Gin Leu Cys Lys lie Thr Leu 370 375 380Glu Glu Phe Asp Leu Gin Phe lie Phe Gin Leu Cys Lys lie Thr Leu 370 375 380

Ser Ala Asp lie Met Thr Tyr lie His Ser Met Asn Pro Ala lie Leu 385 390 395 400Ser Ala Asp lie Met Thr Tyr lie His Ser Met Asn Pro Ala lie Leu 385 390 395 400

Glu Asp Trp Asn Phe Gly Leu Thr Thr Pro Pro Ser Gly Ser Leu Glu 405 410 415Glu Asp Trp Asn Phe Gly Leu Thr Thr Pro Pro Ser Gly Ser Leu Glu 405 410 415

Asp Thr Tyr Arg Phe Val Thr Ser Gin Ala lie Thr Cys Gin Lys Ser 420 425 430Asp Thr Tyr Arg Phe Val Thr Ser Gin Ala lie Thr Cys Gin Lys Ser 420 425 430

Ala Pro Gin Lys Pro Lys Glu Asp Pro Phe Lys Asp Tyr Val Phe Trp 435 440 445Ala Pro Gin Lys Pro Lys Glu Asp Pro Phe Lys Asp Tyr Val Phe Trp 435 440 445

Glu Val Asn Leu Lys Glu Lys Phe Ser Ala Asp Leu Asp Gin Phe Pro 450 455 460Glu Val Asn Leu Lys Glu Lys Phe Ser Ala Asp Leu Asp Gin Phe Pro 450 455 460

Leu Gly Arg Lys Phe Leu Leu Gin Ala Gly Tyr Arg Ala Arg Pro Lys 465 470 475 480Leu Gly Arg Lys Phe Leu Leu Gin Ala Gly Tyr Arg Ala Arg Pro Lys 465 470 475 480

Phe Lys Ala Gly Lys Arg Ser Ala Pro Ser Ala Ser Thr Thr Thr Pro 485 490 495Phe Lys Ala Gly Lys Arg Ser Ala Pro Ser Ala Ser Thr Thr Thr Pro 485 490 495

Ala Lys Arg Lys Lys Thr Lys Lys 500 <210> 5 <211> 34 <212> DNA <213>人工序列 <220> <223> PCR弓丨子 <400> 5 cgtcgacgta aacgtgtatc atattttttt acag <210> 6 <211> 25 <212> DNA <213〉人工序列 4- 95258 1334438 <220> <223> PCR 引子 <400> 6 cagacacatg tattacatac acaac 25 <210> 7 <211> 41 <212> DNA <213>人工序列 <220> <223> PCR弓丨子 <400> 7 ctcagatctc acaaaacaaa atgtctctgt ggcggcctag c 41 <210> 8 <211> 38 <212> DNA <213>人工序列 <220> <223> PCR 弓I 子 <400> 8 gacagatctt actttttagt ttttttacgt tttgctgg 38 95258Ala Lys Arg Lys Lys Thr Lys Lys 500 <210> 5 <211> 34 <212> DNA <213>Artificial Sequence<220><223> PCR Bow Twist <400> 5 cgtcgacgta aacgtgtatc atattttttt Acag <210> 6 <211> 25 <212> DNA <213> artificial sequence 4-95258 1334438 <220><223> PCR primer <400> 6 cagacacatg tattacatac acaac 25 <210><211> 41 <212> DNA <213>Artificial Sequence<220><223> PCR Bower <400> 7 ctcagatctc acaaaacaaa atgtctctgt ggcggcctag c 41 <210> 8 <211> 38 <;212> DNA <213>Artificial sequence<220><223> PCR bow I <400> 8 gacagatctt actttttagt ttttttacgt tttgctgg 38 95258

Claims (1)

1334438 ^ 8； - 3---------- * I年月日修(更}正本 第093124870號專利申請案 中文申請專利範圍替換本(99年8月） 十、申請專利範圍： 1. 一種似病毒粒子（VLP) ’其包含Hpv31之重組匕丨蛋白質或 重組L1+L2蛋白質’其中該重組L1蛋白質或該重組L1+L2 蛋白質係在酵母菌中製備，且其令藉由經密碼子最適化 之HPV31 L1核酸分子來編碼該L1蛋白質，該經密碼子最 適化之核酸分子大體由如SEQ ID N〇:24SEQ m N〇:3所 示之核苷酸序列所組成。 2. 一種製備HPV31似病毒粒子（VLp)的方法，其包含：⑷ 以編碼HPV31 L1蛋白質之核酸分子轉化酵母菌，該 HPV31 L1蛋白質包含如SEQ ID NO:4所示之胺基酸序 列，其中該核酸分子包含為在酵母菌細胞中高水平表現 而經密碼子最適化之核苷酸序列；（b)在許可核酸分子表 現的條件下培養該經轉化之酵母菌細胞以製備重組乳突 狀瘤病毒蛋白質；及（c)分離該重組乳突狀瘤病毒蛋白質 以製備該HPV31 VLP。 3. 如請求項2之方法，其中該酵母菌係由下列各物組成之群 中選出：釀酒酵母、多形漢遜酵母、巴氏畢赤酵母、脆 壁克魯維酵母、乳酸克魯維酵母、及粟酒裂殖酵母菌。 4. 如請求項3之方法，其中該酵母菌係釀酒酵母。 5. 如吻求項2之方法，其中該核酸分子包含如SEq id 2 或SEQ ID n〇:3所示之核苷酸序列。 6_如請求項5之方法，其中該核酸分子大體由如SEQm N0.2或SEQ ID NO:3所示之核苷酸序列所組成。 7·如請求項2之方法，其中步驟⑷進一步包含以編碼Hpv3i 95258-990803.doc 8.1334438 L2蛋白質之核酸分子轉化酵母菌細胞。 一種疫苗’其包含如請求項1之VLp。 9. 10. 一種醫藥組合物，其包含如請求項iiVLp。 於預防哺 -種如請求項8之疫苗之用途，其係用以製備用 乳動物HP V感染之藥物。 其係用以製備用於誘發動 11. 一種如請求項1之VLP之用途 物免疫反應之藥物。 I2· 一種人類乳突狀瘤病毒（HPV)似病毒粒子（VLp)，其包人 hpv31之重組L1蛋白f，其中該重組li蛋白質大體2 SEQ ID NO:4所示之胺基酸序列所組成。 13. 如請求項12之VLP，其中該VLp係於酵母菌中製備。 14. 一種如請求項12iHPV31 VLp之用途，其係用以製備用 於誘發動物免疫反應之藥物。 15. —種核酸分子，其包含編碼如SEQidn〇:4所示之 L1蛋白質的核苷酸序列，該核酸序列為在酵母菌細胞中 高量表現而經密碼子最適化。 16. 如請求項15之核酸分子，其中該核苷酸序列包含如seq IDNO:2或SEQIDN0:3所示之核苷酸序列。 17. —種載體’其包含如請求項15之核酸分子。 18. —種宿主細胞，其包含如請求項17之載體。 19 _如請求項18之宿主細胞，其中該宿主細胞係由下列各物 組成之群中選出：釀酒酵母、多形漢遜酵母、巴氏畢赤 酵母、脆壁克魯維酵母、乳酸克魯維酵母、及粟酒裂殖 酵母菌。 95258-990803.doc 1334438 2 0.如請求項19之宿主細胞，其中該宿主細胞係釀酒酵母。 21. —種載體，其包含如請求項16之核酸分子。 22. —種宿主細胞，其包含如請求項2 1之載體。 95258-990803.doc 1334438 第093124870號專利申請案 中文圖式替換本(99年8月） 十一、圖式. Γ^ΐτ^ΐ- 年月日修(更)正本 31 LI wt 31 LI部分 31 LI全部 31 LI wt 31 L1部分 31 U全部 31 LI wt 31 LI部分 31 LI全部 31 LI wt 31 LI部分 31 L1全部 31 LI wt 31 L1部分 31 LI全部 31 LI wt 31 L1部分 31 L1全部 31 LI wt 31 LI部分 31 LI全部 31 LI wt 31 LI部分 31 LI全部 31 LI wt 31 U部分 31 L1全部 31 LI wt 31 LI部分 31 LI全部 31 LI wt 31 LI部分 31 LI全部 31 LI wt 31 L1部分 31 L1全部 31 LI wt 31 U部分 31 L1全部 31 LI wt 31 L1部分 31 L1全部 111 111 5 5 5 \l# )/ )/ \/ \/ i/ i/ 1 1 1 X X 1 1 «Α 1 1 ooo 5 5 5 ο ο o 5 5 5 111 111 222 222 (301) (301) { 301) (351) (351) (351) o o o 4 4 4 5 5 5 4 4 4 o o o 5 5 5 5 5 5 >5 o o o 6 6 6 5 5 5 6 6 6 31 LI wt 31 U部分 31 L1全部 31 LI wt 0 0 0 5 V /'If %>/ ) \/ *0 / 饭 If t/ \/ \t/ ^1» 1 1 1* 1 1 ii 1 1 1 1 1 1* X 1 1 1x 1 X X 1 ATGTCTCTGTGGCGGCCTAGCGAGGCTACTGTCTACTTACCACCTGTCCC ......T.....A.A. .ATCT. .A.....C........G.....A..... AGTGTCTAAAGTTGTAAGCACGGATGAATATGTAACACGAACCAACATAT ..•C.....G..C. .CTCT· .C..C.....C..C..CA..........C. ATTATCACGCAGGCAGTGCTAGGCTGCTTACAGTAGGCCATCCATATTAT • C..C.....T. -TTC......AT..T.G..mc.....c. .c TCCATACCTAAATCTGACAATCCTAAAAAAATAGTTGTACCAAAGGTGTC ..T..C..A..G........C..A..G..G..C..C..C........C·· AGGATTACAATATAGGGTATTTAGGGTTCGTTTACCAGATCCAAACAAAT T..T..G.....C. .A. .C. .C. .A. .CA.A. .G.....C........G. TTGGATTTCCTGATACATCTTTTTATAATCCTGAAACTCAACGCTTAGTT • C· .T· .C· ·Α· .C_ .C.....C· ·(：· ·(：· ,A.....C...A.A. .G· .C TGGGCCTGTGTTGGTTTAGAGGTAGGTCGCGGGCAGCCATTAGGTGTAGG .....T.....C.....G· .A. .C…Α·Α. ·Τ. .A.....G.....C· · TATTAGTGGTCATCCATTATTAAATAAATTTGATGACACTGAAAACTCTA …CTC......C.....G. .G. -C. .G. .C. .C.....C.......... ATAGATATGCCGGTGGTCCTGGCACTGATAATAGGGAATGTATATCAATG .C.....C. .T........A. .T. -C. .C. .C. .A........C· ·Τ·" GATTATAAACAAACACAACTGTGTTTACTTGGTTGCAAACCACCTATTGG • .C..C..G.....C...T.......GT.G.....T. .G.....A..C.· AGAGCATTGGGGTAAAGGTAGTCCTTGTAGTAACAATGCTATTACCCCTG T. .A. .C........G...TC...A...TC......C.....C.....A. GTGATTGTCCTCCATTAGAATTAAAAAATTCAGTTATACAAGATGGGGAT ____C.....A.....G.....G. ,G. ·0. -T. .C. .C.....C.,T,.C ATGGTTGATACAGGCTTTGGAGCTATGGATTTTACTGCTTTACAAGACAC .....c..mc.,T........C..C..C.....G........ TAAAAGTAATGTTCCTTTGGACATTTGTAATTCTATTTGTAAATATCCAG C· .GTC...C..C..A........C.....C.....C.....G. .C---- ATTATCTTAAAATGGTTGCTGAGCCATATGGCGATACATTATTTTTTTAT ............................C.....C. .C. .G. .C. .C. .C • C-.CT.G..G.....C.....A.....C.....C. .C. .G. .C. -C. .C TTACGTAGGGAACAAATGTTTGTAAGGCATTTTTTTAATAGATCAGGCAC ft 95258-fig-990803.doc 1334438 31 Ll部分 .31 L1全部 31 Ll wt 31 Ll部分 31 Ll全部 31 Ll wt 31 Ll部分 31 L1全部 31 Ll wt 31 L1部分 31 L1全部 31 Ll wt 31 Ll部分 31 L1全部 31 Ll wt 31 Ll部分 31 L1全部 31 Ll wt 31 Ll部分 31 Ll全部 (751) . .G.....A.....G.....C........C-_C..C..C.....C..... (751) . .G.....A.....G.....C........C..C..C"C.....C..... (801) GGTTGGTGAATCGGTCCCTACTGACTTATATATTAAAGGCTCCGGTTCAA (801) C,.A........T.....A. .C. . .C.G. .C* .C. .G...........C. (801) (：··Α........T.....A. .C. . .C.G. .C. .C. .G...........C. { 851) CAGCTACTTTAGCTAACAGTACATACTTTCCTACACCTAGCGGCTCCATG (851) .C.....CC.G......TCC"C.....C. ·Α· ·Τ. ,ATCT......... (851} .C.....CC.G......TCC--C.....C· .A. ·Τ· ·ATCT......... (901) GTTACTTCAGATGCACAAATTTTTAATAAACCATATTGGATGCAACGTGC (901) · .C. ·(：· _C· ·(：. .T. .G· .C. ·<：· .C· _G.....C........G..... (901) . ·(：· ,C, .C· ·<：· ·Τ. .G· .C· U· .G.....C........G..... (951) TCAGGGACACAATAATGGTATTTGTTGGGGCAATCAGTTATTTGTTACTG { 951) A.....T.....ChC."..C........T. .C. · .C.G. .C. .G____ (951) A.....T.....C. -C.....C........T· .C...C.G· .C. .G____ (1001) TGGTAGATACCACACGTAGTACCAATATGTCTGTTTGTGCTGCAATTGCA (1001) . . . · C........G- · .TC......C.....".C....... noni ^ ____c........g___tc......c....... r...........c. .t 111 5 5 5 o o o 111 ^CAGTGATACTACATTTAAAAGTAGTAATTTTAAAGAGTATTTAAGACA • > «TC· · ·0· · · · «C· *C« «GTCCTC· .CC ...TC. . .C.....C. -C. .GTCCTC.. .C· .C* *G.....CC.G..... 31 Ll wt 31 L1部分 31 Ll全铀 31 Ll wt 31 L1部分 31 L1全部 (1101) TGGTGAGGAATTTGATTTACAATTTATATTTCAGTTATGCAAAATAACAT (1101) ............C. . .C.G.....C. .C. .C.....G.....G. .C. ,CC (1101) ............C. . .C.G.....C. .C. .C.....G.....G. .C. .CC (1151) TATCTGCAGACATAATGACATATATTCACAGTATGAATCCTGCTATTTTG (1151) .G.....T.....C.....C..C..C...........C.....C. .CC.. (1151) -G.....T.....C.....C..C..C...........C.....C. .CC.. 31 LI wt 31 L1部分 31 L1全部 31 Ll wt 31 L1部分 31 L1全部 31 Ll wt 31 LI部分 31 Ll全部 31 Ll wt 31 L1部分 31 L1全部 31 Ll wt 31 Ll部分 31 L1全部 31 Ll wt 31 U部分 31 Ll全部 31 Ll wt 31 L1部分 31 Ll全部 (1201) GAAGATTGGAATTTTGGATTGACCACACCTCCCTCAGGTTCTTTGGAGGA {1201) · *G - .C. . . . >Ci >C* · TC · .....T* .A. .T. .C............. n2〇i) . .r. .r.....c..c_tc______ .t. .a. .t. .r:...........a.. f tf - 1* ΙΑ IX Tx -1 1 - 5 5 5 ο o G - 222 333 - «X 1 1 1 1 • /1 /1 fi /1. ( /1 TACCTATAGGTTTGTAACCTCACAGGCCATTACATGTCAAAAAAGTGCCC C.....C· ·Α· .C. _C.....T· ·Α‘ ·Τ· .C. .C........GTC. . .T. CCCAAAAGCCCAAGGAAGATCCATTTAAA6ATTATGTATTTTGGGAGGTT (1351) AATTTAAAAGAAAAGTTTTCTGCAGATTTAGATCAGTTTCCACTGGC5TCG (1351) .................................................. (1351) "C· .G· .G........C.....T. .C. .G. .C. .A. ,C. . .T.....A· (1401) caaatttttattacaggcaggatatagggcacgtcctaaatttaaagcag (1401) .................................................. (1401) A. - G. .C· .G· ,G. ·Α· ·Τ. _T. .C_ ·Α· ·ΤΑ.Α· ·Α. .G. ,C· ‘G· ·Τ. (1451) GTAAACGTAGTGCACCCTCAGCATCTACCACTACACCAGCAAAACGTAAA (1451) .................................................. (1451) ____GA.ATC.“T"A"T..T........C. .C.....T. .GA.A. .G (1501) AAAACTAAAAAGTAA (SEQ ID NO:l) (1501) ............... (SEQ ID NO:2) (1501) ...............{SEQ ID N0:3) 95258-fig-990803.doc 1334438 M S L W R P S E A T V ' Y ' L P ，P_ V P 1 ATGTCTTTGT GGAGACCATC TGAAGCTACC GTCTACTTGC CACCAGTCCC V S K V V S T D E Y V T R T N I Y 51 AGTCTCTAAG GTCGTCTCTA CCGACGAATA CGTCACCAGA ACCAACATCT Y H A G S A R L L T V G H P Y Y 101 ACTACCACGC TGGTTCTGCT AGATTGTTGA CCGTCGGTCA CCCATACTAC S I P K SDN P K K I V V P K V S 151 TCTATCCCAA AGTCTGACAA CCCAAAGAAG ATCGTCGTCC CAAAGGTCTC G L Q Y R V F R V R L P D P N K F 201 TGGTTTGCAA TACAGAGTCT TCAGAGTCAG ATTGCCAGAC CCAAACAAGT G F P D T S F Y N P E T Q R L V 251 TCGGTTTCCC AGACACCTCT TTCTACAACC CAGAAACCCA AAGATTGGTC W A C V G L ε V G R G Q P L G V G 301 TGGGCTTGTG TCGGTTTGGA AGTCGGTAGA GGTCAACCAT TGGGTGTCGG I S G Η P L L N K F DDT E N S N 351 TATCTCTGGT CACCCATTGT TGAACAAGTT CGACGACACC GAAAACTCTA R Y A G G P G T D N R E C ISM 401 ACAGATACGC TGGTGGTCCA GGTACCGACA ACAGAGAATG TATCTCTATG D Y K Q T Q L C L L G C K P PIG 451 GACTACAAGC AAACCCAATT GTGTTTGTTG GGTTGTAAGC CACCAATCGG E H W G K G S PCS N N A I T P G 501 TGAACACTGG GGTAAGGGTT CTCCATGTTC TAACAACGCT ATCACCCCAG D C P P L E L K N S V I Q D G D 551 GTGACTGTCC ACCATTGGAA TTGAAGAACT CTGTCATCCA AGACGGTGAC Μ V D T G F G AMD F T A L Q D T 601 ATGGTCGACA CCGGTTTCGG TGCTATGGAC TTCACCGCTT TGCAAGACAC K S N V P L D I C N SIC K Y P D 651 CAAGTCTAAC GTCCCATTGG ACATCTGTAA CTCTATCTGT AAGTACCCAG Y L K Μ V A E P Y G D T L F F Y 701 ACTACTTGAA GATGGTCGCT GAACCATACG GCGACACCTT GTTCTTCTAC L R R E Q M F V R H F F N R S G T 751 TTGCGTAGAG AACAGATGTT CGTAAGGCAC TTCTTCAACA GATCCGGCAC V G E S V P T D L Y I K G S G S T 801 CGTAGGTGAA TCTGTCCCAA CCGACCTGTA CATCAAGGGC TCCGGTTCCA A T L A N S T Y F P TPS GSM 851 CCGCTACCCT GGCTAACTCC ACCTACTTCC CAACTCCATC TGGCTCCATG V T S D A Q I F N K p y w m Q R A 901 GTCACCTCCG ACGCTCAGAT CTTCAACAAG CCATACTGGA TGCAGCGTGC Q G H N N G I C W G N Q L F V T V 圖2 95258-fig-990803.doc 1334438 951 1001 1051 1101 1151 1201 1251 1301 1351 1401 1451 1501 ACAGGGTCAC AACAACGGTA TCTGTTGGGG TAACCAGCTG TTCGTGACTG VDT TRS TNMS V C A A I A TGGTCGATAC CACGCGTTCT ACCAACATGT CTGTCTGTGC TGCAATCGCT N S D T TFK SSN FKEY L R H AACTCTGACA CTACCTTCAA GTCCTCTAAC TTCAAGGAGT ACCTGAGACA GEE F D L Q FIF QLC KITL TGGTGAGGAA TTCGATCTGC AATTCATCTT CCAGTTGTGC AAGATCACCC SAD I Μ T Y I H S MNP AIL TGTCTGCTGA CATCATGACC TACATCCACA GTATGAACCC TGCCATCCTG EDWN F G L TTP PSGS LED GAGGACTGGA ACTTCGGTCT GACCACTCCA CCTTCCGGTT CTTTGGAAGA TYR FVTS Q A I TCQ K S A P CACCTACAGA TTCGTCACCT CTCAAGCTAT CACCTGTCAA AAGTCTGCTC QKP K E D P F K D Y V F W E V CACAAAAGCC AAAGGAAGAC CCATTCAAGG ACTACGTCTT CTGGGAAGTC NLKE K F S A D L D Q F P L G R AACTTGAAGG AAAAGTTCTC TGCTGACTTG GACCAATTCC CATTGGGTAG KFL LQAG Y R A R P K FKAG AAAGTTCTTG TTGCAAGCTG GTTACAGAGC TAGACCAAAG TTCAAGGCTG K R S APS A S T T T P A K R K GTAAGAGATC TGCTCCATCT GCTTCTACCA CCACCCCAGC TAAGAGAAAG K T K K * (SEQ ID NO:4) AAGACCAAGA AGTAA (SEQ ID NO: 3) 95258-fig-990803.doc -4- 1334438 B3 DM* 工 \mzn ΓΠτ-〇 τ^3~Ό 鹗< φ< Ηΐ< iimlico iialico ιλο |ΙΠφ—l Ρπ^-k fTS_k fia ffii Ππρ·ρ- ΙτΠμ f-* |— —i. _k L1構造 1515 1515 1515 核苷酸 504 504 504 胺基酸 92% 75% 核昔·酸同一性 100% 100% 胺基酸同一性 nt697-1249 之間121個改變 nt1-1515 之間 376個咚寧 備註 95258-fig-990803.doc 13344381334438 ^ 8; - 3---------- * I-year-day repair (more} original patent No. 093124870 patent application replacement scope of Chinese application (August 99) X. Patent application scope: A virion-like particle (VLP) comprising a recombinant prion protein of Hpv31 or a recombinant L1+L2 protein, wherein the recombinant L1 protein or the recombinant L1+L2 protein line is produced in a yeast, and The codon-optimized HPV31 L1 nucleic acid molecule encodes the L1 protein, and the codon-optimized nucleic acid molecule consists essentially of the nucleotide sequence set forth in SEQ ID N: 24 SEQ m N 〇: 3. A method of producing HPV31-like virions (VLp), comprising: (4) transforming a yeast with a nucleic acid molecule encoding a HPV31 L1 protein, the HPV31 L1 protein comprising an amino acid sequence as shown in SEQ ID NO: 4, wherein the nucleic acid The molecule comprises a nucleotide sequence optimized for codon expression at a high level of expression in the yeast cell; (b) culturing the transformed yeast cell under conditions permitting expression of the nucleic acid molecule to produce a recombinant papillomavirus protein ; and (c) The recombinant papillomavirus protein is isolated to prepare the HPV31 VLP. 3. The method of claim 2, wherein the yeast strain is selected from the group consisting of: Saccharomyces cerevisiae, Hansenula polymorpha, Pap. Pichia pastoris, Kluyveromyces cerevisiae, Kluyveromyces lactis, and Schizosaccharomyces pombe. 4. The method of claim 3, wherein the yeast is Saccharomyces cerevisiae. A method, wherein the nucleic acid molecule comprises a nucleotide sequence as set forth in SEq id 2 or SEQ ID n:: 6. The method of claim 5, wherein the nucleic acid molecule is substantially as defined by SEQm N0.2 or SEQ ID NO The method of claim 2, wherein the step (4) further comprises transforming the yeast cell with a nucleic acid molecule encoding the Hpv3i 95258-990803.doc 8.1334438 L2 protein. VLp as claimed in claim 1. 9. A pharmaceutical composition comprising, as claimed in claim iiVLp, for the purpose of preventing the use of a vaccine according to claim 8 for the preparation of a medicament for infection with a milk animal HP V It is used to prepare for inducing movement 11. A medicament for use as an immunological reaction of a VLP of claim 1. I2. A human papillomavirus (HPV)-like virion (VLp), which comprises a recombinant L1 protein f of hpv31, wherein the recombinant li The protein is substantially composed of the amino acid sequence shown in SEQ ID NO: 4. 13. The VLP of claim 12, wherein the VLp is produced in a yeast. 14. Use of claim 12iHPV31 VLp for the preparation of a medicament for inducing an immune response in an animal. A nucleic acid molecule comprising a nucleotide sequence encoding an L1 protein as shown in SEQ ID NO: 4, which nucleic acid sequence is codon-optimized in a high amount in yeast cells. 16. The nucleic acid molecule of claim 15, wherein the nucleotide sequence comprises a nucleotide sequence as set forth in seq IDNO: 2 or SEQ ID NO: 3. 17. A vector comprising the nucleic acid molecule of claim 15. 18. A host cell comprising the vector of claim 17. The host cell of claim 18, wherein the host cell line is selected from the group consisting of: Saccharomyces cerevisiae, Hansenula polymorpha, Pichia pastoris, Kluyveromyces cerevisiae, lactic acid Dictyostelium, and Schizosaccharomyces pombe. The host cell of claim 19, wherein the host cell line is Saccharomyces cerevisiae. 21. A vector comprising the nucleic acid molecule of claim 16. 22. A host cell comprising the vector of claim 21. 95258-990803.doc 1334438 Patent application No. 093124870 Replacement of Chinese schema (August 99) XI. Schema. Γ^ΐτ^ΐ- Year and month repair (more) original 31 LI wt 31 LI part 31 LI all 31 LI wt 31 L1 part 31 U all 31 LI wt 31 LI part 31 LI all 31 LI wt 31 LI part 31 L1 all 31 LI wt 31 L1 part 31 LI all 31 LI wt 31 L1 part 31 L1 all 31 LI wt 31 LI part 31 LI all 31 LI wt 31 LI part 31 LI all 31 LI wt 31 U part 31 L1 all 31 LI wt 31 LI part 31 LI all 31 LI wt 31 LI part 31 LI all 31 LI wt 31 L1 part 31 L1 All 31 LI wt 31 U part 31 L1 all 31 LI wt 31 L1 part 31 L1 all 111 111 5 5 5 \l# )/ )/ \/ \/ i/ i/ 1 1 1 XX 1 1 «Α 1 1 ooo 5 5 5 ο ο o 5 5 5 111 111 222 222 (301) (301) { 301) (351) (351) (351) ooo 4 4 4 5 5 5 4 4 4 ooo 5 5 5 5 5 5 > 5 ooo 6 6 6 5 5 5 6 6 6 31 LI wt 31 U section 31 L1 all 31 LI wt 0 0 0 5 V / 'If %>/ ) \/ *0 / Meal If t/ \/ \t/ ^1» 1 1 1* 1 1 ii 1 1 1 1 1 1* X 1 1 1x 1 XX 1 ATGTCTCTGTGGCGGCCTAGCGAGGCTACTGTC TACTTACCACCTGTCCC ...T.....AA.ATCT. .A.....C........G.....A..... AGTGTCTAAAGTTGTAAGCACGGATGAATATGTAACACGAACCAACATAT ..•C .....G..C. .CTCT· .C..C.....C..C..CA..........C. ATTATCACGCAGGCAGTGCTAGGCTGCTTACAGTAGGCCATCCATATTAT • C..C.. ...T. -TTC...AT..TG.mc.....c. .c TCCATACCTAAATCTGACAATCCTAAAAAAATAGTTGTACCAAAGGTGTC ..T..C..A..G........C ..A..G..G..C..C..C........C·· AGGATTACAATATAGGGTATTTAGGGTTCGTTTACCAGATCCAAACAAAT T..T..G.....C. .A. .C. .C. .A. .CA.A. .G.....C........G. TTGGATTTCCTGATACATCTTTTTATAATCCTGAAACTCAACGCTTAGTT • C· .T· .C· ·Α· .C_ .C.... .C· ·(:··(:· , A.....C...AA .G· .C TGGGCCTGTGTTGGTTTAGAGGTAGGTCGCGGGCAGCCATTAGGTGTAGG .....T.....C.....G· .A .C...Α·Α. ·Τ. .A.....G.....C· · TATTAGTGGTCATCCATTATTAAATAAATTTGATGACACTGAAAACTCTA ...CTC......C.....G. .G. -C. .G. .C. .C.....C.......... ATAGATATGCCGGTGGTCCTGGCACTGATAATAGGGAATGTATATCAATG .C.....C. .T........A. .T. - C. .C. .C. .A........C· ·Τ·" GATTATAAACAAACACAACTGTGTTTACTTGGTTGCAAA CCACCTATTGG • .C..C..G.....C...T.......GT.G.....T. .G.....A..C.· AGAGCATTGGGGTAAAGGTAGTCCTTGTAGTAACAATGCTATTACCCCTG T. .A. .C........G...TC...A...TC......C.....C.....A. GTGATTGTCCTCCATTAGAATTAAAAAATTCAGTTATACAAGATGGGGAT ____C.....A.....G.....G. ,G. ·0. -T. .C. .C.....C.,T,.C ATGGTTGATACAGGCTTTGGAGCTATGGATTTTACTGCTTTACAAGACAC ... ..c..mc.,T........C..C..C.....G........ TAAAAGTAATGTTCCTTTGGACATTTGTAATTCTATTTGTAAATATCCAG C· .GTC...C..C ..A........C.....C.....C.....G. .C---- ATTATCTTAAAATGGTTGCTGAGCCATATGGCGATACATTATTTTTTTAT ............ ................C.....C. .C. .G. .C. .C. .C • C-.CT.G..G.. ...C.....A.....C.....C. .C. .G. .C. -C. .C TTACGTAGGGAACAAATGTTTGTAAGGCATTTTTTTAATAGATCAGGCAC ft 95258-fig-990803.doc 1334438 31 Ll Part .31 L1 All 31 Ll wt 31 Ll Part 31 Ll All 31 Ll wt 31 Ll Part 31 L1 All 31 Ll wt 31 L1 Part 31 L1 All 31 Ll wt 31 Ll Part 31 L1 All 31 Ll wt 31 Ll Part 31 L1 All 31 Ll wt 31 Ll part 31 Ll all (751) . .G.....A.....G.....C........C-_C..C. .C.....C..... (751) . .G.....A.....G.....C........C..C. .C"C.....C..... (801) GGTTGGTGAATCGGTCCCTACTGACTTATATATTAAAGGCTCCGGTTCAA (801) C,.A........T.....A. .C. . . .CG .C * .C. .G...........C. (801) (:··Α........T.....A. .C. . . .CG . C. .C. .G...........C. { 851) CAGCTACTTTAGCTAACAGTACATACTTTCCTACACCTAGCGGCTCCATG (851) .C.....CC.G......TCC"C.... .C. ·Α··Τ. ,ATCT......... (851} .C.....CC.G...TCC--C.....C· .A. ·Τ··ATCT......... (901) GTTACTTCAGATGCACAAATTTTTAATAAACCATATTGGATGCAACGTGC (901) · .C. ·(:· _C· ·(:. .T. .G· .C. ·< :· .C· _G.....C........G..... (901) . ·(:· , C, .C· ·<:· ·Τ. .G · .C· U· .G.....C........G..... (951) TCAGGGACACAATAATGGTATTTGTTGGGGCAATCAGTTATTTGTTACTG { 951) A.....T.....ChC.&quot ;..C........T. .C. · .CG .C. .G____ (951) A.....T.....C. -C.....C ........T· .C...CG· .C. .G____ (1001) TGGTAGATACCACACGTAGTACCAATATGTCTGTTTGTGCTGCAATTGCA (1001) . . . · C........G- · .TC... ...C.....".C....... noni ^ ___ _c........g___tc......c....... r...........c. .t 111 5 5 5 ooo 111 ^CAGTGATACTACATTTAAAAGTAGTAATTTTAAAGAGTATTTAAGACA • &gt «TC· · ·0· · · · «C· *C« «GTCCTC· .CC ...TC. . . C.....C. -C. .GTCCTC.. .C· .C* *G.....CC.G..... 31 Ll wt 31 L1 part 31 Ll all uranium 31 Ll wt 31 L1 part 31 L1 all (1101) TGGTGAGGAATTTGATTTACAATTTATATTTCAGTTATGCAAAATAACAT (1101) ......... ...C. . .CG....C. .C. .C.....G.....G. .C. ,CC (1101) .......... ..C. . .CG....C. .C. .C.....G.....G. .C. .CC (1151) TATCTGCAGACATAATGACATATATTCACAGTATGAATCCTGCTATTTTG (1151) .G..... T.....C.....C..C..C...........C.....C. .CC.. (1151) -G... ..T.....C.....C..C..C...........C.....C. .CC.. 31 LI wt 31 Part L1 31 L1 all 31 Ll wt 31 L1 part 31 L1 all 31 Ll wt 31 LI part 31 Ll all 31 Ll wt 31 L1 part 31 L1 all 31 Ll wt 31 Ll part 31 L1 all 31 Ll wt 31 U part 31 Ll all 31 Ll Wt 31 L1 part 31 Ll all (1201) GAAGATTGGAATTTTGGATTGACCACACCTCCCTCAGGTTCTTTGGAGGA {1201) · *G - .C. . . . >Ci >C* TC · .....T* .A. .T. .C............n2〇i) . .r. .r.....c..c_tc______ . T. .a. .t. .r:...........a.. f tf - 1* ΙΑ IX Tx -1 1 - 5 5 5 ο o G - 222 333 - «X 1 1 1 1 • /1 /1 fi /1. ( /1 TACCTATAGGTTTGTAACCTCACAGGCCATTACATGTCAAAAAAGTGCCC C.....C· ·Α· .C. _C.....T· ·Α' ·Τ· .C. .C. .......GTC. . .T.CCCAAAAGCCCAAGGAAGATCCATTTAAA6ATTATGTATTTTGGGAGGTT (1351) AATTTAAAAGAAAAGTTTTCTGCAGATTTAGATCAGTTTCCACTGGC5TCG (1351) ............................. ..................... (1351) "C· .G· .G........C.....T. C. .G. .C. .A. ,C. . .T.....A· (1401) caaatttttattacaggcaggatatagggcacgtcctaaatttaaagcag (1401) ................... ............................... (1401) A. - G. .C· .G· , G. ·Α · ·Τ. _T. .C_ ·Α· ·ΤΑ.Α· ·Α. .G. ,C· 'G· ·Τ. (1451) GTAAACGTAGTGCACCCTCAGCATCTACCACTACACCAGCAAAACGTAAA (1451) ............ ...................................... (1451) ____GA.ATC. "T"A" T..T........C. .C.....T. .GA.A. .G (1501) AAAACTAAAAAGTAA (SEQ ID NO:l) (15 01) ............... (SEQ ID NO: 2) (1501) ...............{SEQ ID N0:3) 95258 -fig-990803.doc 1334438 MSLWRPSEATV 'Y' LP, P_ VP 1 ATGTCTTTGT GGAGACCATC TGAAGCTACC GTCTACTTGC CACCAGTCCC VSKVVSTDEYVTRTNIY 51 AGTCTCTAAG GTCGTCTCTA CCGACGAATA CGTCACCAGA ACCAACATCT YHAGSARLLTVGHPYY 101 ACTACCACGC TGGTTCTGCT AGATTGTTGA CCGTCGGTCA CCCATACTAC SIPK SDN PKKIVVPKVS 151 TCTATCCCAA AGTCTGACAA CCCAAAGAAG ATCGTCGTCC CAAAGGTCTC GLQYRVFRVRLPDPNKF 201 TGGTTTGCAA TACAGAGTCT TCAGAGTCAG ATTGCCAGAC CCAAACAAGT GFPDTSFYNPETQRLV 251 TCGGTTTCCC AGACACCTCT TTCTACAACC CAGAAACCCA AAGATTGGTC WACVGL ε VGRGQPLGVG 301 TGGGCTTGTG TCGGTTTGGA AGTCGGTAGA GGTCAACCAT TGGGTGTCGG ISG Η PLLNKF DDT ENSN 351 TATCTCTGGT CACCCATTGT TGAACAAGTT CGACGACACC GAAAACTCTA RYAGGPGTDNREC ISM 401 ACAGATACGC TGGTGGTCCA GGTACCGACA ACAGAGAATG TATCTCTATG DYKQTQLCLLGCKP PIG 451 GA CTACAAGC AAACCCAATT GTGTTTGTTG GGTTGTAAGC CACCAATCGG EHWGKGS PCS NNAITPG 501 TGAACACTGG GGTAAGGGTT CTCCATGTTC TAACAACGCT ATCACCCCAG DCPPLELKNSVIQDGD 551 GTGACTGTCC ACCATTGGAA TTGAAGAACT CTGTCATCCA AGACGGTGAC Μ VDTGFG AMD FTALQDT 601 ATGGTCGACA CCGGTTTCGG TGCTATGGAC TTCACCGCTT TGCAAGACAC KSNVPLDICN SIC KYPD 651 CAAGTCTAAC GTCCCATTGG ACATCTGTAA CTCTATCTGT AAGTACCCAG YLK Μ VAEPYGDTLFFY 701 ACTACTTGAA GATGGTCGCT GAACCATACG GCGACACCTT GTTCTTCTAC LRREQMFVRHFFNRSGT 751 TTGCGTAGAG AACAGATGTT CGTAAGGCAC TTCTTCAACA GATCCGGCAC VGESVPTDLYIKGSGST 801 CGTAGGTGAA TCTGTCCCAA CCGACCTGTA CATCAAGGGC TCCGGTTCCA ATLANSTYFP TPS GSM 851 CCGCTACCCT GGCTAACTCC ACCTACTTCC CAACTCCATC TGGCTCCATG VTSDAQIFNK pywm QRA 901 GTCACCTCCG ACGCTCAGAT CTTCAACAAG CCATACTGGA TGCAGCGTGC QGHNNGICWGNQLFVTV FIG 2 95258-fig-990803.doc 1334438 951 1001 1051 1101 1151 1201 1251 1301 1351 1401 1451 1501 ACAGGGTCAC AACAACGGTA TCTGTTGGGG TAACCAGCTG TTCGTGACTG VDT TRS TNMS VCAAIA TGGTCGATAC CACGCGTTCT ACCAACATGT CTGTCTGTGC TGCAATCGCT NSDT TFK SSN FKEY LRH AACTCTGACA CTACCTTCAA GTCCTCTAAC TTCAAGGAGT ACCTGAGACA GEE FDLQ FIF QLC KITL TGGTGAGGAA TTCGATCTGC AATTCATCTT CCAGTTGTGC AAGATCACCC SAD I Μ TYIHS MNP AIL TGTCTGCTGA CATCATGACC TACATCCACA GTATGAACCC TGCCATCCTG EDWN FGL TTP PSGS LED GAGGACTGGA ACTTCGGTCT GACCACTCCA CCTTCCGGTT CTTTGGAAGA TYR FVTS QAI TCQ KSAP CACCTACAGA TTCGTCACCT CTCAAGCTAT CACCTGTCAA AAGTCTGCTC QKP KEDPFKDYVFWEV CACAAAAGCC AAAGGAAGAC CCATTCAAGG ACTACGTCTT CTGGGAAGTC NLKE KFSADLDQFPLGR AACTTGAAGG AAAAGTTCTC TGCTGACTTG GACCAATTCC CATTGGGTAG KFL LQAG YRARPK FKAG AAAGTTCTTG TTGCAAGCTG GTTACAGAGC TAGACCAAAG TTCAAGGCTG KRS APS ASTTTPAKRK GTAAGAGATC TGCTCCATCT GCTTCTACCA CCACCCCAGC TAAGAGAAAG KTKK * (SEQ ID NO: 4) AAGACCAAGA AGTAA (SEQ ID NO: 3) 95258-fi G-990803.doc -4- 1334438 B3 DM*工\mzn ΓΠτ-〇τ^3~Ό 鹗<φ<Ηΐ< iimlico iialico ιλο |ΙΠφ—l Ρπ^-k fTS_k fia ffii Ππρ·ρ- ΙτΠμ f -* | — —i. _k L1 Construction 1515 1515 1515 Nucleotide 504 504 504 Amino acid 92% 75% Ricoh acid identity 100% 100% Amino acid identity nt697-1249 Between 121 changes nt1 Between -1515, 376 Suining Remarks 95258-fig-990803.doc 1334438 圖4 95258-fig-990803.doc 1334438Figure 4 95258-fig-990803.doc 1334438 95258-fig-990803.doc LI齑碲 1 31譯瞜 1 31-^w?t 2 ΰ $β0 2 31 呤-WJi f〇N> t〇N>N>N> Γ^Γ** b 〇 〇> co to n> οι b> <» σ> φ -si ·〇 〇» go *>i cn -sj ct> n> vs> ^ 3901 4081 7135 4428 6628 8461 4M ω87 904 844 5061 5091 晌m濉 (mg/ml ) ίΠ 蒸1S_N-RIA (cpm/ml) 3202 40S7 3S-7 (31卟哲/31琪冷) -λ r〇 _k 00 CD -si ^ 〇) CD CD -ft. 〇〇 ns <〇 Mos 1 (31鹄冷/31雜岑) s§ gisss 31500s (31 鹄次/31 wt) 460 1 {31 wt/31 wt) 31 U VLP*/mg ^^^^pm/mg) 书这商 tbs (cpm/mg)(书这ίΐ / 书达it) 13,3443895258-fig-990803.doc LI齑碲1 31瞜1 31-^w?t 2 ΰ $β0 2 31 呤-WJi f〇N>t〇N>N>N> Γ^Γ** b 〇〇 > co to n> οι b><»σ> φ -si ·〇〇»go *>i cn -sj ct>n>vs> ^ 3901 4081 7135 4428 6628 8461 4M ω87 904 844 5061 5091 晌m濉(mg/ml ) Π 蒸 1S_N-RIA (cpm/ml) 3202 40S7 3S-7 (31卟哲/31琪冷) -λ r〇_k 00 CD -si ^ 〇) CD CD -ft. 〇〇 Ns <〇Mos 1 (31鹄冷/31杂岑) s§ gisss 31500s (31 //31 wt) 460 1 {31 wt/31 wt) 31 U VLP*/mg ^^^^pm/mg) Book this business tbs (cpm/mg) (book this ΐ / book up it) 13,34438 圖6 95258-fig-990803.docFigure 6 95258-fig-990803.doc