TW587942B - Polynucleotide vaccine composition against canine pathogens, in particular respiratory and digestive pathogens - Google Patents

Polynucleotide vaccine composition against canine pathogens, in particular respiratory and digestive pathogens Download PDF

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TW587942B
TW587942B TW086111546A TW86111546A TW587942B TW 587942 B TW587942 B TW 587942B TW 086111546 A TW086111546 A TW 086111546A TW 86111546 A TW86111546 A TW 86111546A TW 587942 B TW587942 B TW 587942B
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plastid
pathogens
gene
bamhi
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Jean-Christophe Audonnet
Annabelle Bouchardon
Michel Riviere
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Rhone Merieux
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Description

587942 五、發明説明（9 =疫苗調配物來進行如同上述之第—次預防接種和補 在根據本發明之方法的較佳具體實施例中，在第 例中對動物投予有效劑量之傳統疫苗，特別是失活的活 f的二減毒的或重組的類型’或另外的亞單元疫苗，以便 k供第’人的預防接種，並在較佳的2到6週期間之後， 投予根據本發明之多價或單價疫苗。 本發明之主題亦包括預防接種工具組，將如同上述之第 /人預防接種疫苗和作爲補強用的根據本發明之疫苗調 配物集合在-起。亦關於根據本發明之疫苗調配物，伴隨 著一張指示利用該調配物來補強如同上述之第一次預防接 種的傳單。 ' 本發明亦關於製備該疫苗調配物的方法，也就是由本説 明來製備病毒價及其混合物。 現在將利用本發明之具體實施例的協助，並參考附圖， 更詳細地説明本發明。 圖片一覽表 圖1 圖2 圖3 圖4 圖5 圖6 圖7 經濟部中央標準局員工消費合作社印製 質體 PVR1012 質體PAB044 質體PAB036 質體PAB024 質體PAB021 質體PAB022 質體PAB037 -12- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） 587942 A7 B7 五、發明説明（1Q ) 經濟部中央標準局員工消費合作社印製 (請先閱讀背面之注意事項再填寫本頁) 圖8 :質體pAB038 圖9 :質體pAB017 圖10 :質體pAB041 序列識別編號之序列一覽表 序列識別編1號：寡核甞酸ABO 1 7 序列識別編2號：寡核甞酸ABO 1 8 序列識別編3號：寡核甞酸AB085 序列識別编4號：寡核甞酸AB086 序列識別編5號··寡核茹酸AB053 序列識別編6號：寡核苷酸AB054 序列識別編7號：寡核甞酸AB045 序列識別編8號：寡核甞酸AB048 序列識別編9號：寡核甞酸AB049 序列識別編10號：寡核苷酸AB050 序列識別編11號：寡核铝酸AB087 序列識別編12號：寡核苷酸AB088 序列識別編13號：寡核甞酸AB089 序列識別編14號：寡核苷酸AB090 序列識別編15號：寡核苷酸AB038 序列識別編16號：寡核棼酸AB039 序列識別編1 7號：寡核苷酸ABO 11 序列識別編18號：寡核甞酸ΑΒ0 12 實例 實例1 ··病毒的培養 -13- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 587942 經濟部中央標準局員工消費合作社印製 A7 _B7___五、發明説明（12 ) 萃取病毒的DNA，再以2份體積之無水乙醇使其沉澱。在 -20 °C下靜置過夜之後，在+4 °C下以10,000g離心DNA 15 分鐘。將DNA小球脱水，然後溶解於最少體積之無菌的超 純水中。然後可利用限制酶消化它。 實例4 :病毒基因組之RNAs的分離 根據熟諳此藝者已熟知的技術純化RNA病毒。然後利用 P. Chomczynski 和 N· Sacchi (Anal· Biochem.，1987，162 ，156-159)描述的”硫氰酸瓠/酚-氣仿”萃取技術，分離每個 病毒的基因組病毒RNA。 實例5 :分子生物技術 利用由 J. Sambrook 等人（Molecular Cloning : A Laboratory Manual，第 2 版，Cold Spring Harbor Laboratory ’ Cold Spring Harbor ，New York，1989)描述的標準分 子生物技術來完成所有的質體建構。所有本發明使用的限 制片段均使用'’Geneclean”工具組（BIO 101 Inc.，La Jolla ，CA)來分離。 實例6 ·· RT-PCR技術 合成專一性的寡核甞酸（包括在其5,端的限制位置，以促 進擴大片段的選殖），使得它完全覆蓋待擴大之基因的密碼 區域（參見特定的實例）。根據標準技術（Sarnbrook J.等人， 1989)¾成逆轉錄（rt)反應和聚合酶連鎖反應（pc r)。以一 對特定的擴增引子（amplimer)完成每個rt_Pcr反應，並萃 取作爲模板的病毒基因組RNA。在以限制酶消化之前，先 利用紛/氣仿/異戊醇（25 : 24 : 1)萃取經過擴大的互補 -15-
(請先閱讀背面之注意事 —0 項再填· :寫本頁) 裝· 訂 会 經濟部中央標準局員工消費合作社印製 587942 A7 B7 五、發明説明（13 ) DNA 〇 實例7 :質體pVR1012 從 Vical Inc·，San Diego，CA，USA 獲得質體 pVR1012 (圖 1 )。已經在 J· Hartikka 等人（Human Gene Therapy，1996 ，7，1205-12 17)中描述了它的結構。 實例8 :建構質體pAB044 (CDV HA基因） 利用根據實例4之技術，並以下列的寡核甞酸： AB017 (35體）（序列識另J 1號）： 5 丨 AAAACTGCAGAATGCTCCCCTACCAAGACAAGGTG 3 丨 AB018 (37體）（序列識另2號）： 5' CGCGGATCCTTAACGGTTACATGAGAATCTTATACGG 31 製備之卡瑞氏病病毒（CDV) (Onderstepoort品系）基因組 RAN (M. Sidhu 等人，Virology，1993，193，66-72)， 來完成根據實例6之技術的RT-PCR反應，以便分離出以 Pstl-BamHI片段之形式存在，編碼CDV HA糖蛋白的基因 。在純化之後，以PstI和BamHI消化1 835個鹼基對之 RT-PCR產物，以便分離出1817個鹼基對之Pstl-BamHI片 段。利用先前以PstI和BamHI消化過的載體pVRl012 (實 例7)連結該片段，得到質體pAB044 (6676個鹼基對）（圖 2) 〇 實例9 :建構質體pAB036 (CDV F基因） 利用根據實例4之技術，並以下列的寡核甞酸： AB085 (40體）（序列識另J 3號）： 5，ATAAGAAGCGGCCGCACATGCACAAGGGAATCCCCAAAAG 3， -16 - 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） ^--·---------IT------0 (請先閱讀背面之注意事項再填寫本頁) 經濟部中央標準局員工消費合作社印製 587942 A7 __B7_ 五、發明説明（14 ) AB086 (32體）（序列識別4號）： 5 丨 CGCGGATCCACTTCAGTGTGATCTCACATAGG 3’ 製備之卡瑞氏病病毒（CDV) (Onderstepoort品系）基因組 RNA (R. Driellen ，基因銀行（Genbank)序列登錄編號 = X65 5 09)，來完成根據實例6之技術的RT-PCR反應，以 便分離出以Notl-BamHI片段之形式存在，編碼CDV F糖 蛋白的基因。在純化之後，以NotI和BamHI消化201 8個 鹼基對之RT-PCR產物，以便分離出2000個鹼基對之Notl -BamHI片段。利用先前以Notl和BamHI消化過的載體 pVR1012 (實例7)連結該片段，得到質體PAB036 (6893個 鹼基對）（圖3)。 實例10 :建構質體PAB024 (犬小病毒VP2基因） 利用根據實例3之技術，並以下列的寡核苷酸·· AB03 5 (33體）（序列識另丨J 5號）： 5' ACGCGTCGACATGAGTGATGGAGCAGTTCAACC 3' AB054 (33體）（序列識別6號）： 5, CGCGGATCCTTAATATAATTTTCTAGGTGCTAG 3, 製備之犬小病毒（CPV) (CPV-b品系）基因組DNA (C· Parrish ，基因銀行序列登錄編號=M 19296)，來完成PCR反應，以 便分離出以Sall-BamHI片段之形式存在，編碼VP2衣殼蛋 白質的基因（CPV VP2)。在純化之後，以Sail和BamHI消 化1773個鹼基對之PCR產物，以便分離出1760個鹼基對 之Sall-BamHI片段。利用先前以Sail和BamHI消化過的載 體pVR1012 (實例7)連結該片段，得到質體PAB024 (6629 -17· 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐) I η ^^衣-- (請先閱讀背面之注意事項再填寫本頁) 訂 A7 B7 7、發明説明（15 ) 修I鹼基對）（圖4)。 實例11 :建構質體PAB021 (CCV S基因） 利用根據實例4之技術，並以下列的寡核甞酸： AB〇45 (32體）（序列識別7號）： 5，ACGCGTCGACATGATTGTGCTTACATTGTGCC 31 AB〇48 (35體）（序列識別8號）： 5» CGCGGATCCTCAGTGAACATGAACTTTTTCAATAG 3’ 製備之犬冠狀病毒（CCV)基因組RNA (B.Horsburgh等人 ，J. Gen· Virol· 1992，73，2849-2862)，來完成根據實 例6之技術的RT_PCR反應，以便擴大43 74個鹼基對之片 探，其含有以SalI_BamHI片段之形式存在，編碼CCV S糖 爹白的基因。在純化之後，以Sail和BamHI消化RT-PCR產 物，得到4361個鹼基對之Sall-BamHI片段。 利用先前以Sail和BamHI消化過的載體pVR1012 (實例 連結該片段，得到質體PAB021 (9230個鹼基對）（圖5)。 實例12 :建構質體pAB022 (CCV Μ基因） 利用根據實例4之技術，並以下列的寡核甞酸： ΑΒ〇49 (34體）（序列識別9號）： 5，AAAACTGCAGAAATGAAGAAAATTTTGTTTTTAC 3， 經濟部中央標準局員工消費合作社印製 (請先閲讀背面之注意事項再填寫本頁) ΑΒ050 (33體）（序列識別10號）： 5，CGCGGATCCTTATACCATATGTAATAATTTTTC 3*
製備之犬冠狀病毒（CCV)基因組RNA (B.Horsburgh等 人，J. Gen. Virol. 1992，73，2849-2862)，來完成根據 實例6之技術的RT-PCR反應，以便分離出以Pstl-BamHI -18- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 經濟部中央標準局員工消費合作社印製 587942 A7 B7 五、發明説明（16 ) 片段之形式存在，編碼Μ糖蛋白的基因（CCV M)。在純化 之後，以PstI和BamHI消化809個鹼基對之PT-PCR產物 ，以便分離792個驗基對之PstI -BamHI片段。利用先前以 PstI和BamHI消化過的載體pVRl012 (實例7)連結該片段 ，得到質體PAB022 (5651個鹼基對）（圖6)。 實例13 :建構質體pAB037 (CHV gB基因） 利用根據實例3之技術，並以下列的寡核苷酸： AB087 (34體）（序列識別11號）： 5，AAAACTGCAGAAGTATGTTTTCATTGTATCTATA 3' AB08 8 (34體）（序列識別12號）： 5' CTAGTCTAGATTATTAAACTTTACTTTCATTTTC 3， 製備之犬癌瘆病毒（CHV) (Carmichael品系）基因組DN A (Κ· Limbach 等人，J. Gen· Virol. 1994，75，2029-2039) ，來完成PCR反應，以便分離出以PstI_XbaI片段之形式存 在，編碼CHV病毒gB糖蛋白的基因。在純化之後，以PstI 和Xbal消化2667個鹼基對之PCR產物，以便分離2648個 鹼基對之PstI - Xbal片段。利用先前以Pstl-Xbal消化過的 載體PVR1012 (實例7)連結該片段，得到質體PAB037 (7523 個鹼基對）（圖7)。 實例14 :建構質體pAB038 (CHV gD基因） 利用根據實例3之技術，並以下列的寡核甞酸： AB089 (34體）（序列識別13號）： 5' AAAACTGCAGAAAATGATTAAACTTCTATTTATC 3* AB090 (35體）（序列識別14號）： -19- 本紙張尺度適用中國國家標準（CNS ) A4規格（210 X 297公釐） 衣IT (請先閲讀背面之注意事項再填寫本頁) 587942 A7 ____B7_ 五、發明説明（17 ) 5f ATAAGAATGCGGCCGCAAAGGCTAAACATTTGTTG 3* 製備之犬疱疹病毒（CHV) (Carmichael品系）基因組DNA (K. Limbach 等人，J. Gen· Virol· 1994，75，2029-2039) ，來完成PCR反應，以便分離出以Pstl-Notl片段之形式存 在，編碼CHV病毒gD糖蛋白的基因。在純化之後，以PstI 和Notl消化1072個鹼基對之PCR產物，以便分離1049個 鹼基對之PstI Notl片段。利用先前以PstI和Notl消化過 的載體PVR1012 (實例7)連結該片段，得到質體PAB038 (5930個鹼基對）（圖8)。 實例15 :建構質體pAB017(伯格多弗雷螺旋體ospA基因） 利用根據實例2之技術，並以下列的寡核苷酸： AB03 8 (37體）（序列識另丨J 15號）： 5, ACGCGTCGACTATGAAAAAATATTTATTGGGAATAGG 3, AB039 (34體）（序列識別16號）： 5' CGCGGATCCCTTATTTTAAAGCGTTTTTAATTTC 3f 經濟部中央標準局員工消費合作社印裝 (請先閱讀背面之注意事項再填寫本頁) 製備之伯格多弗雷螺旋體（B31品系）基因組DNA (S· Bergstrom 等人，Mol. Microbiol. 1989，3，479-486)， 來完成PCR反應，以便分離出以SalI_BamHI片段之形式存 在，編碼OspA膜蛋白的基因。在純化之後，以Sail和BamHI 消化842個鹼基對之PCR產物，以便分離829個鹼基對之 Sall-BamHI片段。利用先前以Sail和BamHI消化過的載體 pVR1012 (實例7)連結該片段，得到質體pAB017 (5698個 鹼基對）（圖9)。 實例16 :建構質體pAB041 (狂犬病病毒G基因） -20- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公董1 " 587942 A7 B7 五、發明説明（18 ) 利用根據實例4之技術，並以下列的寡核甞酸： AB011 (33體）（序列識別17號）： 5，AAAACTGCAGAGATGGTTCCTCAGGCTCTCCTG 3' AB012 (34體）（序歹U线另U 1M虎）： 5，CGCGGATCCTCACAGTCTGGTCTCACCCCCACTC 3， 製備之狂犬病病毒（ERA品系）基因組RNA (A.Anilionis 等人，Nature， 1981，294，275-278)，來完成根據實 例6之技術的RT-PCR反應，以便擴大含有編碼狂犬病病 毒G糖蛋白之基因的1589個鹼基對片段。在純化之後，以 PstI和BamHI消化RT-PCR產物，得到1578個鹼基對之 PstI_BamHI片段。利用先前以PstI和BamHI消化過的載體 PVR1012 (實例7)連結該片段，得到質體pAB041 (6437個 鹼基對）（圖10)。 實例17 :質體之產製和純化 經濟部中央標準局員工消費合作社印製 (請先閱讀背面之注意事項再填寫本頁) 爲了製備企圖用來預防接種動物之質體，可以使用任何 能夠獲得經過純化、主要以超螺旋形式存在之質體懸浮液 的技術。熟諳此藝者已熟知這些技術。特別提及鹼性溶解 技術，接著是在氣化铯梯度上，在溴化乙錠存在下的兩次 連續超離心作用，如同在 J· Sambrook等人（Molecular Cloning : A Laboratory Manual，第 2 版，Cold Spring Harbor Laboratory，Cold Spring Harbor，New York， 1989)中的描述。也可以參考專利申請案PCT WO 95/2丨25〇 和PCT WO 96/02658，其描述以工業規模產製可用於預防 接種之質體的方法。爲了製造疫苗（參見實例18)，將經過 -21 - 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 經濟部中央標準局員工消費合作社印製 587942 A7 ----- -B7__ 五、發明説明（~ 純化之質體再懸浮，以便獲得高濃度的溶液（>2亳克/亳升） ’其亦適於儲存。將質體再懸浮於超純水或TE緩衝溶液 (10mM Tris-HCl ; imM EDTA，pH 8.0)中來進行之。 實例18 :製造聯合的疫苗 以其濃縮溶液開始（實例16)，將製造聯合疫苗所需的各 種質體混合。製備混合物使得每個質體的終濃度相當於各 質體的有效劑量。可用來調整該疫苗之終濃度的溶液，可 以是0.9 % NaCl溶液或PBS緩衝溶液。 也可以使用特殊的調配物，像是微脂粒或陽離子性之脂 質來製造該疫苗。 "" 實例19 :狗的預防接種 以每種質體10微克、50微克或250微克之劑量來接種 狗。 可利用針頭藉著肌肉内之途徑完成注射。在本案例中， 投予預防接種的狗1或2毫升之體積。可利用針頭藉著肌 肉内之途徑完成注射。在本案例中，以丨〇個部位各〇丨毫 升或在20個部位各〇·〇5亳升之總體積i毫升來投予預防接 種的狗。在剃光皮膚之後完成皮内注射（一般在胸部側面） ，或是在平滑解剖區之平面，例如大腿内側。關於皮内注 射，也可以使用液體射出之注射裝置。 •22- 本紙張尺度適用中國國家標準（CNS ) A4規格（21〇XΜ?公董） ： •t-------IT------ (請先閲讀背面之注意事項再填寫本頁)

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  1. 587942 第086111546號專利申請案 中文申請專利範圍替換本(93年3月） A8 B8 C8 D8 利範圍 祭月曰 览 3.31 修正j 補充! 1. 一種用於犬類之疫苗，其包括一質體，該質體包含並於 活體内表現一編碼犬小病毒VP2醣蛋白之核酸，及選自 CMV-IE啟動子、SV40早期啟動子、SV40晚期啟動子、 勞氏内瘤病毒L T R啟動子、肌間絲蛋白（d e s m i η )啟動 子及肌動蛋白啟動子之啟動子。 2. —種用於犬類之疫苗，其包括一質體，該質體包含並於 活體内表現一編碼狂犬病 G醣蛋白之核酸，及選自 CMV-IE啟動子、SV40早期啟動子、SV40晚期啟動子、 勞氏内瘤病毒LTR啟動子、肌間絲蛋白（desmin )啟動 子及肌動蛋白啟動子之啟動子。 本紙張尺度適用中國國家標準(CNS) A4規格(210X 297公釐）
TW086111546A 1996-07-19 1997-08-12 Polynucleotide vaccine composition against canine pathogens, in particular respiratory and digestive pathogens TW587942B (en)

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FR9609401A FR2751227B1 (fr) 1996-07-19 1996-07-19 Formule de vaccin polynucleotidique contre les pathologies canines, notamment les pathologies respiratoires et digestives

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EP0954332B2 (fr) 2014-12-31
BR9710509A (pt) 1999-08-17
PL331246A1 (en) 1999-07-05
CZ300385B6 (cs) 2009-05-06
PL190150B1 (pl) 2005-11-30
WO1998003199A1 (fr) 1998-01-29
NZ333780A (en) 2000-10-27
EP0954332B1 (fr) 2004-10-20
DE69731309T2 (de) 2005-11-17
CA2260273C (en) 2010-12-21
US6586412B2 (en) 2003-07-01
RU2319504C2 (ru) 2008-03-20
KR20060013432A (ko) 2006-02-09
EP0954332A1 (fr) 1999-11-10
KR20000067866A (ko) 2000-11-25
CZ15899A3 (cs) 1999-05-12
JP2000515521A (ja) 2000-11-21
RU2002132833A (ru) 2005-01-20
US6228846B1 (en) 2001-05-08
FR2751227A1 (fr) 1998-01-23
KR100620302B1 (ko) 2006-09-06
AR034997A1 (es) 2004-04-14
DE69731309D1 (de) 2004-11-25
FR2751227B1 (fr) 1998-11-27
CA2260273A1 (en) 1998-01-29
AU3699397A (en) 1998-02-10
AU733563B2 (en) 2001-05-17
KR20050087885A (ko) 2005-08-31
ZA976284B (en) 1999-01-19
DE69731309T3 (de) 2015-06-03
US20010009959A1 (en) 2001-07-26

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