TW201618805A - 合成胜肽在製備用以促進甲殼綱生物生殖的製劑之用途 - Google Patents
合成胜肽在製備用以促進甲殼綱生物生殖的製劑之用途 Download PDFInfo
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Abstract
本發明提供一種促進甲殼綱生物性腺發育的合成胜肽、包含該合成胜肽的製劑以及促進甲殼綱生物性腺發育的方法。該促進甲殼綱生物性腺發育的方法係將一有效劑量的合成胜肽投藥至一甲殼綱生物,以促進該甲殼綱生物性腺發育,其中,該合成胜肽包含如序列辨識編碼:2所示序列。
Description
本發明是關於一種促進甲殼綱生物生殖的合成胜肽、製劑以及方法,特別是關於一種源自於脊椎動物之促性腺激素釋素(Gonadotropin releasing hormone)的合成胜肽與製劑,以及利用該合成胜肽促進甲殼綱生物生殖的方法。
目前對無脊椎動物,如甲殼綱(Crustaceans)的生殖內分泌瞭解程度極其有限,且普遍認為無脊椎動物的生殖內分泌種類與機制皆與脊椎動物頗具差異。以甲殼綱生物為例,其生殖內分泌機制較為明確的是由眼柄分泌的神經胜肽(Neuropeptide)中,卵黃生成抑制激素(Vitellogenesis-inhibiting hormone,VIH)會抑制甲殼綱生物卵黃前質的生合成及被胞飲入卵細胞串堆積,而胸部神經節(Thoracic ganglion)會產生卵黃生成促進激素(Vitellogenesis stimulating hormone,VSH),其會促進卵黃前質的生合成及進入卵細胞中堆積,但該卵黃生成促進激素的成分及結構仍未明瞭。另一方面,關於精莢生成發育的調控機制則瞭解更少,目前只知道由雄性腺(Androgenic gland)分泌的雄性腺激素(Androgenic gland hormone)可決定性別的分化以及生殖腺的發育,但至今仍未能具體瞭解完整的作用機制。
現有技術雖有利用剪除眼柄或是注射生物胺(Biogenic
amine,BA),例如是血清素(Serotonin,5-HT),等方式,刺激甲殼綱生物的生殖性腺發展,但其促進效果仍有限,並且現有剪除眼柄技術催熟存在有可能造成甲殼綱生物的生理反應失衡,促進已交配的雌性個體脫殼以致精莢脫落無法受精,以及高死亡率的風險,多數具高經濟價值的甲殼綱生物迄今仍無法有效培植生殖腺發育成熟的種源,因此,在該些甲殼綱生物的人工養殖性腺成熟的種源量產種苗,或生產性腺發育成熟的產品上仍存在諸多限制。
本發明之目的在於提供一種促進甲殼綱生物生殖的合成胜肽及製劑,可有效且長時間地促進甲殼綱生物的雌雄成體性腺發育,並誘發產卵。
本發明之又一目的在於提供一種促進甲殼綱生物生殖的方法,以利甲殼綱生物的繁殖與種苗量產,生產生殖腺發育成熟的甲殼綱生物供產業之用。
為達上述目的,本發明之一實施例提供一種促進甲殼綱生物性腺發育的合成胜肽,其具有如序列辨識編碼:2所示序列。
為達上述目的,本發明之一實施例更提供一種促進甲殼綱生物性腺發育的製劑,包含前述的合成胜肽以及至少一賦形劑。
為達上述目的,本發明之一實施例另提供一種促進甲殼綱生物性腺發育的方法,係將具有一定有效劑量的該合成胜肽投藥至一甲殼綱生物,以促進該甲殼綱生物性腺發育。
A‧‧‧合成胜肽
B‧‧‧對照組
第1圖繪示本發明的合成胜肽對於雄性白蝦(Litopenaeus vannamei)的精莢生成指數影響的曲線示意圖。
第2圖繪示本發明的合成胜肽對於雄性白蝦(Litopenaeus vannamei)的精莢生成指數影響的長條示意圖。
為使熟習本發明所屬技術領域之一般技藝者能更進一步了解本發明,下文特詳細說明本發明的構成內容及所欲達成之功效,俾使該領域之一般技藝人士得以具以實施。如下所述之較佳實施方式與圖式僅供參考與說明用,並非用來對本發明加以限制者,本發明中亦可採行其他的實施例,或是在不悖離文中所述實施例的前提下作出任何邏輯性的改變。
本發明的一較佳實施例提供一種促進甲殼綱生物性腺發育的方法,首先提供一合成胜肽,其包含如序列辨識編碼:2所示序列,再將一有效劑量的該合成胜肽投藥至一甲殼綱生物,以促進該甲殼綱生物的性腺發育。該合成胜肽可促進雄性甲殼綱生物的精莢成熟並且提昇成熟精莢的質量與精子數量,另一方面,該合成胜肽也可促進雌性甲殼綱生物的卵巢發育並增加產卵次數。
本發明的方法主要是應用於甲殼綱(Crustaceans)生物,特別是其中具有高經濟價值的十足目(Decapoda)生物。十足目生物包含短尾亞目(Brachyuran),例如是毛蟹、陸蟹、旭蟹及大閘蟹等;長尾亞目(Macrura),例如是對蝦類(Penaeus)等,及異尾亞目(Anomura)等,例如是寄居蟹及椰子蟹,該些十足目生物的體內組織及生理機制極其相近,彼此間僅具有外部型態的差異。具體來
說,該些十足目生物皆具有相同或相近的幼蟲期(Zoea)而造成外部型態差異的主要原因在於甲殼綱生物成長期間須歷經變態期,造成部分物種發生腹節萎縮的情況,但其生殖內分泌系統則是具有高度相關性。
在本實施例中,該合成胜肽係源自於脊椎動物的第二型促
性腺激素釋素(Gonadotropin releasing hormone,GnRH-Ⅱ),其具有如序列辨識編碼:1所示序列,其中,前述之脊椎動物可以是雞、鴨、鵝等禽類。該合成胜肽可以是包含10個胺基酸的胜肽片段,其是依據雞的第二型促性腺激素釋素人工合成而製得,例如是利用固相胜肽合成(Solid-phase peptide synthesis)的方式進行合成。相較於該雞的第二型促性腺激素釋素,該合成胜肽兩端的胺基酸並未經過任何修飾,因此並未被阻斷。也就是說,位於該合成胜肽胺基端(N端)的麩胺酸(Glutamine,Glu)並未被環化成pyro-Glu,並且位於該合成胜肽羧基端(C端)的甘胺酸(Glycine,Gly)也未被醯胺化而形成Gly-NH2。
接著,將該有效劑量的該合成胜肽投藥至一甲殼綱生物,
其中,每次投藥的有效劑量例如是每單位生物體重公克(g)投藥10至1000奈克(ng),較佳是50奈克。而為了增加養殖該甲殼綱生物的便利性,在本發明一實施例中,可選擇採用一種長效型的投藥方式,預先利用一賦型劑將高量的該合成胜肽配製成一緩釋劑型,例如是將100至2000奈克的該合成胜肽配製於50至200微升(μl)之包含油脂的賦型劑中,再投藥至該甲殼綱生物體內,使該合成胜肽可以在該甲殼綱生物體內漸進地釋放,以延長其作用效果,在經過3至6個月,較佳是4個月後則可續進行下次投藥。然而本發明
的投藥方式並不以此為限。在其他實施例中,該合成胜肽也可選擇先以生理食鹽水配製成溶液劑型,再與該賦型劑混合,其中該溶液劑型與該賦型劑的混合體積比例則可以是1:1至1:3。該賦型劑包含之油脂可選擇自礦物油,例如是甘油或不完全佐劑;植物油,例如是花生油、橄欖油或椰子脂;油脂黏著劑,例如是甲基纖維素(Methylcellulose)或者是由前述油脂組成之複合物。
值得注意的是,整體投藥至該甲殼綱生物的藥劑體積,大
體上為50微升(μl)至200微升,投藥頻率大體上為每3-4個月注射一次,但不以此為限。此外,該合成胜肽較佳是投藥至該甲殼綱生物的體腔中,例如是長尾類生物(Macrura),例如為蝦類,額劍基部頭胸甲的空腔中,或者是短尾類生物(Brachyura),例如是螃蟹,頭胸甲腹側空腔部位。藉此,可一次性地投藥較高的劑量與體積,並且避免造成甲殼綱生物的緊迫效應。
由上述實施例可得知,本發明的一種促進甲殼綱生物生殖
的方法,主要是依據雞的第二型促性腺激素釋素,以人工合成方式製得一合成胜肽,該合成胜肽包含如序列辨識編碼:2所示序列,再將該合成胜肽投藥至一甲殼綱生物,以促進該甲殼綱生物的性腺發育。值得特別說明的是,本發明是跨越脊椎動物與無脊椎動物的屏界,將源自於脊椎動物之促性腺激素釋素類似物,投藥至屬於無脊椎動物之列的甲殼綱生物,以促進雄性甲殼綱生物的精莢成熟,同時更進一步提升成熟精莢的質量與精子數量;以及促進雌蝦的卵巢發育,並進一步使雌蝦產卵的次數以及卵巢維持在生殖發育狀態的期間皆獲得有效提升。
a卵巢發育期是依據卵巢的外部特徵及成熟度而區分為4期,成熟度介於兩期之間者則可再進一步畫分為0.5期、1.5期及2.5期等,養殖的雌蝦卵巢發育到第3期之後即達可產卵狀態。
b佔整體受測南美白蝦之百分比。
b佔整體受測南美白蝦之百分比。
c產卵次數是在投藥(合成胜肽)的8-16週內所測試的產卵次數,為避免外在因素干擾,而投藥後7週之數據已排除。
以下將進一步說明以本發明的合成胜肽促進甲殼綱生物性腺發育的效果。本試驗係採用十足目中屬長尾類的南美白蝦(Litopenaeus vannamei)作為實際實施樣態進行說明,但本發明並不以此為限。本試驗採用的合成胜肽是配製成前述的緩釋劑型再投藥至各南美白蝦,其中雄蝦於投藥後每4天逐隻觀察精莢外觀以判定其發育期數,至第44天時取出精莢量測其質量及精子數量;而雌蝦則於2至4個月內,每週定期觀察各南美白蝦的卵巢外觀、成熟度並記錄產卵次數。請參閱表1至表3及第1至2圖,表1、第1及2圖代表本發明的合成胜肽投藥於雄性南美白蝦,該雄性南美白蝦所呈現的精莢成熟(例如精莢發育程度及質量的提升、精子數量的增加等)促進效果,其中,對照組代表未經任何處理的雄性南美白蝦。表2代表本發明的合成胜肽投藥於雌性南美白蝦,該雌性南美白蝦所呈現的卵巢成熟效果,其中,對照組代表未處理的雌性南美白蝦。表3代表本發明的合成胜肽投藥於雌性南美白蝦,該雌性南美白蝦的產卵次數,其中,對照組代表未處理的雌性南美白蝦。
由表1、第1及2圖可知,以雄性甲殼綱生物為例,本發明的合成胜肽可有效促進其精莢成熟,相較於對照組,本發明的合成
胜肽可使雄性甲殼綱生物的精子數量及精莢質量大幅提升,同時可使精莢發育程度在44天即達指數2.5以上,也就是說精莢的囊狀構造已明顯可辨,呈現淺褐色。另一方面,如表2所示,以雌性甲殼綱生物為例,本發明的合成胜肽可有效促進卵巢發育,相較於對照組僅有佔全體數目40%的南美白蝦卵巢發育至產卵期,本發明的合成胜肽可使80%的雌性甲殼綱生物的卵巢發育至產卵期。另一方面,如表3所示,本發明施以合成胜肽的南美白蝦,80%進入產卵期,其中有20%可產卵一次,20%可產卵兩次,40%可產卵四次,相較於對照組中僅可產卵一次,本發明之合成胜肽顯然可使產卵次數大幅增加。
此外,相較於習用技術中利用剪除眼柄或是注射生物胺或血清素的方式,本發明的合成胜肽可達到較長的有效促進期,例如利用眼柄剪除或是注射生物胺或血清素的方式僅能誘發雌性甲殼綱生物較少次的產卵,誘發卵巢發育的有效期僅約1個月左右。然而,本發明的合成胜肽可誘發4次的產卵,使誘發卵巢發育的有效期長達2至4個月左右,並且產卵量亦可獲得巨幅的提升。
因此,本發明的合成胜肽可應用於促進各種雄性及雌性之甲殼綱生物的性腺發育及成熟,培育成熟種源,特別是具有高經濟價值的十足目生物,例如是蝦類跟蟹類等,應用於人工繁殖生產種苗,或供產業之用,或做為高級食材。前述各實施例雖皆係長尾類中的南美白蝦作為實際實施樣態進行說明,但本領域技術人員應可理解本發明亦可以同理性地適用於其他甲殼綱生物,而該些實施例仍應屬本發明所涵蓋的範圍。
以上所述僅為本發明之較佳實施例,凡依本發明申請專利範圍所做之均等變化與修飾,皆應屬本發明之涵蓋範圍。
<110> 國立屏東科技大學
<120> 促進甲殼綱生物生殖的合成胜肽、製劑以及方法
<130> NST-P0001-TWN
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 10
<212> PRT
<213> Gallus gallus
<400> 1
<210> 2
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> unblock N-terminal and C-terminal
<400> 2
A‧‧‧合成胜肽
B‧‧‧對照組
Claims (10)
- 一種促進甲殼綱生物性腺發育的方法,係將一有效劑量的合成胜肽投藥至一甲殼綱生物,以促進該甲殼綱生物性腺發育,其中,該合成胜肽包含如序列辨識編碼:2所示序列。
- 如申請專利範圍第1項所述之方法,其中,該合成胜肽的有效劑量為每單位體重公克(g)投藥10-1000奈克(ng)。
- 如申請專利範圍第1項所述之方法,其中,該合成胜肽係先混合一賦形劑,再投藥至該甲殼綱生物,該賦形劑包含一油脂。
- 如申請專利範圍第3項所述之方法,其中,該合成胜肽係先以一生理食鹽水配製成溶液,再混合該賦型劑。
- 如申請專利範圍第4項所述之方法,其中,該溶液與該賦型劑的混合體積比例是1:1至1:3。
- 如申請專利範圍第3項所述之方法,其中,該油脂為礦物油、植物油、油脂黏著劑或由前述油脂組成之複合物。
- 一種促進甲殼綱生物性腺發育的合成胜肽,具有如序列辨識編碼:2所示序列。
- 一種促進甲殼綱生物性腺發育的製劑,包含:一合成胜肽,包含如序列辨識編碼:2所示序列;以及至少一賦形劑。
- 如申請專利範圍第8項所述之製劑,其中,該合成胜肽的有效劑量為每單位體重公克(g)投藥100至2000奈克(ng)。
- 如申請專利範圍第8項所述之製劑,其中,該賦形劑包含一油脂,該油脂為礦物油、植物油、油脂黏著劑或由前述油脂組成之複合物。
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