Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/30473
標題: Preliminaty study on expression of polistes jadwigae dalla torre venom cDNA, polistes mastoparan
家馬蜂(Polistes adwigae Dalla Torre)蜂毒polistes mastoparan cDNA轉殖及表現之初探
作者: Mars, J.Yang
楊景岳
關鍵字: 家馬蜂;Polistes jadwigae;蜂毒蛋白;venom peptide
出版社: 昆蟲學系
摘要: 
建立蜂毒蛋白polistes mastoparan(polistes MP)微生物表現系統,在大腸桿菌(Escherichia coli)中選擇pET expression system,利用大腸桿菌常用之遺傳密碼子,將家馬蜂(Polistes jadwigae Dalla Torre)毒液中所含之polistes mastoparan胺基酸序列VDWKKIGQHILSVL,譯成為適合大腸桿菌表現之polistes mastoparan cDNA序列GTGGACTGGAAGAAAATCGGCCAACATATCCTCAGCGTG
CTC,並配合NdeI及EcoRI之限制酵素切位,成功的將cDNA的insertion fragment接入含lac operon調控因子之pET21b質體中,得到含polistes mastoparan的cDNA之置換載體pETMP。將構築成功之置換載體轉型至BL21品系之大腸桿菌中,並以Isopropyl-1-thio-β-D-galactoside(IPTG)對lac operon進行polistes mastoparan cDNA表現之調控。在加入誘導表現物質IPTG之後,菌株的生長明顯受抑制。將含置換載體之菌株於生長旺盛之指數生長期進行誘導表現時,約於誘導後四十分鐘至一小時間,受誘導表現之菌株呈現明顯的生長遲滯現象。而將此時生長明顯異常之菌株,進行掃描式電子顯微鏡觀察時發現,其菌體表面有許多明顯的凹陷或穿孔現象,並呈現出正常菌體所不常見的不正形長條狀。而將受誘導表現菌株之培養液及其胞質萃取液加入正常菌株時,其生長情況並不會受到影響。

The pET expression system of Escherichia coli was established in vespid peptide venom cDNA expression. The polistes mastoparan(polistes MP)venom cDNA insertion fragment of Polistes jadwigae Dalla Torre was constructed into pET21b vector with NdeI and EcoRI restriction enzyme cutting sites, named pETMP vector. The pETMP vector was transformed into the BL21 strain of E. coli and was regulated polistes MP peptide expression by Isopropyl-1-thio-β-D-galactoside(IPTG), the lactose analogy. After IPTG treatment, the growing of BL21 strain of E. coli that contain pETMP vector was inhibited by polistes MP cDNA expression. The growth inhibition occurred after polistes MP cDNA was expressed about 40 min IPTG was added, but the inhibition were resolved unreasonable at 4 hours later. The surface of E. coli that polistes MP was expressed induced IPTG showed abnormal dents or perforation with examining by scanning electron microscope. Nevertheless, the growing of JM109 and BL21 strain of E. coli was not inhibited by adding medium and cytoplasmic extract of BL21 strain of E. coli that contain pETMP vector after polistes MP cDNA was expressed.
URI: http://hdl.handle.net/11455/30473
Appears in Collections:昆蟲學系

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