本研究使用本土光桿菌Photorhabdus luminescens 0805-P5G 以聚合?連鎖反應(PCR)進行目標基因prtA之擴增,在將目標基因片段與載體pET29b接合,轉型至大腸桿菌Escherichia coli Rosetta中,再以IPTG進行大量誘導,可表現出PrtA目標蛋白約53 kDa,經由核甘酸序列轉譯分析後可推衍出474個胺基酸序列,經由MS/MS分析,部分胺基酸片段與先前由核?酸序列推衍之胺基酸序列相吻合。將純化後的PrtA蛋白酵素進行生化特性分析,當溫度於50℃、pH值為10.5時,此酵素具有最高的活性,於28-37℃時有較佳的熱穩定性,並在pH 5-9有較好的pH值安定性;而對金屬離子的影響,1 mM之ZnSO4及ZnCl2對失活的PrtA能恢復50%的相對活性,生物檢定結果顯示,轉殖株純化之PrtA蛋白?經注射後對七齡大蠟蛾具有殺蟲力,LC50為32.70 μg/mL,但對餵食三齡美國及本土小菜蛾致死率卻相當的低。
A prtA gene of Photorhabdus luminescens 0805-P5G was amplified by polymerase chain reaction (PCR),and cloned into expressed vector pET29b. Sequence analysis of the prtA gene showed an open reading frame of 1422 bp, encoded a protein of 474 amino acid and had a molecular mass of 53 kDa. The expression plasmid pET29b was transformated into Escherichia coli Rosetta (DE3). The recombinant strain was induced by IPTG to produce PrtA protease. Moreover, the optimal pH and temperature of the purified enzyme were 28-37 ℃ and 5.0-9.0, respectively. LC50 of purified PrtA protease of P. luminescens against 7th instar larvae of Galleria mellonella by injection was 32.7 μg/mL. However, the LC50 of purified PrtA protease of P. luminescens aginst 3rd larvae of Plutella xyllostella per os was not obtained.