嗜水性產氣單胞桿菌 Aeromonas hydrophila prtS1 基因缺損株之製作及相關蛋白之探討

Fabrication for Deficient Strain of Aeromonas hydrophila Serine Protease Gene prtS1 and Analysis of Interaction Protein

賴怡岑(Yi-Tsen Lai)

指導教授 ： 劉俊民

國立臺灣大學/生命科學院/微生物與生化學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.10129

摘要

本研究室由成奶j學取得自病人中分離之 Aeromonas hydrophila CKH29 菌株。在先前之研究中由本菌株中選殖出絲胺酸蛋白酶表現基因 prtS1，此 prtS1 基因及表現之產物與 HtrA (DegP) / DegQ / DegS family serine protease 有極高的同源性，同屬於 trypsin-like protease。HtrA (high-temperature-requirement protein A) 最早在 E. coli 中發現與菌株在高溫下之生存有關 (Lipinska et al., 1990)，在 S. typhimurium 之突變株研究中又發現 htrA 除了與溫度相關外也可能與其致病性有關 (Johnson et al., 1991)。
本研究中利用製作 prtS1 基因缺損株，探討其表現產物及該產物在菌株內可能扮演的角色與弁遄C採用 conjugation 及 electroporation 兩種方式，送入在 prtS1 基因上嵌入 kanamycin 抗性基因之 prtS1 基因破壞質體，使其進行同源性基因重組後篩選 prtS1 突變株，初步篩選得到七株蛋白酶活性缺損株。但經 PCR 確認後，未能獲得 prtS1 基因突變的缺損株。推測可能在 CKH29 上不只有一個 prtS1 基因位置，所以再選擇 KpnI、SalI、PvuI、PstI、PvuII 限制酶作用 CKH29 染色體後進行 Southern blot，確認 prtS1 基因在 CKH29 染色體上只有一個。
將帶有 His-tagged 之完整 PrtS1 融合蛋白質表現於 E. coli JM109之中，尋找在 E. coli 中可能與之結合的相關蛋白質，藉此以瞭解 PrtS1 蛋白酶所扮演的角色及作用機制。結果得知，採用一步純化的方式，無法避免非專一性蛋白質的干擾，需再進一步改良融合蛋白質於宿主細胞中之表現，再探尋相關結合之蛋白。
針對 PrtS1 蛋白酶活性部位進行討論，發現其中之 PDZ domain 與蛋白酶活性的調節有關，當兩個 domain 缺失時會使 PrtS1 失去調節，導致菌體趨向死亡。PDZ domain II 之缺失會使蛋白酶失去活性，PDZ domain I 之缺損則會使活性下降，研判 PDZ domain II 與辨認特定受質蛋白有關，而 PDZ domain I 與形成適當之聚合體有關。與 PrtS1 活性相關之絲胺酸的活性中心，於第 214 個絲胺酸進行點突變後，仍具有蛋白酶活性，發現於第 211 個絲胺酸有相近之序列，亦將其進行點突變後，則失去蛋白酶活性，證實 PrtS1 有兩個絲胺酸的活性中心。

關鍵字

絲胺酸蛋白

並列摘要

We obtained Aeromonas hydrophila CKH29 from the patients in National Cheng Kung University. In the previous study, we have cloned and sequenced a proteolytic gene prtS1 of the serine protease, which was highly homologous to HtrA (DegP)/DegQ/DegS family serine protease. Both of the two belonged to trypsin-like protease. In E.coli, HtrA (high-temperature-requir
-ement protein A) is the first known to relate with existence under high temparature (Lipinska, 1990). Moreover, it was observed in the study of S. typhimurium htrA mutant that htrA might also correlate with pathogenicity (Johnson, 1991).
The current study aimed to explore prtS1 and the role and function of its products in two respects. By conjugation and electroporation, we transformed pSup2021-PrtSM into A. hydrophila to perform homologous recombination, so as to find out the influence of prtS1 absence. According to the result, seven serine protease mutants were obtained; while a later PCR indicated that they were not response to prtS. Therefore, we selected KpnI、SalI、PvuI、PstI、PvuII to react upon CKH29 chromosome, and then performed Southern blot to make sure that prtS had only one copy in CKH29.
The behavior of His-tag PrtS1 fusion protein pPDZ03 in JM109 was used to find out whether there is any protein related to PrtS1 and the role it plays. According to the result, the interference of nonspecific proteins was irresistible by the one-step purification being applied. So further improvement and fusion of the proteins in the host cell is needed in order to explore the related interactive proteins.
In the discussion of the protease active region of PrtS1, it was found that the PDZ domain had something to do with the regulator of protease activity. That is, the lack of two domains would cause PrtS1 to fail to regulate, and then the host was inclined to death. When PDZ domain II was missing, the protease would lose the activity; when PDZ domain I was missing, the activity would decrease. We conclude that PDZ domain II might correlate to the recognition of specific substrates and that PDZ domain I might correlate to the formation of appropriate polymer. The center of serine protease related to PrtS1 activity still had protease activity after site mutating at the 214th serine protease. Besides, a similar sequence was observed to occur at the 211th serine protease, and it might be the center of another PrtS1.

並列關鍵字

Aeromonas hydrophila ； serine proteas

參考文獻

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