幽門螺旋桿菌蛋白酶的選殖與功能分析

Abstract

幽門螺旋桿菌 (Helicaobacter pylori) 寄生於人類胃中，目前已知與許多胃部疾病相關，但詳細的致病機制與毒性仍需更進一步的釐清和證明。在微生物中，蛋白酶除了在生存上是必備的成分外，為了能夠寄生在宿主體內，微生物將會發展出針對宿主產生破壞與毒性的功能，例如在營養的取得、毒性因子的合成、分解宿主細胞特定蛋白以便控制宿主的反應以及菌體本身蛋白量與質的控制等。幽門螺旋桿菌26695菌株中有33個基因已被預測出具有蛋白酶的功能，其中我們挑選了tagE、htrA這兩個基因進行探討，這是因為之前的研究並未在蛋白酶功能部分多所著墨，此外也發現這兩個基因轉錄出來的mRNA在酸性環境刺激下都有上升的趨勢。我們在幽門螺旋桿菌NTUH-C1菌株選殖這兩個基因，由於htrA基因在幽門螺旋桿菌中已被證明是essential gene，所以我們僅製作tagE突變株來看對幽門螺旋桿菌的影響。初步發現tagE突變株在生長複製上的表現與野生株無太大的差異，但是經由初步的統計結果及穿透式電子顯微鏡的觀察，我們可以看到突變株的菌體長度較野生株短。之後，為了進一步確定其蛋白酶的活性與功能，我們在大腸桿菌中使用不同的載體，經過表現和純化的過程後得到TagE與HtrA重組蛋白。最後，我們以zymogram這個技術確定HtrA蛋白的確具有蛋白酶的活性，能夠分解受質β-casein ﹔而TagE蛋白則沒有活性的表現。

Helicobacter pylori colonizes in human stomach and is recognized as a cause of many gastric diseases. However, the mechanism of pathogenesis and related toxic factors need further study and identification. In microbes, proteases play important roles in many parts of their life cycles. In order to colonize the host, microbes often use their protease activity to be virulence factors, including the acquisition of nutrient, biosynthesis of pathogenetic factors, cleavage key host proteins for mediation of the host response, and the protein quality control. There are 33 genes which have putative protease function in Helicobacter pylori 26695. In this study, we tried to characterize the TagE、HtrA protein functions, because their protease activity is not yet clarify and mRNA expression in high level under acidic stress. We cloned the tagE、htrA genes from Helicobacter pylori NTUH-C1. htrA is proved as an essential gene, so only the tagE-knockout strain was constructed. Then, we compared the wild type and tagE-knockout strain, it’s no distint difference in the growth and replication, but under the transmission electron microscopy and statistics results we observed the phenotype of the tagE-knockout strain has decreased elongation. Moreover, to identify the protease activity, we expressed and purified the TagE and HtrA recombinant protein. Finally, zymogram confirmed that the htrA had the protease activity to degrade β-casein, but tagE didn’t.