能有效調控大腸桿菌感染的OmpA 蛋白片段之探討

細菌性腦膜炎是世界各地的新生兒和兒童死亡的重要原因。依據2001 年一月至2007 年十二月法國的統計資料中，大腸桿菌引起的新生兒細菌性腦膜炎，其致死率為28%，高居第二名。台灣地區台北馬偕醫院自1984 至2012 共二十九年的統計研究中，發現新生兒細菌性腦膜炎的病患有1/7 會死亡，2/5 的病患則在患病後有後遺症。雖然有抗生素的治療，其致病率、致死率及後遺症仍造成許多國家衛生支出上的重大負擔，然而細菌性腦膜炎的致病機制尚未完全了解，因此，極需要深入地研究。大腸桿菌屬於革蘭氏陰性桿菌，為引起新生兒細菌性腦膜炎的重要致病菌。Outer membrane protein A(OmpA)是大腸桿菌上常在且穩定的重要外膜蛋白，參與細菌的接合反應，與細菌的致病性和毒力有關。全長的OmpA 大小為1038 bp (38 KD)、8 stranded β-barrel、而且從演化的角度觀察似乎OmpA 是被高度保留，而先前Vogel H 的實驗模型中，將OmpA 區分為兩個主要domain，其中胺基酸殘基1-171 的這個domain 主要位於膜的外層，構造上這個domain又可以分為四個loop，這四個loop 都是朝向細胞膜外，在Ravi 及K. S. Kim 的文章中指出有loop 1 和2 的細菌在血清中的存活率約70%-80%，藉著loop 1，2，3 細菌得以入侵血腦屏障，另外在本實驗室先前的實驗中証實完整片段的OmpA 確實有調控E. coli與宿主細胞間的交互作用，並且大幅降低受試C57BL/6 老鼠的死亡率。所以我們希望能夠利用molecular cloning 的方式表現出OmpA 中各重組蛋白片段L1-4、L1-2、L3-4、L-3、L2-3、L2-4，再經由動物實驗來確認這些蛋白片段是否具有保護的作用，以作為日後藥物開發以及疫苗發展的先導實驗。

關鍵字

大腸桿菌；感染； OmpA 蛋白片段

並列摘要

Bacterial meningitis is one of the important causes of neonatal and child mortality around the world. According to the French statistics data from January 2001to December 2007, the mortality rate was 28% and ranked the second. According to the survey from Mackay Memorial Hospital, in Taipei, Taiwan, a twenty-nine years statistical study from 1984 to 2012, indicates patients with neonatal bacterial meningitis, will die in the portion of 1/7, and 2/5 of the patients will have sequelae. Despite of antibiotic treatment, the morbidity, mortality and the sequelae remain as a heavy burden of national health insurance system. However, the pathogenesis of bacterial meningitis hasn’t been fully understood. Therefore, further study is in demand. E. coli belongs to gram-negative bacteria, and is an important pathogen, causing neonatal bacterial meningitis. Outer membrane protein A (OmpA) is a stable and common protein in the E. coli outer membrane. It involves the bacterial conjugation, and associates with bacterial pathogenesis and virulence. The size of full length of OmpA is 1038 bp (38 KD), with 8 stranded β-barrel structure. From the perspective of evolution, it’s highly conserved. According to the experimental models made by Vogel H, OmpA is divided into two main domains. The domain containing amino acid residues 1-171 is mainly located in the outer membrane. This domain can be divided into four loops, and the four loops are towards outside of the cell membrane. The article published by Ravi and K. S. Kim pointed out with loop1 and 2, in serum, the E. coli survive rate could be up to 70%- 80%. E. coli invades blood brain barrier by loop1, 2, and 3. Additionally, in our previous report demonstrated that full-length OmpA does regulate the interaction with the host cells, and significantly reduces the mortality of C57BL/6 mice after intracerebral infection by E. coli. In this study, we express recombinant protein of OmpA fragments- L1-4, L1-2, L3-4, L-3, L2-3, L2-4 using molecular cloning. Then base on the animal experiments, we can evaluate whether these protein fragments play protective roles in the animal meningitis model. As a result, the research can be a pilot experiment both in drug and vaccine development in the future.