以基因重組方式生產Aeromonas hydrophila毒力因子aerolysin與其特性之探討

Preparation and Characterization of the Recombinant Aerolysin of Aeromonas hydrophila

黃卓男(Cho-Nan Huang)

《中華民國獸醫學會雜誌》 27卷4期 (2001/12) Pp. 273-287

https://doi.org/10.7009/JTCSVS.200112.0273

摘要

香魚潰瘍病之病原菌Aeromonas hydrophila的致病性是由多種分泌性細胞外毒力因子所造成，aerolysin是其中主要的毒力因子之一，本實驗利用基因重組的方式大量製備本毒力因子，以作為瞭解aerolysin扮演的角色及其致病機制的基礎。將分離自台灣野外株之親水性產氣單胞菌A. hydrophila其毒力因子aerolysin之基因加以選殖與表現。得到preproaerolysin基因的序列，為含有1461個核酸之DNA片段，可以表現出486個氨基酸，在接入表現載體後，產生分子量約為59 kDa的preproaerolysin重組蛋白。與國外分離株中已發表的preproaerolysin與hemolysin比對，核酸序列相似性為86%以上，氨基酸序列相似則為90%以上，由基因種系親疏關係分析，顯示台灣分離株與中國大陸分離株相似度最高，而與日本分離株的相似度最低。由基因選殖所製備出來的preproaerolysin重組蛋白，經trypsin活化以後，呈現溶血活性，會對血液培養基產生溶血的現象，且對於vero細胞株有細胞溶解的毒性。在動物試驗中，會造成老鼠的死亡，在兔迴腸試驗中，會造成兔子迴腸的出血水腫。本實驗發現天然與基因重組aerolysin之生物特徵皆十分相似，故可利用基因重組的方式大量生產基因重組aerolysin，以供相關研究之用。

關鍵字

基因重組

並列摘要

The pathogen of ulcerative disease of ayu is several extracellular virulence factors secreted by Aeromonas hydrophila. Aerolysin is one of the main virulence factors. We prepare aerolysin to be the foundation to realize the role and mechanism of pathogenicity by gene recombination. Preproaerolysin gene isolated from the wild type strain of Aeromonas hydrophila in Taiwan was cloned and expressed. The cloned preproaerolysin gene included 1461 nucleotides, which encode 487 amino acids. After insertion into an expression vector, the recombinant preproaerolysin with molecular weight of about 59 kDa was expressed. The homology is higher than 85% in nucleotide sequences and higher than 90% in amino acids sequences in comparison between this cloned preproaerolysin and the other ones. Phylogenetically, the preproaerolysin gene from Taiwan is most close to the one from China and relatively distal to that from Japan. The recombinant preproaerolysin retained its toxicity after activation by trypsin. It's hemolytic ability on blood agar and cytolytic ability in vero cell were demonstrated. In the animal test, it caused the death of mice and hematoma in the rabbit ileum loop test. We found the natural and recombinant aerolysin are very similar in biological characteristics. For the future studies, preparation the recombinant aerolysin by recombination is valuable.

並列關鍵字

Recombinant ； Aerolysin ； Aeromona hydrophila