探討腸炎弧菌幾丁質酶A所扮演的角色及功能

Abstract

腸炎弧菌胞外絲胺酸蛋白酶PrtA已證實為一個可能的致病因子，本篇研究首先於養分成分不同之培養基中比較腸炎弧菌野生株與PrtA蛋白酶缺陷株的基本生理特性，包括菌株生長速率、膠原蛋白酶及幾丁質酶酵素活性以及上述三種蛋白質的轉錄情形。結果發現野生株及PrtA蛋白酶缺陷株之菌體生長速率於二種培養基中無明顯差異，腸炎弧菌幾丁質酶及膠原蛋白酶的基因及其酵素活性會經由海洋類似成份的培養基誘導而明顯表現。其中之VPA1598幾丁質酶基因表現，會由於prtA基因缺失的影響而表現量減少。經由胺基酸序列比對發現編號VPA1598之幾丁質酶與霍亂弧菌的GlcNAc-binding protein (GbpA, 編號VCA0811)有高達百分之七十的高相似度。由於GbpA已證實為霍亂弧菌附著於浮游生物幾丁質外骨骼，及人體小腸上皮細胞之蛋白質醣基的主要因子，故本篇研究擬繼續探討腸炎弧菌VPA1598 (命名為chitinase A)，是否為環境生存及參與人體感染的重要因子。ChiA基因長為1,464 bp，可轉譯成53 kDa幾丁質酶。選殖chiA基因至pET-T7啟動子表現系統，在30℃以0.5 mM IPTG誘導10小時由大腸桿菌大量表現His-tagged ChiA重組蛋白，分子量為56.6 kDa，純化後取2 mg作為抗ChiA抗體製作的抗原。製備一株chiA 基因缺陷株，以序列in-frame的方式建構，目的為踢除chiA基因而不影響前後基因序列，經由定序確認所建構之缺陷株序列無誤。此外亦建構一株ChiA補償株，目的為欲證實ChiA是否為單一影響菌體對環境中幾丁質附著及人體小腸附著的因子，供日後與ChiA缺陷株對照研究幾丁質酶A之功能。本研究所建構的缺陷株及補償株的ChiA存在情形亦經由ChiA抗體偵測證實之。

Vibrio parahaemolyticus no. 93 extracellular serine protease PrtA has been considered as a putative virulence factor. Accordingly, we compared basic physical properties including growth rates, protein activities and specific genes transcriptional level of wild-type and the prtA mutant. Results showed that nutrient differences had no siginificant effect on the growth of wild-type and the prtA mutant. On the contrary, madeium containing marine alike ingredients, such as marine broth, not only apparently induced chitinase and collagenase activities from both of the strains but indeed had effect on those gene transcriptional expressions. It was notced that gene expression of VPA1598 and that of collagenase prtV were extremely low in the prtA deletion mutant when incubated in marine broth medium. The amino acid sequence of VPA1598, encoding a chitinase, had 70% similarity with that of VCA0811, a V. cholerae GlcNAc-binding protein (GbpA). We considered that VPA1598 may have similar properties to GbpA of being a colonization factor that links environmental survival and human infection. The chitinase gene named chiA was cloned from V. parahaemolyticus no. 93 into pET-T7 promoter system and expressed as a His-tagged fusion protein in Escherichia coli BL21star (DE3). The chiA gene, 1,464 bp in length, could be translated to a molecular weight 53 kDa protein. By adding 0.5 mM of IPTG to the culture and inducted at 30℃ for 10 hours, His-tagged ChiA was purified with affinity column and 2 mg of protein was acquired to immunize rabbit for preparing anti-ChiA antibody. Thereafter, we successfully constructed a chiA in-frame deletion mutant and a complement strain of ΔchiA; both sequences were confirmed by nucleotide sequencing. ChiA of wild-type, deletion mutant and complement strains have been detected by using anti-ChiA antibody.