腸炎弧菌絲胺酸蛋白酶在大腸桿菌中的表現

Abstract

以自腸炎弧菌胞外蛋白酶純化到之絲胺酸蛋白酶A的N端序列比對已經完成染色體定序的RIM2001633株的資料庫，可得到１個相符合的蛋白序列VPA0227，其位於較小的chromosome 2上，其ORF全長為2034 bp，可轉錄轉譯成677 amino acid，其中前29個胺基酸為signal peptide而成熟態之蛋白酶由第122個胺基酸絲胺酸開始，因此絲胺酸蛋白酶A為pre-pro 型態之蛋白酶。以VPA0227序列設計多組引子來進行不同片段長度的prtA基因的選殖，並送至大腸桿菌系統中大量表現該絲胺酸蛋白酶。選殖方式中包含signal peptide sequence的選殖方式能使蛋白酶A分泌至大腸桿菌細胞外，其中以pET21a系統表現之蛋白酶活性為最高，其粗酵素液之活性為18 U/mg。在去除signal peptide sequence但仍保留pro-region方式的選殖，與GST融合之蛋白質經由GST親合管柱純化後所得到的GST融合蛋白酶A無需切去GST融合蛋白質仍可形成具活性的蛋白酶比活性為5.24 U/mg。在只保留mature形式的蛋白酶選殖方式，所表現之GST融合蛋白酶A經由GST親合管柱純化，此融合蛋白酶A無論是否加入thrombin切去GST結合蛋白皆是無蛋白酶活性呈現。以PCR檢測方式檢測38株腸炎弧菌其中包含16株環境株及22致病株的實驗結果中，97% (37/38) 的腸炎弧菌都存在prtA基因。西方漬片反應之結果証實臨床菌株與環境菌株中存在著絲胺酸蛋白酶A。在點墨漬片實驗中，10株腸炎弧菌都顯示具有蛋白酶A基因，而其他弧菌屬則有V. alginolyticus, V. costicola, V. mimicus, V. natriegens與645 bp之prtA探針具有微弱雜交訊號。

Blast against Vibrio parahaemolyticus genome database with the N-terminal sequence of mature protease A purified from V. parahaemolyticus No.93 and the ORF VPA0227 located on chromosome 2 was matched. The ORF VPA0227 has 2,034 base pairs in nucleic acid and 677 amino acids in protein. To study over-expression of protease A, several primers were designed for cloning different length of prtA. The entire prtA containing signal peptide sequence was cloned, and the recombinant proteases were secreted out of E. coli cells. When used azocoll as substrate, the protease activity of the crude enzyme was 18 U/mg. The clones harboring pro-region of PrtA without signal peptide sequence appeared protease activities in its soluble fraction. Using GST-affinity column, the purified GST-pro-PrtA were obtained and the recombinant protease activity were 5.24 U/mg. Clones without signal and pro-regions also can be purified by GST-FF column, but no protease activity. Using PCR and dot blot to detected the distributions of prtA gene in Vibrio spp. , the results showed that 97% of Vibrio parahaemolyticus strains containing prtA gene. The result of western blot confirmed the clinical strains and environmental strains processed PrtA serine protease. The genomes of V. alignolyticus, V. costicola, V. mimicus , V. natrigens had weak hybridization signals when using the probe of the 645bp fragment of prtA.