臺灣博碩士論文加值系統

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.

目錄
　　　　　　　　　　　　　　 頁次
壹、 前言
一、 腸炎弧菌（Vibrio parahaemolyticus）……………….………….1
二、 腸炎弧菌胞外致病因子之探討………………………………….2
1. 溶血素…………………………………………………………………2
2. 致死毒素………………………………………………………………4
3. 尿素水解酶……………………………………………………………5
4. 胞外蛋白酶……………………………………………………………5
5. DNA 甲基轉換酶……………………………………………………..7
三、 致病途徑的探討………………………………………………….7
四、 腸炎弧菌之基因體..................................................................8
五、 蛋白質的大量表現系統...........................................................8
六、 研究緣起與目的…………………………..…………………….12

貳、 實驗方法及步驟
Ⅰ、實驗材料
一、菌株與質體. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . ... .13
二、培養基. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. . . . . .13
三、藥品及試劑. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . … . . .13
四、實驗中使用之套組(kit) . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . .14
五、試劑、緩衝溶夜. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . …..14
Ⅱ、實驗方法：
一、一般DNA技術
1．菌株、質體與引子. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17
2．DNA瓊脂膠體電泳. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17
3．細菌染色體DNA的製備. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17
4．質體的製備. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17
5．引子設計. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18
6．聚合酶連鎖反應. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18
7．選殖DNA與載體的製備. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18
8．接合作用. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18
9．勝任細胞之製備. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
10．轉形作用. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19
11．基因定序及序列分析. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19
12．篩選選殖株. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19
13．Probe的製備. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20
14．點墨漬片. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20
二、prtA於大腸桿菌中的表現及培養條件的探討
1．培養溫度及表現蛋白分佈的探討. . . . . . . . . . . . . . . . . . . . . . . 21
2．IPTG的誘導. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21
3．誘導時間. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21
三、蛋白質技術
1．蛋白質濃度的測定. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21
2．SDS-PAGE蛋白質膠體電泳. . . . . . . . . . . . . . . . . . . . . . . . . . .22
3．活性電泳. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22
4．CBR染色. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .23
5．偶氮基膠原活性測試. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .23
6．抗體製作…………………………………………………………….23
7．GST融合蛋白質的純化……………………………………………24
8．西方漬片….. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .24
9．In-gel digestion……………………………………………………..25


參、實驗結果
一、 prtA基因的選殖. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
二、大腸桿菌辨識prtA的signal peptide………………….………….27
三、不同溫渡、IPTG濃度對表現蛋白酶的影嚮………………….….27
四、表現重組蛋白分子大小. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
五、表現GST結合蛋白的純化及活性. . . . . . . . . . . . . . . . . ………29
六、prtA gene在Vibro spp.的分布情形. . . . . . . . . . . . . . . .... . . .30

肆、討論
1.重組蛋白質的表現…………………………………..…………31
2.重組蛋白酶的純化及活性……………………………………..31
3.PCR檢測prtA基因的分佈……………………………..…….33
伍、結論…………………………………………………………….…34

陸、參考文獻. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . .35









表次
表一、本研究所使用的菌株與質體. . . . . . . . . . . . . . . . . . . . . . . . . .45
表二、本研究所使用的引子. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .48
表三、E. coli BL21 (DE3) (pMHK1) 於不同溫度下經1mM
IPTG 誘導其蛋白酶表現分布比較. . . . . . . . . . . . . . . . . . ..49
表四、 E. coli BL21 (DE3) (pMHK1) 於25℃以不同濃度IPTG
誘導下胞外蛋白酶表現之活性. . . . . . . . . . . . . . . . . . . . . . .50
表五、E. coli novablue (pMHK2) 於不同溫度下經1mM
IPTG誘導其蛋白酶表現分布比較. . . . . . . . . . . . . . . . . . . . 51
表六、E. coli novablue (pMHK2) 於25℃以不同濃度IPTG
誘導下胞外蛋白酶表現之活性. . . . . . . . . . . . . . . . . . . . . . .52
表七、E. coli Top10 (pMHK3) 不同溫度下經1mM IPTG誘導其
蛋白酶表現分布比較. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
表八、E. coli Top10 (pMHK3) 於25℃以不同濃度IPTG誘導
下胞外蛋白酶表現之活性. . . . . . . . . . . . . . . . . . . . . . . . . . .54
表九、探針偵測腸炎弧菌prtA存在情形. . . . . . . . . . . . . . . . . . . . . .55
表十、建構之各種表現株之表現活性. . . . . . . . . . . . . . . . . . . . . . . 57




圖次

圖一、 利用aligment預測PrtA活性位置. . . . . . . . . . . . . . . …….58
圖二、V. parahaemolyticus N093之prtA核酸及胺基酸序利……61
圖三、不同的引子以PCR方式增幅prtA基因……………………65
圖四、pET21a表現系統之prtA基因之選殖與建構.....................66
圖五、pQE表現系統之prtA基因之選殖與建構. . . . . . . . . . . . . .67
圖六、pTrcHis表現系統之prtA基因之選殖與建構. . . . . . . . . . . ..68
圖七、pET41a表現系統之prtA基因之選殖與建構. . . . . . . . . . . 69
圖八、建構之各表現質體的比較示意圖…………………………..70
圖九、轉殖株於2% gelatin medium上之活性測試. . . . . . . . . . . .71
圖十、pMHk1胞外蛋白質之表現. . . . . . . . . . . . . . . . . . . . . . . .72
圖十一、pMHK1胞內蛋白質之表現. . . . . . . . . . . . . . . . ………73
圖十二、pMHK1形態之蛋白表現及活性測試. . . . . . . . . . . . . . . .74
圖十三、LC-Mass-Mass檢測pMHK4表現蛋白質之位置. . .. .. 75
圖十四、pMHK5及pMHK6的蛋白質表現及純化. . . . . . .. . . . .76
圖十五、GST-fusion PrtA活性測試. . . . . . . . . . . . . . . . . . . . 77
圖十五、以prtA片段基因為探針之點墨雜交. . . . . . . . . . . . . . .78


圖十六、PCR檢測prtA gene 在V. parahaemolyticus strains
的分佈情形. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ….79
圖十七、西方漬片…….. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . .80

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