表現納豆激之重組大腸桿菌的冷凍保存條件探討

Studies on cryopreservation conditions of nattokinase-expressing recombinant E. coli

詹惟婷(Wei-Ting Chan)

指導教授 ： 官宜靜

大同大學/工程學院/生物工程學系所/碩士(2011年)

摘要

生物與物理、化學實驗的差異在於生物材料不易保存，故早在西元前2000年即已出現了以低溫保存生物材料來延長實驗時間的做法。隨著科學不斷的推進，此技術發展到今日，已被廣泛應用於微生物、各種動植物細胞、組織及器官上。抗凍劑的發明更讓不同種類的細胞可依其特性來做保存，但是細胞在冷凍過程中，仍無可避免的會有某些損傷，故須藉由微調冷凍的各種條件，例如如抗凍劑的種類、濃度，及冷凍、回溫的速度等，使冷凍保存程序最適化。本研究分別以含有三種不同質體：pNT、pVN及pdVN之E. coli BL-21 (DE3)來進行測試。pNAT具外源nattokinase基因，而pVN及pdVN則是除了nattokinase基因外，還分別建構了Vitreoscilla hemoglobin (VHb) 和double Vitreoscilla hemoglobin基因(由兩個VHb基因前後串聯而成)。在OD600=0.5及2時，各以0、5、10、15 % glycerol來保存菌種，並於冰凍1個月後計算存活菌量。帶有pNT之重組E. coli在OD600=0.5，以10% glycerol進行保存時的細胞存活率最高，達98 %。不論攜帶何種質體，重組E. coli於OD600=0.5保存時的存活率較在OD600=2.0時為佳。在高菌體濃度(OD600=2.0)下，帶有pNT的重組E. coli之細胞存活率明顯較帶有pVN或pdVN的細胞為高。重組質體種類、甘油和保存菌種濃度並無明顯關聯性。

關鍵字

低溫生物學 ； 重組大腸桿菌

並列摘要

The differences among biological, physical and chemical experiments mainly result from that it is not easy to preserve the biological materials. Before 2000 B.C., it is known to extend the experiment period by keeping biological materials at low temperatures. As the advance of science continues, the cryopreservation technology have been extensively used for microorganisms, a variety of tissues and organs of plants and animals nowadays. The invention of cryoprotectants (anti-freezing agents) allows the preservation of various cells according to their specific properties. During cryopreservation process, it is inevitable to have certain damages on cells. Thus, it is necessary to micro-adjust the cryopreservation conditions such as the kinds and concentrations of cryoprotectants, freezing and thawing speed etc. to optimize the freezing process. In this study, E. coli strain BL-21 (DE3) recombinants bearing pNT、pVN or pdVN were used for testing. pNT owed the exogenous nattokinase gene, while pVN and pdVN carried Vitreoscilla hemoglobin (VHb) and double Vitreoscilla hemoglobin genes (two VHb genes connected in tandem), respectively, besides the nattokinase gene. When OD600=0.5 and 2, the cells were cryopreserved in the presence of 0、5、10、15 % glycerol and examined for the survival rate after frozen for 1 month. Recombinant E. coli carrying pNT exhibited the highest survival rate of 98% when cryopreserved at OD600=0.5 in the presence of 10% glycerol. No matter carrying which plasmid, recombinant E. coli showed better survival rate at OD600=0.5 than at OD600=2. At high cell concentration (OD600=2), the survival rate for recombinant E. coli having pNT was higher than those for E. coli with pVN or pdVN. No apparent correlation among the variety of recombinant plasmids, glycerol and cell concentration for cryopreservation was found.

並列關鍵字

cryobiology ； cryopreservation ； nattokinase ； recombinant E. coli

參考文獻

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