【作者】 杨书慧；

【导师】 赵胜军；

【作者基本信息】 武汉工业学院 ， 动物营养与饲料科学， 2008， 硕士

【摘要】 本研究设计单链monellin基因,利用大肠杆菌表达系统,构建高表达的Monellin重组大肠杆菌工程菌,从而提高Monellin的稳定性;并用乳糖代替IPTG作为诱导剂,以降低生产成本。同时对两种载体(pET22b和pET28a)构成的大肠杆菌表达系统的表达效果进行了比较。1根据monellin基因的序列,按照Coden Usage Database数据库(NCBI-GenBank)中大肠杆菌密码子的偏爱性,人工设计并合成monellin基因。并在合成基因的5’端添加NcoⅠ酶切位点,在基因的3’端添加BamHⅠ酶切位点,将合成好的全基因通过NcoⅠ/BamHⅠ位点克隆到pET22b载体上。构建重组载体pET22b-Mon,将重组载体转化大肠杆菌BL21(DE3),得到表达monellin的大肠杆菌工程菌株DE3-pET22b-Mon。再用NcoⅠ、BamHⅠ从含甜蛋白monellin基因的pET22b上切下monellin基因,并将其插入到pET28a的NcoⅠ、BamHⅠ位点之间,构建重组载体pET28a-Mon,将重组载体转化大肠杆菌BL21(DE3),得到表达Monellin的大肠杆菌工程菌株DE3-pET28a-Mon。2以摇瓶发酵为基础,探讨了不同载体构成的大肠杆菌工程菌在异丙基硫代-β-D-半乳糖苷(IPTG)诱导下的表达,研究了诱导起始生长量、诱导培养温度、诱导培养时长及接种量对目的蛋白表达的影响。对于大肠杆菌工程菌株DE3-pET22b-Mon,其诱导优化条件如下:在装液量为20mL/100mL摇瓶中,按1%的接种量接种,当OD600值达到0.73时,添加诱导剂IPTG至终浓度为1.0mmol/L,37℃继续培养6h后,Monellin的表达量达到细菌总蛋白的22.26%。对于大肠杆菌工程菌株DE3-pET28a-Mon,其诱导优化条件如下:在装液量为20mL/100mL摇瓶中,按1%的接种量接种,当OD600值达到1.0时,添加诱导剂IPTG至终浓度为1.0mmol/L,37℃继续培养6h后,Monellin的表达量高达细菌总蛋白的34.08%。3以摇瓶发酵为基础,探讨了不同载体构成的大肠杆菌工程菌在乳糖诱导下的表达,研究了诱导剂浓度、诱导起始生长量、诱导培养温度、诱导培养时长及接种量对目的蛋白表达的影响。对于大肠杆菌工程菌株DE3-pET22b-Mon,其诱导优化条件如下:在装液量为20mL/100mL摇瓶中,按3%的接种量接种,当OD600值达到1.0时,添加诱导剂乳糖至终浓度为10g/L,37℃继续培养7h后,Monellin的表达量占细菌总蛋白的19.86%。对于大肠杆菌工程菌株DE3-pET28a-Mon,其诱导优化条件如下:在装液量为20mL/100mL摇瓶中,按3%的接种量接种,当OD600值达到1.0时,添加诱导剂乳糖至终浓度为11g/L,37℃继续培养8h后,Monellin的表达量占细菌总蛋白的33.09%。4在摇瓶发酵的基础上,以DE3-pET28a-Mon为研究对象,选用终浓度为11g/L的乳糖作为诱导剂,采用5L的发酵罐进行小试。发酵终产物OD600=6.02,菌体湿重=18.5g,得到的纯化Monellin为0.06g。性质较天然Monellin有显著改善:中性条件下,60℃下保温50min仍具有强烈的甜味,70℃保温5min仍有甜味;在pH=3,50℃下仍有甜味。更多还原

【Abstract】 According to the monellin amino acid sequence and Escherichia coli preference codon usage, the monellin gene was designed and synthesized. Then it was cloned into vector pET22b and pET28a, and the recombined plasmid pET22b-Mon and pET28a-Mon were transformed into E. coli BL21(DE3). The recombinant might improve monellin stability upon temperature or pH changes. Lactose was used as an inducer instead of IPTG, which might reduce the production cost. And compared the influences of two different vectors(pET22b/pET28a) on the expression of Monellin.The synthesized monellin gene was cloned in the pET22b vector by using NcoⅠ/BamHⅠcutting sites. Recombinant plasmid pET22b-Mon was gotten, which was then transformed into E. coli BL21(DE3) to obtain the recombinant strain- DE3-pET22b-Mon.The monellin gene was cut from pET22b-Mon by NcoⅠ/BamHⅠ, and was ligated in pET28a. Recombinant plasmid pET28a-Mon was gotten, which was then transformed into E. coli BL21(DE3) to obtain the recombinant strain- DE3-pET28a-Mon.The induction conditions on the expression of Monellin were optimized when inducing by IPTG. As for DE3-pET22b-Mon, it was grown in 100 mL shake flask with 20mL LB at 37℃. Vaccination quantity was 1%. When the culture OD600nm reached 0.73, IPTG was added to give a final concentration of 1mmol/L. The culture was then incubated at 37℃for 6h and the Monellin content reach 22.26% of total proteins. As for DE3-pET28a-Mon, it was grown in 100mL shake flask with 20mL LB at 37℃. Vaccination quantity was 1%. When the culture OD600nm reached 1.0, IPTG was added to give a final concentration of 1mmol/L. The culture was then incubated at 37℃for 6h and the Monellin content reach 34.08% of total proteins.The induction conditions on the expression of Monellin were optimized when inducing by lactose. As for DE3-pET22b-Mon, it was grown in 100mL shake flask with 20mL LB at 37℃. Vaccination quantity was 3%. When the culture OD600nm reached 1.0, lactose was added to give a final concentration of 10g/L. The culture was then incubated at 37℃for 7h and the Monellin content reach 19.86% of total proteins. As for DE3-pET28a-Mon, it was grown in 100mL shake flask with 20mL LB at 37℃. Vaccination quantity was 3%. When the culture OD600nm reached 1.0, lactose was added to give a final concentration of 11g/L. The culture was then incubated at 37℃for 8h and the Monellin content reach 33.09% of total proteins.To exploite feasibility of producing Monellin in large scale, we chose DE3-pET28a-Mon and lactose for fermention. The germ with OD600nm=0.8 was poured into the pilot scale fermentor(working volume is 5L), and cultured for 21h to give a final optical density of 6.02 at 600nm, the purified expressed protein yield reached 0.06g. And the newly designed protein is the same sweet as the nature one. It is more stable upon temperature or pH changes, and retains sweetness after heating to 70℃for 5min, even heating to 50℃at low pH(pH=3).更多还原