紫花地丁一种新型环肽的制备工艺及功能研究

发布时间：2018-05-12 22:16

本文选题：大环肽 + 包涵体　；参考：《广东药科大学》2017年硕士论文

【摘要】：本课题主要内容为大环肽241b的重组表达、纯化工艺与生物功能的研究。涉及对目的大环肽241b的表达与富集、包涵体处理条件与大环肽纯化过程优化;采用抑菌试验、细胞实验和秀丽隐杆线虫等不同方法检测目的大环肽具有的生物学功能;对大环肽发酵过程中质量控制及对污染菌株进行鉴定,并基于秀丽隐杆线虫检测其对241b生物学活性的影响。通过对上述几方面内容研究,获得如下结果:1、在实验室前期表达优化后的基础上,获得可溶性目的大环肽241b。并对沉淀包涵体中目的大环肽的释放条件进行优化,获得一组包涵体释放的工艺条件,即:使用8 mol/L的尿素溶解包涵体后,获得的上清液在含有1 mmol/L GSH,0.1 mmol/L GSSG,0.8 mmol/L L-Arg的透析液中,于25°C条件下进行梯度透析,最终每克包涵体可获得约1.4 mg大环肽。2、采用C18固相萃取对可溶性蛋白进行脱盐处理,70%ACN进行洗脱;脱盐后大环肽经程序为:0-5 min(88%A相:5%ACN+0.05%TFA,12%B相:90%ACN);5.01-10 min(40%A相,60%B相);10.01-30(10%A相,90%B相);30-30.10(88%A相,12%B相);30.10-40(88%A相,12%B相)的RP-HPLC进行洗脱;经MOLDI-TOF-MS鉴定所得目的大环肽为241b。3、MIC法测定大环肽241b对G-菌,大肠杆菌、绿脓杆菌最小抑菌浓度分别为0.2、1.0 mg/m L;对G+菌枯草芽孢杆菌和金黄色葡萄球菌的最小抑菌浓度均为0.2mg/m L。CCK-8法检测241b在低浓度时对MCF-7生长具有促进作用,高浓度具有抑制作用:C241b=0.03 mg/m L时,较对照组细胞活性增加38.44%;C241b=1mg/m L时达到最大抑制效果,达95%,IC50值为0.225 mg/m L。当241b与Cd Cl2共同作用于MCF-7时,展现出明显的不规律性,说明两者在适当的浓度条件下可发生络合作用。对L1秀丽隐杆线虫饲喂241b培养48 h,C241b=0.5 mg/m L时,对线虫生长抑制效果达100%;浓度为1 mg/m L时,线虫全部死亡。当C241b=0.05mg/m L时,线虫中值寿命降低36.36%;随着大环肽浓度的增加而逐渐减少;当C241b=2 mg/m L时,中值寿命降低90.9%;继续增加大环肽浓度,已无明显影响。该结果说明大环肽对线虫的生长具有较强的抑制作用。4、对比相同条件下,摇瓶和搅拌式发酵罐发酵方式对大环肽的产量影响。优化条件后,确定:241b菌液接种量为5%,加入葡萄糖终浓度为10 g/L LB中,培养至OD600≈1.2时,IPTG诱导表达8 h后,摇瓶与发酵罐中菌体量分别为3.5 g/L和5.38 g/L,菌体质量提高了34.76%;包涵体中目的蛋白含量减少30.43%;最终摇瓶中每升发酵液可获得44.91 mg大环肽241b,而发酵罐中可获得68.57 mg,产量提高34.5%。说明在原有表达条件基础上进一步扩大培养,可提高大环肽产量。5、采用16S r DNA技术,鉴定污染的241b保存菌种241b-5和241b-7为粘质沙雷氏菌的两个亚型。基于秀丽隐杆线虫模型,检测其对241b生物学活性影响。发现单独添加241b-5、241b-7时,均会降低线虫的生长速率、寿命及身体摆动频率;而与241b-1混合添加时,两者对线虫的生长速度均具有明显影响,而对头部摆动频率影响较小;且241b-5与241b-1菌液混合时对线虫寿命的影响与241b-1无明显差别;当241b-7与241b-1混合时,线虫寿命相比于241b-1单独添加时降低了16%。为保证发酵质量,应在实验操作过程控制好实验卫生条件。综上所述,本文通过重组表达技术获得具有生物活性的大环肽,为重组大环肽的工业化生产提供了理论依据,也为本实验室后期突变体库的构建奠定了基础。
[Abstract]:The main content of this project is the recombinant expression of macrocyclic peptide 241b, the purification process and the biological function research. It involves the expression and enrichment of the target macrocyclic peptide 241b, the conditions of inclusion body treatment and the optimization of the purification process of macrocyclic peptide, and the biological work of the macrocyclic peptide detection by the different methods of bacteriostasis test, cell experiment and Caenorhabditis elegans. Quality control and identification of contaminated strains in the fermentation process of macrocyclic peptide and the detection of the effect of Caenorhabditis elegans on the biological activity of 241b. The following results were obtained by studying the contents of these aspects. 1, on the basis of the optimization of the early expression of the laboratory, the soluble target macrocyclic peptide 241b. was obtained and the inclusion inclusion inclusion inclusion was obtained. The release conditions of the target macrocyclic peptide in the body were optimized, and a group of process conditions for the release of inclusion bodies were obtained, that is, after the inclusion body was dissolved with 8 mol/L urea, the obtained supernatant was in the dialysate containing 1 mmol/L GSH, 0.1 mmol/L GSSG, 0.8 mmol/L L-Arg, and at 25 degree C, the inclusion body could get about 1.4 m. G macrocyclic peptide.2 was desalted by C18 solid phase extraction and eluted by 70%ACN; after desalination, the macrocyclic peptide was programmed to be 0-5 min (88%A phase: 5%ACN+0.05%TFA, 12%B phase: 90%ACN); 5.01-10 min (40%A phase); The purpose of MOLDI-TOF-MS identification was 241b.3. The minimum inhibitory concentration of macrocyclic peptide 241b for G-, Escherichia coli and Pseudomonas aeruginosa was 0.2,1.0 mg/m L, and the minimum inhibitory concentration for Bacillus subtilis and Staphylococcus aureus were 0.2mg/m L.CCK-8 method, which promoted the growth of 241b at low concentration. Effect, high concentration has inhibitory effect: C241b=0.03 mg/m L, the cell activity of the control group increased by 38.44%, C241b=1mg/m L reached the maximum inhibitory effect, reached 95%, IC50 value was 0.225 mg/m L. when 241b and Cd Cl2 acted on MCF-7, showing obvious irregularity, suggesting that the two could have complex action under the appropriate concentration conditions. L1 Caenorhabditis elegans feeding 241b culture 48 h, C241b=0.5 mg/m L, the inhibitory effect on the growth of nematodes reached 100%; when the concentration was 1 mg/m L, the nematodes all died. When C241b=0.05mg/m L, the median life of nematodes decreased by 36.36%; as the concentration of macrocyclic peptides increased, the median life expectancy decreased by 90.9%; continue to increase large rings. The results showed that the peptide concentration had no obvious influence. The results showed that the macrocyclic peptide had a strong inhibitory effect on the growth of nematode.4. Under the same condition, the production of macrocyclic peptide was influenced by the fermentation mode of shake flask and stirred fermenting tank. The optimum conditions were determined: the inoculation amount of 241b bacteria was 5%, and the final concentration of glucose was 10 g/L LB, and it was cultured to OD600 1.2 When IPTG was induced to express 8 h, the amount of bacteria in the shake flask and the fermenting tank were 3.5 g/L and 5.38 g/L, respectively, the mass of the bacteria increased by 34.76%, the content of the target protein in the inclusion body decreased by 30.43%, and the final fermentation liquid in the shake flask could obtain 44.91 mg macrocyclic peptide 241b, and the fermentation tank could obtain 68.57 mg, and the yield increased 34.5%. in the original expression basis. On the basis of further expansion, the yield of macrocyclic peptide could be increased by.5, and 16S R DNA technology was used to identify the two subtypes of 241b-5 and 241b-7 contaminated 241b species, 241b-5 and 241b-7. Based on the Caenorhabditis elegans model, the effects on the biological activity of 241b were detected. It was found that the growth rate of the nematode could be reduced when 241b-5241b-7 was added alone. Life and the frequency of body wobble; when mixed with 241b-1, both of them have an obvious effect on the growth speed of the nematode, but less on the frequency of the head swing, and there is no significant difference in the effect of the 241b-5 and 241b-1 mixture on the life of the nematode when mixed with the 241b-1. When 241b-7 and 241b-1 are mixed, the life of the nematode is added to the 241b-1 alone. In order to reduce the quality of 16%. to ensure the quality of fermentation, we should control the experimental sanitary conditions in the process of experimental operation. In summary, the recombinant expression technology is used to obtain macrocyclic peptides with biological activity, which provides a theoretical basis for the industrial production of recombinant macrocyclic peptide and the foundation for the construction of the later mutant library of the laboratory.

【学位授予单位】：广东药科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R945

【参考文献】