【作者】 成岩；

【导师】 颜炜群；

【作者基本信息】 吉林大学 ， 医学生物工程学， 2007， 硕士

【摘要】 海葵毒素(anthopleurin)是一类由海葵毒液中分离到的能增强哺乳动物心肌收缩能力的多肽类毒素,相对分子质量在5kD左右,其作用于钠离子通道,延长去极化过程中钠通道的开放时间,致使钠离子内流增加,增强心肌收缩。海葵毒素对心肌产生生理活性的过程中,对实验哺乳动物的心率和血压都没有不利的影响,可望开发成为新一代安全有效的强心药物。在所有的海葵毒素中,anthopleurin B(Ap-B)型表现出最强的心肌收缩活性。本研究按照毕氏酵母的偏爱密码子,设计并合成了Ap-B的基因序列,将合成基因序列导入毕氏酵母表达载体pPICZαC中,以电穿孔方法转化入毕赤酵母X33,并进行转化子的表型及高拷贝筛选,通过PCR鉴定和SDS-PAGE测定,筛选出高表达重组Ap-B(rAp-B)的酵母菌株。建立大规模(80L发酵罐)发酵体系,优化发酵条件。本实验的创新之处在于:在毕赤酵母中高效表达了rAp-B;利用80L发酵罐,通过改变发酵液的pH值、DO值(溶解氧)、培养液成分、加入甲醇速度等多个影响发酵过程的因素,确定了适合工业化生产rAp-B的最佳发酵条件,为rAp-B的生产提供了实验依据。更多还原

【Abstract】 Shibata at theUniversity of Hawaii isolated the neuro toxins Ap-A,Ap- B from sea anemonein the1976 and d scovered the toxins were highly heartst imulant.Following this discovery, a variety of neuro toxins were characterized and their amino acids sequences, secondary structure and tertiary structure were also characterized. These toxins are polypeptides of single chain and consist of 46-49 amino acids, cross-linked by threed isulfideb ridges.The ordered secondary structure has been determined on the basis of NMR studies. The dominant feature is a four-stranded anti-parallelβ-sheet,linked with three loop.Sea anemone neurotoxins act by binding to specific receptor sites on the voltage-gated sodium channel of excitable tissue.They can prolong action potentials due to their action in slowing down the inactivation of theNa+ channel.As a result the toxins cause intracellular Na+ and Ca2+ increasing,so the toxins can enhance heart contraction.Its activities are not associated with any significante ffects on heart rate or blood pressure in vivo.On the other hand,the toxins have been shown to increase action potential duration of cardiocytes and belong to class III antiarrhythmic effects. So the toxins may serve as a valuable lead in the development of new therapeutic agent for treatment of the heart failure.Recently, we are interested in studies on Ap-B by genic engineering technology. Ap-B can be expressed using many expression systems such as Pichia pastoris and E.coli expression systems.Studies were done as follows:①Constructed expression plasmid pPICZαC-Ap-B and transformed it into Pichia pastoris via electroporation.We obtained r Ap-B secreted into the culture supernatant by the Pichia pastoris;②Studied the large-scale fermentation process of r Ap-B in the New Brunswick Scientific bioflow 5000 fermentor;1. Construction of Pichia pastoris expression system for r Ap-B①Synthesis of Ap-B gene sequence introducts restriction enzyme adhesive end.After .reannealing, the sequence was inserted into pPICZαC vector which has been endonuclease digestation It showed that the recombinant. After confirmation by endonuclease digestation assay and sequencing, linearized the expression vector pPICZαC-Ap-B and transformed it into Pichia pastoris via electroporation.②Screening of the transformed Pichia pastoris: Extracted the genomic DNA of the transformed yeasts to perform PCR using the expression primers.③Expression and assay of rAp-B Proliferated the PCR tested positively yeast clones, then induced the expression of rAp-B with 0.5% methanol in BMMY media. Analyzed the expression products by activity determination and SDS-PAGE. In conclusion, we obtained the protein with a Mr of 5kD and it could inhibited PPE.2. Studies on large-scale fermentationStudies on large-scale fermentation process of rAp-B: Pichia pastoris has many advantages as a kind of expression host, and it is very suitable for large-scale expression of the extraneous proteins. Accordingly, it is very important to study on large-scale fermentation of rAp-B. So after identifying the bioactivities of the rAp-B, we explored the large-scale fermentation process of rAp-B and found that the best pH was pH4.0, DO between 20%～30% and the supplied speed of methanol was 7.2ml/h/L initial fermentation volume. The concentration of r-Ap-B in the broth can reached 80mg·L-1 .In conclusion, our studies on rAp-B had very important meaning, which established significant foundation for the further study.更多还原