精氨酸脱酰酶的克

发布时间：2018-03-11 05:16

本文选题：精氨酸脱酰酶　切入点：克隆　出处：《中国医科大学》2007年硕士论文　论文类型：学位论文

【摘要】： 目的合成编码支原体精氨酸脱酰酶(Arginine Deiminase，ADI)的基因，构建原核表达载体，获得高水平表达支原体精氨酸脱酰酶的工程化菌种；探索用聚乙二醇修饰支原体精氨酸脱酰酶的反应条件，建立PEG化支原体精氨酸脱酰酶(PEG-ADI)的制备工艺；探索修饰混合物的分离纯化工艺，获得PEG-ADI纯品；建立活性测定方法，并研究修饰物活性。方法采用化学合成方法和DNA聚合酶链式反应(PCR)合成编码支原体精氨酸脱酰酶的基因。采用pBV220质粒构建原核表达载体，转化大肠杆菌DH5α，筛选氨苄青霉素抗性克隆。采用5升发酵系统进行发酵研究。采用分子量20kD的聚乙二醇修饰精氨酸脱酰酶。探索温度、搅拌、反应时间等因素对反应效率的影响。采用凝胶层析等方法对修饰产物进行分离纯化。采用聚丙烯酰胺凝胶电泳检测蛋白质分子量；采用C18反向高效液相色谱分析修饰产物纯度；采用体外精氨酸降解方法研究修饰产物的活性。结果我们获得了编码支原体精氨酸脱酰酶的全长DNA，成功构建了原核表达载体pBV220-ADI，获得了表达支原体精氨酸脱酰酶的大肠杆菌菌种，，目标蛋白表达量占全菌蛋白的30％以上；表达产物主要以包含体形式存在；发酵实验表明工程菌能够大规模制备，表达稳定；建立了聚乙二醇修饰精氨酸脱酰酶的化学反应工艺；建立了修饰产物的分离纯化工艺。通过二次凝胶排阻层析方法可以充分分离单一修饰产物，获得了单修饰产物纯品；成功建立了精氨酸降解法来测定产物活性的质量控制方法，未修饰ADI与PEG-ADI的比活性分别为40IU/mg和30IU/mg；聚丙烯酰胺凝胶电泳检测ADI和PEG-ADI的分子量分别约为45kD和65kD；高效液相检测结果显示PEG-ADI纯度大于95％。结论本研究通过原核表达系统获得了支原体精氨酸脱酰酶的高效表达。表达产物以包含体形式存在于细胞质内。小试发酵表明该菌种可以进行放大生产，这为继续进行ADI深层次研究奠定了基础；建立了优化的聚乙二醇修饰精氨酸脱酰酶的反应条件，明确了最佳反应参数，为大规模制备PEG-ADI提供了前提；研究还建立了简便、高效的反应混合物分离纯化工艺路线，为聚乙二醇修饰物的制备提供了坚实的下游工程技术保障；研究还建立了采用体外精氨酸降解法来测定产物活性的质量控制方法，为该活性物的深入研究提供了方便的检测和控制手段。本研究为肝癌治疗提供了一个新的活性物质，具有重要的学术意义和临床应用价值。
[Abstract]:Purpose. The gene encoding arginine deacylase (Arginine Deiminase) was synthesized, the prokaryotic expression vector was constructed, the engineering strain expressing arginine deacylase with high level was obtained, and the reaction conditions for modification of arginine deacylase by polyethylene glycol were explored. To establish the preparation process of PEG arginine deacylase (PEG-ADI); to explore the separation and purification of modified mixture to obtain pure PEG-ADI; to establish a method for the determination of activity; and to study the activity of the modified compound. Method. The gene encoding arginine deacylase of mycoplasma was synthesized by chemical synthesis and DNA polymerase chain reaction. The prokaryotic expression vector was constructed by using pBV220 plasmid. Transformation of Escherichia coli DH5 伪, screening of ampicillin resistant clones. Fermentation of ampicillin by 5 liter fermentation system. 20kD polyethylene glycol modified arginine deacylase. The effect of reaction time and other factors on the reaction efficiency. The modified product was separated and purified by gel chromatography. The molecular weight of the modified product was detected by polyacrylamide gel electrophoresis, and the purity of the modified product was analyzed by C18 reverse high performance liquid chromatography. The activity of the modified product was studied by arginine degradation in vitro. Results. The full-length DNA encoding mycoplasma arginine deacylase was obtained, and the prokaryotic expression vector pBV220-ADIwas constructed successfully. The E. coli strain expressing mycoplasma arginine deacylase was obtained, and the target protein expression amount was more than 30% of the whole bacterial protein. The expression products mainly existed in the form of inclusions, the fermentation experiments showed that the engineering bacteria could be prepared on a large scale and expressed stably, and the chemical reaction process of polyethylene glycol modified arginine deacylase was established. The purification process of the modified product was established. The single modified product could be fully separated by secondary gel exclusion chromatography, and the pure product of the single modified product could be obtained, and the arginine degradation method was successfully established to determine the quality control method for the activity of the product. The specific activities of unmodified ADI and PEG-ADI were 40 渭 / mg and 30 渭 / mg respectively, the molecular weight of ADI and PEG-ADI by polyacrylamide gel electrophoresis were about 45kD and 65kDrespectively, and the purity of PEG-ADI was higher than 95kD by HPLC. Conclusion. In this study, a prokaryotic expression system was used to obtain the high expression of arginine deacylase from mycoplasma. The expressed product existed in the cytoplasm in the form of inclusion body. The small scale fermentation showed that the strain could be amplified to produce mycoplasma arginine deacylase. This laid a foundation for the further study of ADI, established the optimized reaction conditions of polyethylene glycol modified arginine deacylase, determined the optimum reaction parameters, provided the premise for the large-scale preparation of PEG-ADI, and established a simple and convenient method for the preparation of PEG-ADI. The efficient separation and purification process of reaction mixture provides a solid downstream engineering guarantee for the preparation of polyethylene glycol modifiers, and also establishes a quality control method for the determination of product activity by in vitro arginine degradation method. This study provides a new active substance for the treatment of liver cancer and has important academic significance and clinical application value.
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R341

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