原创《重组人超氧化物岐化酶基因工程菌稳定性》：此文是一篇岐化酶论文范文,为你的毕业论文写作提供有价值的参考。
摘 要：目的：研究重组人超氧化物岐化酶毕赤酵母基因工程菌的遗传稳定性.方法：基因工程菌连续传代50次,每10代取样保藏菌种,通过对菌株形态、菌落形态、质粒稳定性、目的基因的PCR鉴定,蛋白表达的SDS-PAGE鉴定来评价工程菌的稳定性.结果：在连续传代50代的过程中,菌株、落形态和原始工程菌相比无明显差异,质粒保有率高,PCR鉴定结果显示,重组载体携带目的基因传至50代未消失,SDS-PAGE结果表明,传至50代蛋白表达量无明显差异.结论：该工程菌有良好的遗传稳定性.
关键词：重组人超氧化物岐化酶；毕赤酵母工程菌；遗传稳定性
中图分类号：Q78 文献标志码：A 文章编号：2095-2945（2018）04-0011-02
Abstract： Objective： to study the genetic stability of recombinant human superoxide dismutase Pichia pastoris. Methods： the genetically engineered bacteria were subcultured for 50 times, and the preservation strains were sampled every 10 generations. The morphology of the strain, colony morphology, plasmid stability and target gene were identified by PCR. SDS-PAGE analysis of protein expression was used to evaluate the stability of engineering bacteria. Results： there was no significant difference in the strain and colony morphology between the original engineering strain and the original engineering strain in the course of successive subculture for 50 generations. The plasmid retention rate was higher than that of the original engineering strain. The results of PCR identification showed that the plasmid retention rate was high. The result of SDS-PAGE showed that there was no significant difference in the expression of protein from the recombinant vector to the 50th generation. Conclusion： the engineering strain has good genetic stability.
Keywords： recombinant human superoxide dismutase； engineered Pichia pastoris； genetic stability
超氧化物歧化酶是生物體防御氧化损伤的一种十分重要的生物酶,具有清除体内氧自由基的能力,能较好地抵御氧自由基和其他氧化物自由基对细胞质膜的毒性[1],在机体保护方面起重要作用,在医药和化妆品方面有很好的应用前景.巴斯德毕赤酵母具有许多其他蛋白表达系统所不具备的优点,在表达产物的加工、外分秘、翻译后修饰以及糖基化修饰等方面有明显的优势,现已广泛用于外源蛋白的表达[2-3].由于巴斯德毕赤酵母自身分泌的蛋白非常少,且培养基中不含其他蛋白,分泌的外源蛋白占了培养液中蛋白的绝大部分,十分有利于蛋白的分离和纯化[4-6].实验室以pPIC9K为载体,成功将人超氧化物歧化酶基因转入巴斯德毕赤酵母GS115,构建了人超氧化物岐化酶毕赤酵母工程菌株GS115-9K-SOD,该实验旨在考察工程菌株的遗传稳定性.
1 材料和方法
1.1 材料
1.1.1 菌株
GS115-9K-SOD,由本公司构建和保存.
1.1.2 试剂
标准相对分子量DNA,购自Thermo Fisher Scientific；标准相对分子量蛋白质,26632,购自Thermo Fisher Scientific；G418,购自MP BIOmedicals；丙烯酰胺、甲叉丙烯酰胺,购自上海生化试剂抚生有限公司；酵母粉,蛋白胨,购自Oxoid；YNB,购自上海伟进生物科技有限公司.
1.1.3 仪器
PCR仪,BIO-RAD公司；垂直电泳仪DYCZ-24DN型,水平电泳仪DYCP-31DN型,北京六一仪器厂.
1.1.4 培养基
YPD液体培养基：酵母粉10g/L,蛋白胨20g/L,葡萄糖20g/L；YPD固体培养基：酵母粉10g/L,蛋白胨20g/L,葡萄糖20g/L,琼脂粉20g/L；MD培养基：葡萄糖20g/L,蛋白胨20g/L,琼脂粉20g/L,YNB13.4g/L,生物素（4×10-4）g/L；G418-YPD培养基：酵母粉10g/L,蛋白胨20g/L,葡萄糖20g/L,琼脂粉20g/L,G4184g/L；BMGY培养基：酵母粉10g/L,蛋白胨20g/L,YNB 13.4g/L,0.1mol/L pH6.0磷酸钾缓冲液,生物素（4×10-4）g/L,甘油10g/L；BMMY培养基：酵母粉10g/L,蛋白胨20g/L,YNB 13.4g/L,0.1mol/L pH6.0磷酸钾缓冲液,生物素（4×10-4）g/L,甲醇30g/L.
岐化酶论文参考资料：
结论：重组人超氧化物岐化酶基因工程菌稳定性为大学硕士与本科岐化酶毕业论文开题报告范文和相关优秀学术职称论文参考文献资料下载，关于免费教你怎么写氧化物歧化酶的作用方面论文范文。
