论文部分内容阅读
苯丙氨酰-tRNA合成酶是布氏锥虫蛋白合成过程中的一类重要酶,以其为靶点的抑制剂可能发展成为新一代的抗锥虫药物,但此前并没有分离锥虫苯丙氨酸-tRNA合成酶的报道。本研究用大肠杆菌成功克隆表达并纯化了布氏锥虫苯丙氨酰-tRNA合成酶并进行了活性测定。首先通过PCR方法从布氏锥虫细胞基因组中分别扩增出苯丙氨酰-tRNA合成酶的α亚基、β亚基的基因,依次克隆入pCOLADuet共表达载体,然后在大肠杆菌BL21(DE3)RIPL中进行了成功表达,并采用Ni-Bind亲和层析对其进行了纯化,最后用免疫印迹进行了鉴定。此外还采用放射性同位素方法进行了酶活性测定,这为下一步进行布氏锥虫苯丙氨酰-tRNA合成酶抑制物的设计和体外筛选奠定了良好的基础。
Phenylalanyl-tRNA synthetase is a type of important enzyme in the process of Trypanosoma brucei protein synthesis. The target inhibitors may develop into a new generation of trypanosomiasis, but trypanosinate Alanine-tRNA synthetase reported. In this study, Clostridium butyricum phenylalanyl-tRNA synthetase was successfully cloned and expressed and purified. Firstly, the α-subunit and β-subunit gene of phenylalanyl-tRNA synthetase were amplified from the genome of Trypanosoma brucei by PCR and cloned into pCOLADuet co-expression vector, then expressed in E. coli BL21 ) RIPL was successfully expressed and purified by Ni-Bind affinity chromatography and finally identified by immunoblotting. In addition, the radioactive isotope method was also used for the determination of enzyme activity, which laid a good foundation for the design and screening of Trypanosoma brucei phenylalanyl-tRNA synthetase inhibitor in the next step.