CAJ | 학술논문

目的　利用融合表达载体pThioHisA的重组质粒pThioHisA-Tα1④，转化大肠杆菌TOP10得到的工程菌，来分离纯化表达的胸腺素α1（thymosin alpha 1，T α1）. 方法　将高效表达融合蛋白的工程菌用细胞破碎器裂解，经80℃热处理，离心上清采用Q-Sepharose FF阴离子交换色谱纯化融合蛋白. 融合蛋白经CNBr裂解后用常压离子交换色谱纯化Tα1单体. 应用SDS-PAGE，2L-Tricine-SDS-PAGE和HPLC进行鉴定. 结果　[ HTSS SDS-PAGE分析表明菌体表达的融合蛋白占菌体总蛋白的400 g*kg-1. 经2L-Tricine-SDS-PAGE分析证实,融合蛋白可裂解出Tα1单体，裂解物经简单的离子色谱可以纯化出Tα1单体，HPLC鉴定纯化出的Tα1纯度可达950 g*kg-1以上. 利用3H-TdR进行的生物活性测定表明，在致有丝分裂原ConA存在的条件下，融合蛋白和Tα1单体均具有刺激小鼠脾淋巴细胞分裂增殖的能力，与化学合成的Tα1相比具有相似的生物活性. 结论　该方法是分离纯化Tα1 简便易行，经济有效的方法；同时也为Tα1的分离纯化和大规模的开发生产奠定了基础.
목적　이용융합표체재체pThioHisA적중조질립pThioHisA-Tα1④， 전화대장간균TOP10득도적공정균，래분리순화표체적흉선소α1（thymosin alpha 1，T α1）. 방법　장고효표체융합단백 적공정균용세포파쇄기렬해，경80℃열처리，리심상청채용Q-Sepharose FF음리자교환색보순화융합단백. 융합단백경CNBr렬 해후용상압리자교환색보순화Tα1단체. 응용SDS-PAGE，2L-Tricine-SDS-PAGE화HPLC진행감정. 결과　[ HTSS SDS-PAGE분석표명균체표체적융합단백점균체총단백적400 g*kg-1. 경2L-Tricine-SDS-PAGE분석증실,융합단백가렬 해출Tα1단체，렬해물경간단적리자색보가이순화출Tα1단체，HPLC감정순화출적Tα1순도가체950 g*kg-1이상. 이용3H-TdR진행적생물활성측정표명，재치유사분렬원ConA존재적조건하，융합단백화Tα1단체균구유자격소서비림파세포 분렬증식적능력，여화학합성적Tα1상비구유상사적생물활성. 결론　해방법시분리순화Tα1 간편역행，경제유효적방법；동시야위Tα1적분리순화화대규모적개발생산전정료기출.
AIM　To purify and prepare thymosin alpha 1 (Tα 1). METHODS　The engineering bacteria TOP10 including recombinant plasmid of a fusion expression vector pThioHisA with the tandem Tα 1 gene of 4 repeats (Tα1④) was used. after bacter ia lysated by Bionee Cell Disruoter, the lysate was incubat ed in the temperature of 80℃ for 10 minutes, and cooled quickly. By Q-Sepharose Fast Flow chromatography, the fusio n protein was purified from the supernatant of the lysate. At the concentrion of 0.5 mol*L-1 CNBr cleavage of the fusion protein in 700 mL*L-1 formic acid, Tα1 was purified by ion exchange methods of SP-Sepharose Fast Flow and Q-Sepharose Fast Flow. T hese were identified by SDS-PAGE, 2L-Tricine-SDS-PAGE and HPLC. RESULTS　SDS-PAGE analysis showed an induced expression fusion protein band which consititued 400 g*kg-1 of the total bacterial proteins. After CNBr cleava ge of the fusion protein, Tα1 was obtained by ion exchange methods and proved by 2L-Tricine-SDS-PAGE analysis. HPLC showed the purity of Tα1 was 950 g*kg-1. Their biological activity was analysed by 3H-TdR incorparation. They results sh owed that the fusion protein and Tα1 had the similar biological activity to that of the synthesized Tα1. They could increase the p roliferative response of the mitogen ConA stimulated spleen lymphocytes. CONCLUSION　Method is convenient and simple for purification and preparation of Tα1 in large scale.