摘要
目的:构建、表达重组猪源胰蛋白酶原,并对其进行分离纯化和酶活性分析。方法:利用大肠埃希菌表达系统(p ET-28a,宿主菌BL21DE3)优化天然猪源胰蛋白酶原基因,经乳糖诱导表达重组胰蛋白酶原蛋白,采用阴离子交换色谱法分离纯化,以SDS-PAGE电泳鉴定目标蛋白,以紫外分光光度法检测重组蛋白的酶活力。结果:测序结果表明,重组猪源胰蛋白酶原基因工程菌构建成功,SDS-PAGE检测显示目的蛋白条带位置与标准猪源胰蛋白酶条带位置一致,且酶活力可达513.09U·mg-1。结论:成功获得了重组猪源胰蛋白酶原,且酶活力较好,为进一步研究生产提供了基础。
Objective: To optimize and express the gene of recombinant porcine trypsinogen, and to detect the specific activity after purification. Methods: The aynthetic porcine-recombination trypsinogen gene, of which the rare codon was substituted, was inserted into p ET28 a and then transformed into E.coli BL21(DE3). Porcine-recombination trypsinogen was obtained in the inducement of lactose. The target protein by using anion exchange chromatography was purified and its activity was detected with UV spectrophotometer. Results: The inactive inclusion body of porcinerecombination trypsinogen was obtained. From the result of SDS-PAGE, its relative molecular mass met the expectation, and the specific activity achieved 513.09 U·mg-1 after purification. Conclusion: The porcine-recombination trypsin was successfully obtained, which had the better specific activity. It is important to raise awareness of recombination trypsin and industrial production.
出处
《药学进展》
CAS
2014年第12期916-921,共6页
Progress in Pharmaceutical Sciences
基金
国家级大学生创新创业训练计划项目(No.J1030830)
国家自然科学基金(No.81172973
No.81373232)
关键词
猪源胰蛋白酶原基因
包涵体变性
乳糖诱导
稀释复性
recombinant trypsin
denaturing of inclusion body
induction of lactose
dilution method