重组猪源胰蛋白酶原的构建、表达与活性分析

Cloning and Expression of Porcine-recombination Trypsinogen Gene and Its Activity Analysis

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.