目的·探讨睾丸素（testisin）酶原在体外的活化方式及活性调控机制，为进一步研究testisin在生物体内的生理功能奠定基础。方法·通过全基因合成，构建鼠源testisin（mTN）蛋白酶原的真核表达质粒，并将其转染至真核HEK293S细胞中；通过镍离子亲和层析等方法获得纯化重组testisin蛋白；将酶原激活位点和活性中心位点进行定点突变，利用酶活性检测等手段分析testisin的活化机制，通过改变酶原的孵育条件确定蛋白的活化速率；利用Edman降解法及定点突变确定蛋白酶的自剪切位点；通过检测蛋白对底物的分解速率分析蛋白酶的活性调控机制。结果·重组mTN酶原在真核HEK293S细胞中高效表达，通过一步纯化可以获得纯度较高且性质均一的重组蛋白。该蛋白在酸性条件下稳定，在中性或碱性环境下会发生自激活，其激活速率受到酸碱度和温度等因素的影响，且这种自激活依赖于该酶激活位点Arg⁴⁶和酶活性中心Ser²⁴⁰的完整性。重组mTN酶原的活化常伴随分子表面170/175肽环的自剪切，但该自剪切并不影响其蛋白酶的活性。活化的mTN最适反应pH值为8.0，且在其最适pH值附近较为稳定；最适反应温度为50 ℃，且蛋白适宜保存在低于30 ℃的环境中；Zn²⁺和Ca²⁺对mTN的活性有明显的抑制作用。结论·成功建立了一种有效制备重组mTN酶原的方法，发现重组mTN在体外可以发生自激活和自剪切。

Objective ·To explore the activation methods and activity regulation mechanisms of the testisin zymogen in vitro, laying a foundation for further research on the physiological functions of testisin in organisms. Methods ·The eukaryotic expression plasmid for the mouse-derived testisin (mTN) zymogen was constructed through full-gene synthesis and subsequently transfected into eukaryotic HEK293S cells for expression. Purified recombinant testisin protein was obtained through methods such as nickel ion affinity chromatography. Site-directed mutagenesis was performed on the activation site and active center of the zymogen, and the activation mechanism of testisin was analyzed through enzymatic activity assays. Additionally, the activation rate of the protein was determined by altering the incubation conditions of the zymogen. The self-cleavage site of the protease was identified by using Edman degradation and site-directed mutagenesis. Finally, the regulatory mechanism of protease activity was examined by analyzing the proteolytic rate of the protein towards its substrates. Results ·The recombinant mTN zymogen was expressed in eukaryotic HEK293S cells, and high-purity, uniform recombinant protein was obtained through a two-step purification process. This protein was found to remain stable under acidic conditions but underwent self-activation in neutral or alkaline environments. The rate of activation was influenced by factors such as pH value and temperature. The self-activation process required on the integrity of the enzyme's activation site, Arg⁴⁶, and its active center, Ser²⁴⁰. Additionally, the self-activation of the recombinant mTN zymogen was often accompanied by the self-cleavage at the 170/175 loop on the molecular surface, but this self-cleavage did not affect the activity of testisin. Furthermore, the optimal reaction pH for activated mTN was 8.0, with the protein remaining relatively stable near this pH. The optimal reaction temperature was 50 ℃, while the protein was best stored at temperatures below 30 ℃. Zn²⁺ and Ca²⁺ were found to significantly inhibit mTN activity. Conclusion ·An effective method for the preparation of recombinant mTN zymogen is successfully established, and it is discovered that the recombinant mTN undergoes self-activation and self-cleavage in vitro.