日本囊对虾Kunitz型蛋白酶抑制剂在毕赤酵母中的表达纯化及活性分析

Expression of a Shrimp Kunitz-type Protease Inhibitor in Pichia pastoris and Activity Analysis

SKPI(shrimp Kunitz-type protease inhibitor)是日本囊对虾(Marsupenaeus japonicus)体内的一个小分子多肽,含有一个Kunitz型结构域,属于丝氨酸蛋白酶抑制剂。目前已知丝氨酸蛋白酶抑制剂在节肢动物免疫系统中起着非常重要的作用,为了了解SKPI在对虾天然免疫系统中的作用,首先对其进行了重组表达。从日本囊对虾肝胰腺中扩增skpi的cDNA片段,插入改造后的pPIC9K酵母表达载体,获得的重组质粒转化至毕赤酵母GS115进行表达。由于改造的pPIC9K载体加入了6-His标签,因此利用Ni Sepharose High Performance对SKPI进行了高效纯化。初步的活性研究表明,重组表达的SKPI能特异性地抑制胰蛋白酶的水解活性。

SKPI （shrimp Kunitz-type protease inhibitor） from Marsupenaeus japonicus is a member of serine protease inhibitors which play an important role in the arthropod immunity. To fully understand its function in the innate immunity of shrimp, the skpi gene was cloned into a modified pPIC9K vector with a 6-His tag and expressed by Pichia pastoris GS115. The secretory SKPI was purified from the medium with high purity by using Ni Sepharose High Performance. This results also indicated that the purified SKPI could inhibit the activity of trypsin specifically.