Immunoreactive Trypsinogen (IRT) is a protein-based pancreatic proenzyme that has an important role in protein digestion in humans. In human body, once IRT present in the small intestine, the proteolytic cleavage activates trypsinogen into trypsin. When IRT is in the active form, it is capable to cleave antibodies, other proteins and even itself while it is desired to use in immunoassays. According to the literature, there are three important IRT isoforms called Immunoreactive Trypsinogen 1 (IRT1), Immunoreactive Trypsinogen 2 (IRT2), and Immunoreactive Trypsinogen 3 (IRT3). However, trypsinogen 1 (cationic trypsinogen, IRT1) and trypsinogen 2 (anionic trypsinogen, IRT2) are the major isoforms in human pancreatic juice and used in the diagnosis of cystic fibrosis (CF). In this study, it is aimed to restrain its proteolytic activity with K23D mutation, which changes lysine (K) residue at the 23rd position to aspartic acid (D). Because we wanted to produce a hassle-free human recombinant immune reactive trypsinogen proenzyme which has similar antigenic properties with the native form. It is also aimed that the mutant IRTs do not exhibit proteolytic activity for the development of durable detection kits with a longer shelf life for both two isoforms. The innovation was actualized in order to use IRTs as a standard antigen in Immunoassays such as ELISA kits. The gene was synthesized as mutated and expressed in P. pastoris X-33 strain. The loss of proteolytic activity has been proven with the BAEE test. Antigenic properties of K23D IRTs and the effect of proteolytic inactivation on their performance in immunoassays were assessed with ELISA and Western Blot. In ELISA results K23D mutated IRTs showed higher signals than Wild-Type forms.

中文翻译：

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开发用于免疫测定的突变型人免疫反应性胰蛋白酶原1（IRT1）和突变型人免疫反应性胰蛋白酶原2（IRT2）。

免疫反应性胰蛋白酶原（IRT）是一种基于蛋白质的胰腺酶，在人类的蛋白质消化中具有重要作用。在人体中，一旦IRT存在于小肠中，蛋白水解酶就会将胰蛋白酶原激活为胰蛋白酶。当IRT呈活性形式时，它能够裂解抗体，其他蛋白质，甚至本身，同时需要用于免疫测定。根据文献，存在三种重要的IRT亚型，分别称为免疫反应性胰蛋白酶原1（IRT1），免疫反应性胰蛋白酶原2（IRT2）和免疫反应性胰蛋白酶原3（IRT3）。但是，胰蛋白酶原1（阳离子胰蛋白酶原，IRT1）和胰蛋白酶原2（阴离子胰蛋白酶原，IRT2）是人胰液中的主要同工型，用于诊断囊性纤维化（CF）。在这个研究中，它旨在通过K23D突变来限制其蛋白水解活性，该突变会将第23位的赖氨酸（K）残基变为天冬氨酸（D）。因为我们想生产一种无麻烦的人类重组免疫反应性胰蛋白酶原酶，它具有与天然形式相似的抗原特性。还旨在突变IRT不表现出蛋白水解活性，从而开发出对两种同工型都具有更长货架期的耐用检测试剂盒。为了将IRT用作免疫测定（例如ELISA试剂盒）中的标准抗原，已实现了创新。该基因被合成为突变体，并在巴斯德毕赤酵母X-33菌株中表达。蛋白水解活性的丧失已通过BAEE测试证明。使用ELISA和Western Blot评估了K23D IRT的抗原特性以及蛋白水解失活对其在免疫测定中的性能的影响。在ELISA结果中，K23D突变的IRT显示出比野生型更高的信号。