旋毛虫两种丝氨酸蛋白酶抑制剂的高效表达及特性分析
High-level expression and characterization of two serine protease inhibitors from Trichinella spiralis.
作者信息
Zhang Zhaoxia, Mao Yixian, Li Da, Zhang Yvhan, Li Wei, Jia Honglin, Zheng Jun, Li Li, Lu Yixin
机构信息
Key Laboratory of Animal Common Disease Prevention, College of Veterinary Medicine, Northeast Agricultural University, 59 Mucai Street, Harbin 150030, PR China.
State Key Laboratory of Veterinary Biotechnology-Michigan State University Joint Laboratory of Innate Immunity, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Maduan Street 427, Nangang District, Harbin 150001, PR China.
出版信息
Vet Parasitol. 2016 Mar 30;219:34-9. doi: 10.1016/j.vetpar.2016.02.003. Epub 2016 Feb 3.
Serine protease inhibitors (SPIs) play important roles in tissue homeostasis, cell survival, development, and host defense. So far, SPIs have been identified from various organisms, such as animals, plants, bacteria, poxviruses, and parasites. In this study, two SPIs (Tsp03044 and TspAd5) were identified from the genome of Trichinella spiralis and expressed in Escherichia coli. Sequence analysis revealed that these two SPIs contained essential structural motifs, which were well conserved within the tumor-infiltrating lymphocytes (TIL) and serpin superfamily. Based on protease inhibition assays, the recombinant Tsp03044 showed inhibitory effects on trypsin, α-chymotrypsin, and pepsin, while the recombinant TspAd5 could effectively inhibit the activities of α-chymotrypsin and pepsin. Both these inhibitors showed activity between 28 and 48 °C. The expression levels of the two SPIs were also determined at different developmental stages of the parasite with real-time PCR. Our results indicate that Tsp03044 and TspAd5 are functional serine protease inhibitors.
丝氨酸蛋白酶抑制剂（SPIs）在组织稳态、细胞存活、发育和宿主防御中发挥着重要作用。到目前为止，已从各种生物体中鉴定出SPIs，如动物、植物、细菌、痘病毒和寄生虫。在本研究中，从旋毛虫基因组中鉴定出两种SPIs（Tsp03044和TspAd5）并在大肠杆菌中表达。序列分析表明，这两种SPIs包含基本的结构基序，在肿瘤浸润淋巴细胞（TIL）和丝氨酸蛋白酶抑制剂超家族中高度保守。基于蛋白酶抑制试验，重组Tsp03044对胰蛋白酶、α-糜蛋白酶和胃蛋白酶有抑制作用，而重组TspAd5能有效抑制α-糜蛋白酶和胃蛋白酶的活性。这两种抑制剂在28至48℃之间均表现出活性。还通过实时PCR测定了这两种SPIs在寄生虫不同发育阶段的表达水平。我们的结果表明，Tsp03044和TspAd5是功能性丝氨酸蛋白酶抑制剂。
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