Zhao Hailong, Qiao Jun, Meng Qingling, Gong Shasha, Chen Cheng, Liu Tianli, Tian Lulu, Cai Xuepeng, Luo Jianxun, Chen Chuangfu
Key Laboratory of Control and Prevention of Animal Disease of Xinjiang Production & Construction Corps, College of Animal Science and Technology, Shihezi University, North Street No.4, Shihezi, 832003, Xinjiang, China.
State Key Lab of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, 730046, Gansu, China.
Antonie Van Leeuwenhoek. 2015 Dec;108(6):1485-1494. doi: 10.1007/s10482-015-0595-z. Epub 2015 Sep 29.
The nematode-trapping fungi possess a unique capability of predating and invading nematodes. As a representative nematode-trapping fungus, Arthrobotrys oligospora has been widely used to study the interactions between nematode-trapping fungi and their hosts. Serine proteinase is one of the important virulence factors during process of invasion of the nematode-trapping fungi into nematodes. In this study, using reverse transcription polymerase chain reaction, we amplified the gene sequence of serine proteinase 186 from A. oligospora, cloned it into pPIC9K vector and expressed it in the yeast Pichia pastoris. The expressed recombinant serine proteinase186 (reP186) was purified via Ni-affinity chromatography. The in vitro nematode-degrading activity of reP186 was analyzed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis revealed that reP186 with molecular weight of 33 kDa was successfully obtained. ReP186 was capable of degrading a series of protein substrates including casein, gelatin, bovine serum albumin, denatured collagen and nematode cortical layer. The reP186 exhibited the maximal activity at pH 8.0 and 55 °C and was highly sensitive to the inhibitor, phenylmethanesulfonylfluoride. Treatment of Caenorhabditis elegans and Haemonchus contortus with reP186 for 12, 24 and 36 h, respectively, resulted in 62, 88 and 100 % of killing rates for C. elegans, and 52, 65 and 84 % of killing rates for H. contortus, respectively, indicating a relatively strong nematode-degrading bioactivity of reP186.
捕食线虫真菌具有捕食和侵入线虫的独特能力。少孢节丛孢菌作为一种典型的捕食线虫真菌，已被广泛用于研究捕食线虫真菌与其宿主之间的相互作用。丝氨酸蛋白酶是捕食线虫真菌侵入线虫过程中的重要毒力因子之一。在本研究中，我们利用逆转录聚合酶链反应扩增了少孢节丛孢菌丝氨酸蛋白酶186的基因序列，将其克隆到pPIC9K载体中，并在毕赤酵母中表达。通过镍亲和层析纯化表达的重组丝氨酸蛋白酶186（reP186）。分析了reP186的体外降解线虫活性。十二烷基硫酸钠聚丙烯酰胺凝胶电泳和蛋白质免疫印迹分析表明，成功获得了分子量为33 kDa的reP186。ReP186能够降解一系列蛋白质底物，包括酪蛋白、明胶、牛血清白蛋白、变性胶原蛋白和线虫皮层。reP186在pH 8.0和55℃时表现出最大活性，并且对抑制剂苯甲基磺酰氟高度敏感。分别用reP186处理秀丽隐杆线虫和捻转血矛线虫12、24和36小时，秀丽隐杆线虫的死亡率分别为62%、88%和100%，捻转血矛线虫的死亡率分别为52%、65%和84%，表明reP186具有较强的降解线虫生物活性。
