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首页> 外文期刊>Journal of Parasitology >SERINE PROTEASE ACTIVITY IN DEVELOPMENTAL STAGES OF EIMERIA TENELLA
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SERINE PROTEASE ACTIVITY IN DEVELOPMENTAL STAGES OF EIMERIA TENELLA

机译：紫茎泽兰发育阶段的丝氨酸蛋白酶活性

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摘要

A number of complex processes are involved in Eimeria spp. survival, including control of sporulation, intracellular invasion, evasion of host immune responses, successful reproduction, and nutrition. Proteases have been implicated in many of these processes, but the occurrence and functions of serine proteases have not been characterized. Bioinformatic analysis suggests that the Eimeria tenella genome contains several serine proteases that lack homology to trypsin. Using RT-PCR, a gene encoding a subtilisin-like and a rhomboid protease-like serine protease was shown to be developmentally regulated, both being poorly expressed in sporozoites (SZ) and merozoites (MZ). Casein substrate gel electrophoresis of oocyst extracts during sporulation demonstrated bands of proteolytic activity with relative molecular weights (Mr) of 18, 25, and 45 kDa that were eliminated by coincubation with serine protease inhibitors. A protease with Mr of 25 kDa was purified from extracts of unsporulated oocysts by a combination of affinity and anion exchange chromatography. Extracts of SZ contained only a single band of inhibitor-sensitive proteolytic activity at 25 kDa, while the pattern of proteases from extracts of MZ was similar to that of oocysts except for the occurrence of a 90 kDa protease, resistant to protease inhibitors. Excretory-secretory products (ESP) from MZ contained AEBSF (4-[2-Aminoethyl] benzenesulphonyl fluoride)–sensitive protease activity with a specific activity about 10 times greater than that observed in MZ extracts. No protease activity was observed in the ESP from SZ. Pretreatment of SZ with AEBSF significantly reduced SZ invasion and the release of the microneme protein, MIC2. The current results suggest that serine proteases are present in all the developmental stages examined.
机译：Eimeria spp涉及许多复杂的过程。存活，包括控制孢子形成，细胞内侵袭，逃避宿主免疫反应，成功繁殖和营养。蛋白酶与许多这些过程有关，但是丝氨酸蛋白酶的发生和功能尚未得到表征。生物信息学分析表明，艾美尔球虫基因组含有几种丝氨酸蛋白酶，它们与胰蛋白酶缺乏同源性。使用RT-PCR，编码枯草杆菌蛋白酶样和菱形蛋白酶样丝氨酸蛋白酶的基因显示受到发育调控，在子孢子（SZ）和裂殖子（MZ）中均表达不佳。卵囊形成过程中卵囊提取物的酪蛋白底物凝胶电泳显示蛋白水解活性带，相对分子量（Mr）为18、25和45 kDa，可通过与丝氨酸蛋白酶抑制剂共孵育消除。通过亲和层析和阴离子交换层析相结合，从未孢子化的卵囊提取物中纯化出具有25 kDa Mr的蛋白酶。 SZ提取物在25 kDa时仅含有一条抑制剂敏感的蛋白水解活性带，而MZ提取物中蛋白酶的形态与卵囊相似，只是出现了90 kDa的蛋白酶抑制剂，对蛋白酶抑制剂具有抗性。 MZ的排泄分泌产物（ESP）含有AEBSF（4- [2-氨基乙基]苯磺酰氟）敏感的蛋白酶活性，比活性比MZ提取物中观察到的活性高约10倍。在来自SZ的ESP中未观察到蛋白酶活性。用AEBSF预处理SZ可以显着减少SZ的侵袭和微粒蛋白MIC2的释放。目前的结果表明丝氨酸蛋白酶存在于所研究的所有发育阶段。

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