Identification and partial characterization of a serine protease inhibitor (serpin) of Eimeria tenella
Identification and partial characterization of a serine protease inhibitor (serpin) of Eimeria tenella
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DOI：
10.1007/s00436-011-2568-0
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发表时间：
2012-02-01
影响因子：
2
通讯作者：
Huang, Bing
中科院分区：
文献类型：
--
作者：
Jiang, Lianlian;Lin, Jiaojiao;Huang, Bing
丝氨酸蛋白酶抑制物(Serpins)介导许多生物过程，包括对病原体感染的免疫反应。本研究通过表达序列标签分析和快速扩增基因末端扩增技术，从家禽常见寄生虫柔嫩艾美耳球虫中分离到一个丝氨酸蛋白超家族成员。该基因全长1,918个碱基，开放阅读框1,248个碱基，编码415个氨基酸的多肽，理论等电点为5.26，预测相对分子质量为45.5 kDa。实时荧光定量聚合酶链式反应分析表明，丝蛋白基因在子孢子中的表达水平高于其他发育阶段(未孢子化卵囊、孢子化卵囊和第二代裂殖子)。通过聚合酶链式反应扩增出该成熟蛋白的编码序列，将其克隆到pET28(A)载体中，并在大肠杆菌中表达。制备了针对重组蛋白的特异性抗血清，并用于检测侵袭抑制能力和定位；结果表明，丝氨酸可能在孢子丝虫在宿主体内的侵袭和存活中发挥重要作用。
Serine protease inhibitors (serpins) mediate many biological processes, including immune responses to pathogenic infection. In this study, a member of the serpin superfamily was identified from the common poultry parasite Eimeria tenella by expressed sequence tag analysis and the rapid amplification of cDNA ends technique. The full-length cDNA was 1,918 bp and had an open reading frame of 1,248 bp encoding a polypeptide of 415 amino acids with the theoretical isoelectric point of 5.26 and predicted molecular weight of 45.5 kDa. Real-time quantitative PCR analysis revealed that the serpin gene was expressed at higher levels in sporozoites than in the other developmental stages (unsporulated oocysts, sporulated oocysts, and second-generation merozoites). The sequence encoding the mature protein was amplified by PCR, cloned into the pET28(a) vector, and expressed in Escherichia coli. Specific antiserum generated against the recombinant protein was prepared and used to determine invasion inhibition capacity and localization; the results suggested that the serpin may play an important role in invasion and survival of the sporoziotes in the host.