Fujian Key Laboratory of Developmental and Neuro Biology, College of Life Science, Fujian Normal University, Fuzhou 350108, Fujian, PR China.
Exp Parasitol. 2012 Mar;130(3):189-94. doi: 10.1016/j.exppara.2012.01.012. Epub 2012 Feb 1.
Cryptocaryon irritans is a ciliated parasite causing cryptocaryosis in marine fish. To isolate functional genes, a cDNA library of C. irritans trophonts was constructed and a gene designated CiSA-32.6 (GenBank ID: JF812643) was cloned and characterized. The full-length cDNA (1158 bp) encoded a deduced polypeptide of 330 amino-acid (aa) with a signal peptide of 22 aa. To express the ciliate gene, a truncated open reading frame (CiSA-32.6t) was synthesized to remove fragments encoding the signal peptide and hydrophobic C-terminal and to modify non-universal genetic codes. CiSA-32.6t was subcloned into Escherichia coli DH5α strain using the pGEX-4T-1 vector and then expressed as a glutathione S transferase fusion protein (rCiSA-32.6t). Western blotting analysis showed that sera from mice immunized with rCiSA-32.6t reacted specifically with a native protein (32.6 kDa) in parasite lysates. Moreover, rCiSA-32.6t reacted specifically with sera from mice immunized with a C. irritans trophont lysate. Expression of the CiSA-32.6 gene in C. irritans was detected at all developmental stages by reverse transcriptase PCR and Western blotting analysis. This study provides the basis of further investigations into the pathogenic biology of C. irritans and the control of cryptocaryosis.
刺激隐核虫是一种纤毛虫寄生虫，会导致海水鱼患隐核虫病。为了分离功能基因，构建了刺激隐核虫营养体的 cDNA 文库，并克隆和鉴定了一个名为 CiSA-32.6（GenBank ID：JF812643）的基因。该基因的全长 cDNA（1158bp）编码一个 330 个氨基酸（aa）的推定多肽，带有 22 个氨基酸的信号肽。为了表达纤毛虫基因，合成了一个截断的开放阅读框（CiSA-32.6t），用于去除编码信号肽和疏水性 C 末端的片段，并修改非通用遗传密码。CiSA-32.6t 被亚克隆到大肠杆菌 DH5α菌株中，使用 pGEX-4T-1 载体，然后表达为谷胱甘肽 S 转移酶融合蛋白（rCiSA-32.6t）。Western blot 分析表明，用 rCiSA-32.6t 免疫的小鼠血清与寄生虫裂解物中的一种天然蛋白（32.6kDa）特异性反应。此外，rCiSA-32.6t 与用刺激隐核虫营养体裂解物免疫的小鼠血清特异性反应。通过逆转录 PCR 和 Western blot 分析，在刺激隐核虫的所有发育阶段都检测到 CiSA-32.6 基因的表达。本研究为进一步研究刺激隐核虫的致病生物学和隐核虫病的防治提供了基础。
