【摘要】 刺激隐核虫(Cryptocaryon irritans)是严重危害海水鱼类的寄生纤毛虫,为了对其进行分子水平的研究,采用SMART技术构建了刺激隐核虫滋养体期的cDNA文库,并随机挑取克隆做EST测序,获得大量克隆的基因。从中挑选ATP/ADP载体蛋白基因同源物(CiAAC)的克隆进一步测序获得全长的cDNA序列。该序列全长为1029bp,包含编码287个氨基酸的开放阅读框。用RT-PCR分析了其在刺激隐核虫各时期的表达情况,并应用生物信息学分析方法,对CiAAC基因进行不同物种的系统进化树分析、功能区分析、物理性质分析、疏水性分析、跨膜结构域预测、亚细胞定位、二级结构预测等等。结果表明:CiAAC蛋白在刺激隐核虫各时期均有表达;据预测,它为六次跨膜蛋白,与卵形肠虫(Nyctotherus ovalis)的ADP/ATP载体蛋白的同源性最高,相似性达65%;蛋白为疏水性,并定位于刺激隐核虫的细胞质中。随后实验中通过对基因的定点诱变,将纤毛虫的非通用密码进行改造后,构建了pGEX-4T-1/CiAAC表达载体。以上实验为病原生物刺激隐核虫CiAAC载体蛋白的研究及应用提供了基础资料。更多还原

【Abstract】 Cryptocaryon irritans is a ciliated protozoa parasiting on marine fishes, which results in severe economic loss in mariculture. ADP/ATP carrier(AAC) plays important roles in living organisms. In this study, a cDNA of AAC homolog(1029 bp) was isolated from a cDNA library of Cryptocaryon irritans(CiAAC). The open reading frame(ORF) of CiAAC was 864 bp encoding a protein of 287 aa. The functional region, physical properties, hydrophobicity, transmembrane domains, sub-cellular localization and protein secondary structure of the putative CiAAC protein were predicted by using the bioinformatics methods. Phylogenetic tree of 16 AAC proteins from different species was constructed. The transcription profiles at different stages of life cycle were analyzed. The results showed that the putative CiAAC protein was highly hydrophobic and contained six transmembrane domains. It was homologous to AAC from Nyctotherus ovalis with an identity of 65%. CiAAC is more likely located in the cytosol. CiAAC gene was expressed at all stages as detected by using reverse transcriptase PCR. After site-directed mutagenesis of the ORF by PCR, the gene was sub-cloned into pGEX-4T-1. As a result, pGEX/CiAAC expression vector was constructed. This study has provided basic information of CiAAC carrier protein for further researches.更多还原