不同基因型芽囊原虫半胱氨酸蛋白酶的全长cDNA的获取及生物信息学分析

Blastocystis sp. is the most common enteric protozoan found in the intestinal tract of humans. The pathogenic potential of this organism in human beings is still controversial, and has become the hot areas of research. In recent years, nine distinct subtypes of Blastocystis have been isolated from humans, but a clear link of subtypes to symptomatology is not well established. Despite accumulating evidence indicating that the parasite is pathogenic and that cysteine proteases are involved in pathogenesis, only genes coding for cysteine proteases in a Blastocystis isolate belonging to subtype 7 have been identified. In our previous study, a correlation between a high level of protease activity and the virulence of the intestinal parasite Blastocystis sp. was proven, and inter-Blastocystis sp. and intra-Blastocystis sp. subtype differences in protease profiles have also been demonstrated by gelatin-SDS-PAGE analysis. The aims of this project are: (1) to use rapid amplification of cDNA ends (RACE) and RT-PCR methods to obtain full-length cDNAs encoding cysteine proteases in six known different genotypes ( subtype 1 to 3, and 5 to 7) strains studied for their protease profiles in our previous study, and in strains within the same subtype showed significant differences in protease activities or/and protease profiles in our previous study; (2) to use bioinformatics means to compare variations in the nucleotide sequences and deduced amino acid sequences among all the obtained cDNAs encoding cysteine proteases from above-mentioned Blastocystis strains as well as those from other parasites. These analyses will helpe us to understand the sequence features and evolving status of various cp genes from Blastocystis and to predict cp genes related with the pathogenic potential of the intestinal parasite. As the study are conducted, the roles of above-mentioned Blastocystis subtype in disease will become clearer. In addition, These research results will lay the foundation for further elucidation of the pathogenic potential of the parasite and understanding its pathogenesis.

芽囊原虫是人最常见的肠道寄生原虫。研究发现该虫的人源株有9种亚型，并推测其某些亚型有致病性，但未得到证实。尽管越来越多的证据表明该虫具有致病性，且证明其半胱氨酸蛋白酶（CPs）在致病过程中起重要作用，但至今仅亚型7虫株的CPs基因被鉴定。我们研究发现，该虫的CPs水解活性强弱与毒力相关，不同亚型甚至相同亚型不同虫株间的CPs酶谱存在明显差异。故本项目拟采用cDNA末端快速扩增(RACE)等技术，对CPs酶谱明显不同的6种亚型（亚型1～3、5～7）虫株及亚型内CPs活性和/或CPs酶谱存在明显差异的不同虫株的各种CP的全长cDNA进行克隆和测序；用生物信息学方法分析它们所含的cp基因数量、序列和结构差异，同时与其它已知寄生虫的CPs进行比较，以了解该虫各cp基因序列特征和进化地位，并预测与致病力相关的cp基因，为进一步弄清不同亚型芽囊原虫在疾病中的地位、阐明该虫的致病性及致病机制奠定基础。

芽囊原虫与多种肠道疾病有关，但其亚型（ST）和疾病之间的相关性尚未明确。半胱氨酸蛋白酶（CP）是大多数致病性原虫的毒力因子。已有研究表明该虫的CP在其致病过程中起重要作用，但仅有ST7虫株的2个CP基因被鉴定。本研究首次完成了人感染率高的ST3（41%）和ST1（32%）虫株的转录组测序，共获得78974个独立基因。用DEGseq进行差异表达分析，有15435个独立基因为差异表达，表明该2种亚型的基因表达谱有显著差异。对所有独立基因进行初步筛选发现，ST3和ST1虫株特有的CP独立基因分别有140个和137个，共有的只有10个。鉴于芽囊原虫的致病性可能是其所分泌的分子能克服宿主免疫应答所致，故通过对该2株不同ST虫株的全部CP独立基因进行信号肽分析，并经基因克隆和qRT-PCR验证，共有14个CP独立基因编码的蛋白预测为分泌蛋白，其中，ST1有4个，ST3有9个，共有的1个。采用DNA walking法扩增未知侧翼序列，在获得扩增序列后，利用FGENESH和BlastX方法查找开放阅读框，去除内含子，获得了14条包含全长CDS的CP cDNA序列。用生物信息学对其相应的每条氨基酸序列进行特性分析、同源序列比对及进化树构建。结果显示：14个编码的蛋白均为分泌性蛋白；9个为糖蛋白；5个含C13结构域，属CD族中的C13家族成员，9个含C1结构域，属半胱氨酸蛋白酶CA族中C1家族成员。同源序列比对表明，亚型内的CPs同源性高，变异较小，亚型间的同源性较低，变异较大。本研究发现2个CP基因为ST7虫株内的已鉴定为毒力因子的2个CP直系同源，可能编码潜在的毒力因子。构建的进化树显示，每个序列均与同属的芽囊原虫聚为一组，与同为SAR 超类群中的囊泡虫类或有孔虫类有较近的亲缘关系。表明物种进化树与其分类体系一致，同时也支持SAR 超类群作为一个单系的原生生物系统的新分类体系。