弓形虫胚层发育相关蛋白基因的克隆和序列分析

Cloning and sequence analysis of embryogenesis-related protein gene of Toxoplasma gondii strains

目的对15个弓形虫虫株的弓形虫胚层发育相关蛋白（TgERP）基因进行克隆和序列分析，了解其变异和进化情况，为评价该蛋白在血清学诊断和疫苗防疫应用上的价值提供理论基础。方法设计TgERP基因的PCR引物，对15个弓形虫虫株进行PCR扩增和序列测定，测序结果利用Puzzle 5．2、Paup 4．0和DNAStar5．0软件进行分析。结果TgERP基因包含的完整开放阅读框（ORF）长度均为315bp，A＋T含量在46．67％～46．98％之间，仅有1个碱基发生变异，变异率为0％～0．32％。编码104个氨基酸，变异率在0％～0．96％之间。系统进化分析显示，TgERP基因序列虽然能够将弓形虫Ⅰ型虫株与Ⅱ／Ⅲ型虫株区分开，但却不能区分所有基因型的虫株。结论TgERP基因在各基因型虫株中十分保守，不适合作为遗传标记分子来区分不同基因型的弓形虫虫株。

The embryogenesis-related protein （ERP） gene of Toxoplasrna gondii was cloned in this study to analyze se- quence variation in T. gondii ERP gene among different T. gondii strains. Genomic DNA from 15 strains of Toxoplasrna gon- dii was extracted. The open reading frame （ORF） of Toxoplasma gondii ERP gene was amplified by PCR and sequenced from 15 T. gondii strains from different geographical locations and hosts. The sequencing results were aligned using the ClustaIX 1.83, with the corresponding sequences of T. gondii ME49 strain and VEG strain which were obtained from http：//toxodb. org. The phylogenetie relationships among T. gondii strains were then constructed using the software Mega 5.0 and Puzzle 5.2. The length of all TgERP sequence was 315 bp, and the A＋T contents were 46.67%-46.98%. The intra-specific varia- tion among the examined T. gondii strains was 0%-0.32 %. The range of amino acid sequences was 0%-0.96%. Phylogenetic analysis revealed that TgERP gene sequences cannot be used as a marker for studying genetic relationships of T. gondii iso- lates.