约氏疟原虫235ku多基因家族的基因克隆和序列分析

Cloning and sequencing of gene encoding Plasmodium yoelii yoelii 235 ku rhoptry proteins

目的克隆约氏疟原虫不同虫期235 ku棒状体蛋白(Plasmodium yoelii yoelii235 ku rhoptry proteins,Py235)的基因,并对其进行序列测定和分析。方法根据疟原虫棒状体235 ku蛋白的基因序列,设计引物。用Tri-pure与氯仿等试剂提取不同时相点的约氏疟原虫总RNA,逆转录合成cDNA,用PCR的方法扩增Py235基因片段。将获得的预期大小的PCR产物插入PMD18-T克隆载体,转化XL1-Blue感受态细胞,蓝白筛选白色克隆,进行体外扩增,提取质粒,进行酶切鉴定,对含有目的插入片段的克隆进行序列测定。结果从不同侵入虫期的疟原虫总RNA中成功克隆出292 bpPy235基因片段,测序结果与GeneBank报道序列具有很高的相似性。结论在疟原虫235 ku多基因家族,不同侵入虫期疟原虫Py235基因的mRNA转录与表达具有一定的遗传稳定性,但表型也有变异,从而获得侵入不同宿主细胞的功能。

Objective To clone and sequence （Py235） in different invasive stages. Methods the gene encoding Plasrnodium yoelii yoelii 235 ku rhoptry proteins The primers was designed according to the gene sequence of Py235. Total RNA was isolated from different stages and the eDNA was synthesized by reverse transcription. The objective fragment was amplified by PCR with the eDNA and cloned into PMD18-T vector. E. coli XL1-Blue was transformed with the recombinant plasmid, and positive clones were selected. The inserted DNA was verified by enzyme digestion and DNA se- quencing. Results A 292 bp length Py235 fragment was successfully cloned and had highly similarity comparing to the sequence reported in the GeneBank. Conclusion The transcriptions of Py235 mRNA in different invasive stages had both some genetic stability and clonal phenotypic variation.