约氏疟原虫红外期yir基因保守区的克隆、表达与表位分析

Cloning,expression and epitope analysis of the conserved yir gene fragment from Plasmodium yoelii during the exoerythrocytic stage

目的对红外期的yir基因进行克隆和表达,探讨疟原虫在红外期的抗原变异。方法针对yir基因家族的保守区设计特异引物,以约氏疟原虫BY265株红外期的mRNA为模板,进行RT-PCR扩增,克隆和测定插入片段,所得序列进行BLAST查询,证实其为yir基因,然后构建表达质粒pQE80L/YIR,并转染到宿主菌DH5α中,对获得的yir保守区进行原核表达,经IPTG诱导后,以SDS-PAGE和WesternBlotting检测表达产物。结果从红外期获得了yir基因的保守区cDNA序列,证实红外期存在yir基因的表达;成功构建了表达质粒pQE80L/YIR,对YIR蛋白的保守区片段进行了表达。结论约氏疟原虫红外期存在yir基因的表达,该基因与红外期抗原变异的关系有待进一步研究;约氏疟原虫红外期yir基因保守区原核表达的成功,为疟原虫的红外期阻断疫苗提供了研究基础。

To clone and express the yir gene from Plasmodium yoelii during the exoerythrocytic stage in order to clarify the variations during this stages, the yir gene was amplified by RT-PCR with primers designed according to the sequences of the conserved region of yir multigene family. Then, the target PCR product was purified and cloned into simple vector pMD18-T for sequencing. After blasted with GenBank sequence database, the expression plasmid pQE80L/YIR was constructed and transfected to host bacteria DH5α The eDNA of the yir conserved region was expressed after induction with 1.0 mmol/L IPTG at 37℃ and the expressed products were identified by SDS-PAGE and Western blotting. In these ways,the inserted fragment was demonstrated to be belonged to the yir multigene family,which was the first time to prove that yir gene could express in the exoerythrocytic stage. The work presented in the present study would help for the further investigation of the function of yir/YIR molecule in Plasmodium yoelii during the exoerythrocytic stage.