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目的构建弓形虫RH株微线体蛋白M2AP和MIC2的真核表达载pGAPZαA-mic2和pGAPZαA-m2ap,为建立同时表达MIC2和M2AP蛋白的重组毕赤酵母表达系统,制备重组粘附蛋白复合体MIC2-M2AP奠定基础。方法提取弓形虫RH株速殖子总RNA,用Oligo dT-Adaptor引物逆转录合成cDNA。根据已知的弓形虫mic2和m2ap基因序列,采用引物设计软件Primer premier5.0自行设计并合成引物,以cDNA为模板,PCR扩增mic2和m2ap基因,克隆入pMD-19-simple-T载体,经酶切和测序鉴定后回收目的片段,分别插入至pGAPZαA内,构建重组毕赤酵母表达载体pGAPZαA-mic2和pGAPZαA-m2ap,转化入E.coli DH5α。提取转化菌质粒,进行酶切和测序鉴定。结果 PCR扩增得到的mic2和m2ap基因分别为2 200bp和1 000bp,与预期大小一致。T-A克隆重组质粒pMD19-T-mic2、pMD19-T-m2ap和重组酵母表达质粒pGAPZαA-mic2、pGAPZαA-m2ap经测序鉴定,与GenBank收录的弓形虫mic2基因和m2ap基因序列同源性为100%,重组毕赤酵母表达载体pGAPZαA-mic2和pGAPZαA-m2ap构建成功。结论成功构建弓形虫重组毕赤酵母表达载体pGAPZαA-mic2和pGAPZαA-m2ap,为进一步研究MIC2和M2AP相互作用机制及其免疫保护效应奠定了基础。
OBJECTIVE: To construct eukaryotic expression vectors of M2AP and MIC2 of RH strain microtubule proteins RHP2, pGAPZαA-mic2 and pGAPZαA-m2ap. To establish a recombinant Pichia pastoris expression system that expresses both MIC2 and M2AP proteins, the recombinant adhesion protein complex MIC2 -M2AP laid the foundation. Methods Toxoplasma gondii RH strain tachyzoites total RNA was extracted and reversely transcribed into cDNA using Oligo dT-Adapter primer. According to the known sequence of mic2 and m2ap genes of Toxoplasma gondii, Primer premier5.0 was designed and synthesized by Primer design software Primer. The cDNA of mic2 and m2ap was amplified by PCR and cloned into pMD-19-simple-T vector. After digestion and sequencing, the target fragment was recovered and inserted into pGAPZαA, respectively. The recombinant Pichia pastoris expression vector pGAPZαA-mic2 and pGAPZαA-m2ap were constructed and transformed into E. coli DH5α. Plasmids were extracted and digested and sequenced. Results The mic2 and m2ap genes amplified by PCR were 200 bp and 1 000 bp, respectively, which were consistent with the expected size. The TA cloning recombinant plasmids pMD19-T-mic2, pMD19-T-m2ap and the recombinant yeast expression plasmids pGAPZαA-mic2 and pGAPZαA-m2ap were identified by sequencing. The recombinant Pichia pastoris expression vectors pGAPZαA-mic2 and pGAPZαA-m2ap were successfully constructed. Conclusion The recombinant plasmids pGAPZαA-mic2 and pGAPZαA-m2ap of T. gondii were successfully constructed, which laid the foundation for further study of the interaction mechanism between MIC2 and M2AP and their immunoprotective effects.