弓形虫微线体蛋白MIC2和M2AP真核表达载体的构建

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。

Construction of eukaryotic expression vectors for the microneme proteins MIC2 and M2AP Toxoplasma gondii

Objective To construct the eukaryotic expression vectors pGAPZαA-mic2 and pGAPZαA-m2 ap in a Pichia pastoris system to study the functions of the microneme proteins MIC2 and M2AP of the RH strain of Toxoplasma gondii Methods Total RNA was extracted from the tachyzoites of the RH strain of T.gondii using a Trizol Kit.mic2 and m2ap cDNA was reverse-transcribed with total RNA as a template and OligodT-Adaptor as a primer.In accordance with the sequence of T.gondii mic2 and m2ap in GenBank,specific primers of mic2 and m2ap were designed with Primer Premier 5.0and then synthesized.The mic2 and m2ap genes were amplified using PCR from the cDNA template and PCR products were purified with a GK2042 Gel DNA Recovery Kit.The genes mic2 and m2ap were separately cloned into a pMD-19-simple-T vector.Positive clones were inserted into pGAPZαA after identification with restriction enzyme digestion and sequencing.Pichiaexpression vectors of pGAPZαA-mic2 and pGAPZαA-m2 ap were constructed and transformed into E.coli DH5α.The recombinant plasmids were extracted from positive transformed E.coli DH5αand identified via restriction enzyme digestion and sequencing. Results The mic2 and m2ap genes that were amplified from mic2 and m2ap cDNA using PCR had a molecular weight of 2,200 bp and 1,000 bp,and BLAST sequencings indicated that the two genes were consistent with the genes mic2 and m2ap in GenBank.T-A cloning of the recombinant plasmids pMD19-Tmic2 and pMD19-T-m2 ap was performed.Sequencing of these plasmids and the recombinant Pichia expression vectors pGAPZαA-mic2 and pGAPZαA-m2 ap verified that these vectors were correctly constructed. Conclusion The recombinant Pichiaexpressing vectors pGAPZαA-mic2 and pGAPZαA-m2 ap were successfully constructed,laying the foundation for the further study of their mechanism of interaction and the immunoprotective effect of mic2 and m2ap.