论文部分内容阅读
目的:构建刚地弓形虫(Toxoplasma gondii)表面抗原1(surface antigen 1,SAG1)及棒状体蛋白18(rhoptry protein 18,ROP18)的真核表达重组质粒,并在真核细胞中表达目的蛋白。方法:设计SAG1、ROP18的特异引物,采用RCR技术从弓形虫RH株基因组DNA中扩增编码SAG1、ROP18基因片段,经克隆至p TG19-T载体后,亚克隆至真核表达载体p3×FLAG-Myc-CMVTM-24,构建真核表达重组质粒p3×FLAG-Myc-CMVTM-24-SAG1和p3×FLAG-MycCMVTM-24-ROP18。将构建好的真核表达重组质粒转染人肾上皮细胞系293-T细胞,分析转染细胞的表达情况。结果:PCR扩增弓形虫SAG1和ROP18基因片段分别为1 011 bp和1 665 bp,与预期大小相符。重组质粒经PCR、酶切和测序鉴定结果均正确,通过RT-PCR和蛋白质印迹技术鉴定出重组质粒p3×FLAG-Myc-CMVTM-24-SAG1和p3×FLAG-Myc-CMVTM-24-ROP18在293-T细胞中表达。结论:成功构建了真核表达质粒p3×FLAG-Myc-CMVTM-24-SAG1和p3×FLAG-Myc-CMVTM-24-ROP18,该重组质粒能够在真核细胞中表达目的蛋白。
Objective: To construct eukaryotic expression recombinant plasmid of Toxoplasma gondii surface antigen 1 (SAG1) and rhoptry protein 18 (ROP18), and to express the target protein in eukaryotic cells. Methods: The specific primers of SAG1 and ROP18 were designed. The SAG1 and ROP18 gene fragments were amplified from genomic DNA of RH strain Toxoplasma gondii by RCR. After cloned into pTG19-T vector, they were subcloned into eukaryotic expression vector p3 × FLAG Eukaryotic expression recombinant plasmids p3 × FLAG-Myc-CMVTM-24-SAG1 and p3 × FLAG-MycCMVTM-24-ROP18 were constructed. The constructed eukaryotic expression recombinant plasmid was transfected into human renal epithelial cell line 293-T and the expression of the transfected cells was analyzed. Results: The fragments of SAG1 and ROP18 amplified by PCR were 1 011 bp and 1 665 bp respectively, which were consistent with the expected size. The recombinant plasmids were identified by PCR, restriction enzyme digestion and sequencing. The recombinant plasmids p3 × FLAG-Myc-CMVTM-24-SAG1 and p3 × FLAG-Myc-CMVTM-24-ROP18 were identified by RT-PCR and Western blot 293-T cells. CONCLUSION: The eukaryotic expression plasmids p3 × FLAG-Myc-CMVTM-24-SAG1 and p3 × FLAG-Myc-CMVTM-24-ROP18 have been successfully constructed, which can express the target protein in eukaryotic cells.