论文部分内容阅读
目的构建刚地弓形虫(Toxoplasma gondii)过氧化物氧化还原酶(Peroxiredoxin,Prx)基因重组真核表达质粒,并在HEK293T细胞中进行表达。方法以重组质粒pET-30a(+)/TgPrx为模板,PCR扩增TgPrx基因片段,插入真核表达载体p3×Flag-CMW-14,构建重组表达质粒p3×Flag-CMW-14/TgPrx,转染HEK293T细胞,RT-PCR和Western blot检测TgPrx基因的转录和蛋白的表达。结果 PCR扩增出591 bp的TgPrx基因片段;重组真核表达质粒p3×Flag-CMW-14/TgPrx经PCR、双酶切及测序证明构建正确;转染重组表达质粒的HEK293T细胞可检测到TgPrx基因的转录和蛋白的表达。结论成功构建了重组真核表达质粒p3×Flag-CMW-14/TgPrx,并在HEK293T细胞中正确表达,为进一步研制核酸疫苗奠定了基础。
Objective To construct a recombinant eukaryotic expression plasmid of Toxoplasma gondii Peroxiredoxin (Prx) gene and express it in HEK293T cells. Methods The TgPrx gene fragment was amplified by PCR using the recombinant plasmid pET-30a (+) / TgPrx as a template, and inserted into the eukaryotic expression vector p3 × Flag-CMW-14 to construct the recombinant plasmid p3 × Flag-CMW-14 / TgPrx HEK293T cells were stained for RT-PCR and Western blot to detect TgPrx gene transcription and protein expression. Results The 591 bp fragment of TgPrx gene was amplified by PCR. The recombinant plasmid p3 × Flag-CMW-14 / TgPrx was confirmed by PCR, double enzyme digestion and sequencing. HEK293T cells transfected with recombinant plasmid could detect TgPrx Gene transcription and protein expression. Conclusion The recombinant eukaryotic expression plasmid p3 × Flag-CMW-14 / TgPrx was successfully constructed and correctly expressed in HEK293T cells, which laid the foundation for the further development of nucleic acid vaccine.