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目的构建刚地弓形虫热休克蛋白70(TgHSP70)真核表达重组质粒p3×Flag-CMW-14-TgHSP70,并在HEK 293T细胞内获得表达,为制备基因疫苗奠定基础。方法扩增TgHSP70基因,双酶切后与真核表达质粒p3×Flag-CMW-14连接,经酶切、PCR及测序鉴定;脂质体法将重组体转染HEK 293T细胞中,TgHSP70的表达产物通过RT-PCR和western blot在基因和蛋白2个水平鉴定。结果 PCR扩增获得约495 bp的HSP70基因片段,p3×Flag-CMW-14-TgHSP70重组质粒构建成功,经双酶切、PCR及测序鉴定,重组质粒基因序列完全正确;将p3×Flag-CMW-14-TgHSP70转染到HEK 293T细胞中,经RT-PCR检测获得预期大小目的基因条带,Western-blot检测表达产物大小约19 kDa。结论成功构建了真核表达重组质粒p3×Flag-CMW-14-TgHSP70,并在HEK 293T正确表达。
Objective To construct the eukaryotic expression plasmid p3 × Flag-CMW-14-TgHSP70 of Toxoplasma gondii heat shock protein 70 (TgHSP70) and obtain its expression in HEK 293T cells, which will lay the foundation for the preparation of gene vaccines. Methods The TgHSP70 gene was amplified by double digestion and ligated with eukaryotic expression plasmid p3 × Flag-CMW-14. The recombinant plasmid was identified by restriction enzyme digestion, PCR and sequencing. The recombinant plasmid was transfected into HEK 293T cells by lipofectamine. The expression of TgHSP70 The product was identified at 2 levels of genes and proteins by RT-PCR and western blot. Results The HSP70 gene fragment of about 495 bp was obtained by PCR. The recombinant plasmid p3 × Flag-CMW-14-TgHSP70 was successfully constructed. The double-digested, PCR and sequencing results showed that the recombinant plasmid was correct. The p3 × Flag- -14-TgHSP70 was transfected into HEK 293T cells. The target gene bands of the expected size were obtained by RT-PCR. The size of the expressed product was about 19 kDa by Western-blot. Conclusion The eukaryotic expression plasmid p3 × Flag-CMW-14-TgHSP70 was constructed successfully and correctly expressed in HEK 293T cells.