Construction, identification and expression of eukaryotic recombinant expression plasmid containing Toxoplasma gondii HSP70

Objective To construct p3×Flag-CMW-14-TgHSP70 from Toxoplasma gondii heat shock protein 70 (TgHSP70) and to express it in HEK 293T cells.Methods The TgHSP70 gene was amplified by PCR.The gene fragments were digested with double enzyme and connected to eukaryotic expression plasmid p3×Flag-CMW-14.The connection products were identified by enzyme digestion,PCR and DNA sequencing.The recombinants were transfected into HEK 293T cells by Entranster TM-D.TgHSP70 expression products were identified based on gene and protein levels with reverse transcription-PCR(RT-PCR) and western blot.Results A total of 495 bp HSP70 fragment genes were amplified by PCR.The recombinant plasmid p3×Flag-CMW-14-TgHSP70 was successfully constructed.The positive clone was identified with enzyme digestion,PCR and DNA sequencing and the gene sequence was completely correct.The p3×Flag-CMW-14-TgHSP70 eukaryotic expression plasmids was transfected into HEK 293T cells.The positive band with expected size was displayed by RT-PCR.The expression products of HSP70 gene were approximately 19 kDa detected with western-blot analysis.Conclusion Recombinant eukaryotic expression plasmid of HSP70 was successfully constructed and correctly expressed in HEK 293T cell.