Effect Of SEA On The TCRζ Chain Gene Expression Of T Cells Stimulated By PML-RARα Peptide In Vitro And Construction Of The Eukaryotic Expression Plasmid Of BCR-ABL Gene And SEA Gene Respectively

Objective:To measure the expression of signaling transduction factor T lymphocyte-TCR chain gene in peripheral blood mononuclear cells which stimulated by PML-RARαfusion peptide or SEA or both of them.To construct the eukaryotic expression plasmid of BCR-ABL gene and SEA gene respectively.Methods:1,Peripheral blood mononuclear cells from normal individual were cultured with PML-RARαpeptide,SEA and both of them,which were induced by PML-RARαpeptide for 20 days,respectively add the SEA at the beginning,the fifth day,and a comparison of SEA was set.2,Real-Time PCR with SYBR GreenⅠtechnique was used for detecting TCRζchain expression level andβ2-microglobulin gene(β2M) was used as an endogenous reference.Relative changes in TCRζchain expression level were used by the 2^{-ΔΔCt} method between each group and the control.3,BCR-ABL fusion gene segments were amplified from K562 leukemia cell by RT-PCR and were inserted into the MCS A of pIRES2-EGFP plasmid to construct a recombinant plasmid pIRES2-EGFP-BCR-ABL.The whole SEA gene were amplified from the DNA of staphyloccocus aureus by PCR and to construct a recombinant plasmid pIRES2-EGFP-SEA.4,To transfect the pIRES2-EGFP-BCR-ABL and the pIRES2-EGFP-SEA into K293 cells respectively,detect the protein expression of EGFP,and to detect the mRNA expression of BCR-ABL genes and SEA genes by RT-PCR.Results:1,The expression level of TCRζchain in groups which were added the SEA at the beginning and the fifth day were over expressed,the other groups were decreased;2,The results of PCR,double restriction endonuclease cutting and DNA sequencing analysis confirmed that the pIRES2-EGFP-BCR-ABL and the pIRES2-EGFP-SEA were successfully constructed;3,Both EGFP protein and mRNA expressed by recombinant plasmids in K293 cell could be correctlly detected.Conclusion:1,The expression of signaling transduction factor T lymphocyte-TCRζchain gene in peripheral blood mononuclear cells,which stimulated by SEA and PML-RARαfusion peptide,could be over expressed.2,Successfully construct the recombinant plasmid of the plRES2-EGFP-BCR-ABL 3,Successfully construct the recombinant plasmid of the pIRES2-EGFP-SEA 4,These plasmids have normal function of transcription and can express the protein of EGFP in eukaryotic cells in vitro.