Construction And Expression Of Eukaryotic Coexpression Plasmid Containing BCR/ABL Gene And SEA Gene And Effection Of Superantigen SEA On The CD3ε Chain Expression Of Cord Blood T Cells Stimulated By K562 Cells

Objective:To construction and expression of eukaryotic coexpression plasmid containing BCR/ABL gene and staphylococcal enterotoxins A (SEA). To investigate effect of staphylococcal enterotoxin A(SEA) on CD3s chain expression of T cells in cord blood, which were stimulated by K562 cells in vitro.Methods:1.BCR/ABL fusion gene was amplified from K562 cell line by RT-PCR, and the whole SEA gene was amplified from Staphylococcus aureus. Both PCR products were cloned into multi-clone site(MCS) A and B of pIRES plamid respectively to construct recombinant plasmids BCR/ABL-pIRES-SEA and SEA-pIRES-BCR/ABL. The plasmid was transfected into K293 cells to detect the expression of BCR/ABL and SEA by RT-PCR and SDS-PAGE electrophoresis respectively.2. The anti-CD3 antibodies, K562 cells, SEA or both SEA and K562 cells were respectively cultured with mononuclear cells (MNC) from four normal human cord blood for 48 hours, Real-Time PCR with SYBR GreenⅠtechnique was used for detecting CD3s chain expression level of T lymphocytes in cord blood. Relative changes in CD3e chain expression level were indicated by the 2-△△Ct method between each group and the control.β2-microglobulin gene(β2-M) was used as an endogenous reference.Results:1.Double enzyme cutting and sequence analyzing confirmed that the length and the sequence of the fragments inserted into pIRES plamids were absolutely correct. Both mRNA and protein expressed of recombinant plasmids in K293 cell lines could be correctly detected.2. The expression level of CD3εchain was slightly decreased in K562 cells group and was increased in the anti-CD3 antibodies,SEA,both of SEA and K562 cells respectively. The expression level of CD3εchain induced by both SEA and K562 was significantly higher than that of the SEA group (P<0.01).Conclusion:The recombinant eukaryotic plasmids BCR/ABL-pIRES-SEA and SEA-pIRES-BCR/ABL were successfully constructed, and they have the function of transcription and translation in eukaryotic cells; Superantigen SEA could enhance the expression level of CD3εchain of T cells stimulated by CML antigen on K562.