Expression of MLAA34-HSP70 fusion gene constructed by SOE-PCR

To construct the pIRES2-MLAA34-HSP70 recombinant vector and express the MLAA34-HSP70 recombinant proteins in Escherichia coli [E. coli]. The MLAA34 and the HSP70 genes were extracted from U937 cells by RT-PCR, and then we amplified the fusion gene MLAA34-HSP70 by SOE-PCR and inserted it into the pIRES2-EGFP vector to construct the pIRES2-MLAA34-HSP70 recombinant vector. We amplified the fusion gene MLAA34-HSP70 successfully and identified the correctness of pIRES2-MLAA34-HSP70 recombinant vector by PCR and restriction endonuclease. Moreover, the MLAA34-HSP70 recombinant proteins expressed in E. coli were consistent with the expected molecular weight. We constructed the pIRES2-MLAA34-HSP70 recombinant vector successfully and the MLAA34-HSP70 recombinant proteins were successfully expressed by the induction of IPTG

Study on immune effect of DNA vaccine pIRES2-MLAA34-HSP70 constructed by SOE-PCR / 重庆医学

Objective To construct the DNA vaccine pIRES2-MLAA34-HSP70,and to detect its immune effect.Methods The acute monocytic leukemia associated antigen gene MLAA-34 and heat-shock protein (HSP)70 gene were extracted by using RT-PCR.The specific overlapping primer was designed,and the fusion gene MLAA34-HSP70 was amplified by using SOE-PCR technique.Then the DNA vaccine pIRES2-MLAA34-HSP70 was constructed,and BALB/c mice were immunized with this DNA vaccine.The splenic lymphocyte killing activity was detected by using MTT,levels of IL-2,IL-4 and IFN-γ were also detected by using ELISA.Results The MLAA34-HSP70 gene (2 956 bp) and the DNA vaccine pIRES2-MLAA34-HSP70 was amplified and constructed successfully.The killing efficiency of DNA vaccine pIRES2-MLAA34-HSP70 in U937 cells was significantly higher than that in other experimental groups and control group (P＜0.01),and levels of IL-4,IL-2 and IFN-γin DNA vaccine pIRES2-MLAA34-HSP70 group were significantly higher than those in the other experimental groups and control group (P＜0.01).Conclnsion The DNA vaccine pIRES2-MLAA34-HSP70 is constructed successfully.It is shown that the DNA vaccine induces strong humoral immunity,which could enhance immune responses to tumor cells and specificlly kill MLAA34 positive cells.