The Construction Of Plasmid PcDNA3.1(+)-IL-24 And The Effects Of RhDCN Cooperate With RhIL-24 On Human PBMC In Vitro

Objective:To investigate the effects of decorin (DCN) and interleukin-24 (IL-24) on the proliferation and secretion of human peripheral blood mononuclear cells (PBMC)Methods:1.DCN and IL-24 were amplified by RT-PCR to construct eukaryotic expression recombination pcDNA3.1 (+)-DCN and pcDNA3.1 (+)-IL-24. After digest identification, PCR identification and DNA sequencing, DCN and IL-24 were expressed in E.coli. DH-5α.2.The plasmids were extracted with Endo-Free Plasmid Mini Kit and transfected into PBMC with LipofectamineTM 2000 respectively.3.MTT was performed to detect the cell proliferation. ELISA was used to detect the IFN-yexpression in the supernatant. Flow cytometry(FCM) assay was used to determine the expression of programmed death-1(PD-1).Results:1.The target band was accordance with the anticipated objective strap size in the digest identification and PCR identification. DNA sequence analysis proved correct reconstruction of IL-24.2. The mRNA of DCN and IL-24 was detected by RT-PCR after transfecting PBMC with LipofectamineTM 2000.3. MTT results:DCN and IL-24 could promote PBMC proliferation more significantly after transfecting 96h,120h,144h. ELISA showed that DCN and IL-24 could promote PBMC IFN-y secretion more significantly(P<0.05). FCM showed that the combined genes could upregulate the expression of PD-1 more obviously than other groups.Conclusion:The recombinant plasmid pcDNA3.1 (+)-IL-24 was constructed successfully. DCN and IL-24 could promote PBMC proliferation and IFN-y secretion more significantly, and could upregulate the expression of PD-1 than the other groups. The double combined genes: DCN and IL-24 could strengthen the function of PBMC in vitro.