The Eukaryotic Expression Vector Construction Of PIRES2-EGFP-hIL-12-HSP70 And Its Influence On The Growth Of 7402 Liver Cancer Cell

Objective:This project is to obtain the fusion protein molecules with activity of hIL-12 and HSP70 by constructing the eukaryotic expression vector of pIRES2-EGFP-hIL-12-HSP70, detecting its expression in 7402 liver cancer cells and observig its influence on the growth of 7402 liver cancer cell.Methods:hIL-12 gene was amplified from pBI121-hIL-12 plasmid by PCR and HSP70 gene was obtained from pShuttle-CMV-HSP70 plasmid, The two target genes were cloned into pIRES2-EGFP to construction recombinant eukaryotic expression vector, pIRES2-EGFP-hIL-12, pIRES2-EGFP-HSP70 and pIRES2-EGFP-hIL-12-HSP70 by gene recombination technology. PCR, Restrictive digestion and DNA sequencing were used to confirm the recombinant plasmid construction. Recombinant eukaryotic expression vectors and pIRES2-EGFP were transfected into 7402 liver cancer cells by Lipofectamine 2000, and the 7402 liver cancer cells without transfecting was set as negative control. Experiment groups were divided as 7402/hIL-12,7402/HSP70,7402/hIL-12-HSP70,7402/PE and 7402/0. The green fluorescent of all groups was captured by inverted fluorescence microscope. The expression of recombinant gene was detected by Real-time PCR. Cell growth was observed by MTT, and the cells apoptosis rate was detected by AnnexinV-FITC/PI.Results:Eukaryotic expression vectors of pIRES2-EGFP-hIL-12, pIRES2-EGFP-HSP70 and pIRES2-EGFP-hIL-12-HSP70 have been successfully constructed. The green fluorescent in transfected groups can be observed at 24h,48h and 72h. The recombinant foreign gene were expressed in 7402 cells. The highest inhibiting rate cells was the group of 7402/hIL-12-HSP70. The inhibiting rate of the cells transfected after 48h was significantly higher than the 24h and 72h. The inhibiting rate of 7402/hIL-12-HSP70 group was significant different with 7402/hIL-12,7402/HSP 70 and 7402/PE (P<0.05). The number of apoptosis in 7402/hIL-12-HSP70 group was more than that of other groups (P <0.05).Conclusion:The eukaryotic expression vector pIRES2-EGFP-hIL-12, pIRES2-EGFP- HSP70 and pIRES2-EGFP-hIL-12-HSP70 were constructed successfully. All eukaryotic expression vector could transfect into 7402 liver cancer cells and effective expression. The recombinant united gene of hIL-12 and HSP70 could significantly promote apoptosis of 7402 liver cancer cells and has synergistic effect.