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Published on: January 25, 2017
[Establishment of AFP promoter operated murine IL-1beta recombinant vector and its expression in H22 cells]
Qing-chun Li1, Shu-juan Liang, Xue-jing Wang
1Department of Immunology in Weifang Medical College, Key Lab for Immunology in Universities of Shandong, Weifang 261042, China. pdli2005@163.com
Aim:
To establish a hepatoma specific murine IL-1beta (mIL-1beta) expression vector operated by AFP promoter and analysis its expression in H22 cell.
Methods:
The chimeric operating sequence composed of the minimal AFP promoter and CMV enhancer(ECMV) was prepared through SOE-PCR. The sequence was inserted to replace the conventional enhancer and promoter in pIRES2-EGFP to establish the novel hepatoma specific vector p(afp)IRES2-EGFP. Full length of murine IL-1beta was amplified through RT-PCR by pfu DNA polymerase followed by cloning to establish the recombinant pIRES2-EGFP-mIL-1beta expression vector verified through PCR, restriction enzyme assay, DNA sequencing and cell transfection. p(afp)IRES2-EGFP-mIL-1beta was tranfected into H22 hepatoma cells and YAC-1 lymphoma cells in a transient transfection system mediated by jetPEI. Expression of the vector was observed under fluorescent microscope 48 h after transfection. Expression level of mIL-1beta was detected by RT-PCR.
Results:
A 537 bp chimeric AFP promoter and ECMV was yield and inserted to establish a novel hepatoma specific vector p(afp)IRES2-EGFP proved by restriction enzyme assay, DNA sequencing and transfection. Full length murine IL-1beta was then amplified and cloned to establish the recombinant expression vector p(afp)IRES2-EGFP-mIL-1beta verified through repeated clony PCR, restriction enzyme assay by EcoR I and Xho I, DNA sequencing and transfection. Purified p(afp)IRES2-EGFP-mIL-1beta was transiently transfected into H22 cells and YAC-1 cells by jetPEI, and bright green fluorescence was only seen on the surface of H22 cells, indicating that p(afp)IRES2-EGFP-mIL-1beta can specifically express target gene within the murine hepatoma cells. Simutaneously, the expression level of mIL-1beta was markedly elevated in H22/mIL-1beta in RT-PCR assay.
Conclusion:
We successfully prepared a hepatoma specific expression vector named p(afp)IRES2-EGFP-mIL-1beta that could expression high level of murine IL-1beta in a transient transfection system.
