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[Establishment of a stable and regulable 293 cell line expressing mycoplasma hyorhinis protein P37]
Man-man Gong1, Lin Meng, Wen-bin Liu
1Department of Biochemistry and Molecular Biology, Peking University School of Oncology, Peking University Centre for Cancer Research, Beijing 100034, China.
Objective:
To establish a stable cell line, which can express P37 protein of mycoplasma hyorhinis and be regulated by tetracycline, for investigating the effect of p37 on phenotype of cells and its mechanism.
Methods:
Recombinant plasmid PcDNA5/FRT/TO-p37 was constructed and cotransfected with pOG44 into Flp-In-T-REx-293 cells by lipofectamine. Positive clones were screened with Hygromycin and Blasticidin. RT-PCR and Western blot were used to exam the mRNA and protein expression in selected clones. The expression level at different inducing times and concentrations of tetracycline were examined. MTT assay was used to observe the effect of P37 on proliferation of 293 cells.
Results:
P37 protein, which is 43.5x10(3), was expressed in the selected clone as well as secreted from cells. Tetracycline showed a good regulation on the expression of P37 protein, which was not detectable without tetracycline induction. When induced with 2 mg/L tetracycline for 60 hours, the P37 protein expression reached maximum level. Cell growth was promoted after being transfected with p37.
Conclusion:
A stable cell line expressing P37 regularly was established, which provides a good cell model for studying p37 function and its molecular mechanism.
