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[Cloning and eukaryotic expression of murine beta-defensin-2(mBD-2)]
Xiao-li Wei1, Qiao-fa Shi, Hong Li
1Department of Microbiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China. xiaoli1909@126.com
Aim:
To clone murine beta defensin-2 gene (mBD2) and to express the mBD2 protein eukaryotically.
Methods:
Total RNA was isolated from the lungs of BALB/c mice which were injected with LPS in advance. The DNA fragment encoding mBD2 was amplified by RT-PCR and inserted into the plasmid pcDNA3.1(+), which was then digested with EcoR I and Xho I to construct the recombinant plasmid, pcDNA3.1(+)/mBD2. The pcDNA3.1(+)/mBD2 was identified by endonuclease digestion, PCR, and sequencing analysis. The SiHa cells were transfected with pcDNA3.1(+)/mBD2 plasmid and screened by G418 of 100 mg/L over 20 days. Steady expression of mBD2 was confirmed by immunofluorescent staining and RT-PCR.
Results:
About 250 bp DNA fragment was amplified by RT-PCR from lung total RNA of the mice injected with LPS. The eukaryotic expression vector, pcDNA3.1(+)/mBD2, was successfully constructed after inserting the mBD2 fragment into pcDNA3.1(+). Most of SiHa cells transfected with pcDNA3.1(+)/mBD2 and screened by G418 could express the mBD2 protein, confirmed by immunofluorescent staining and RT-PCR.
Conclusion:
The eukaryotic vector of pcDNA3.1(+)/mBD2 was successfully constructed and transfected into SiHa cells, which established a solid foundation for further study on the biological characteristics and anti-tumor mechanisms of the mBD2 protein.
