Cloning and eukaryotic expression of murine betaª²defensinª²2(mBDª²2)

    WEI Xiaoª²li, SHI Qiaoª²fa, LI Hong, LI Wanª²yi, JIANG Zhongª²hua, LI Mingª²yuan*

    Department of Microbiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China

    \[Abstract\] AIM: To clone murine beta defensinª²2 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 RTª²PCR and inserted into the plasmid pcDNA3ª±1(+), which was then digested with EcoR¢ñand Xho I to construct the recombinant plasmid, pcDNA3ª±1(+)/mBD2. The pcDNA3ª±1(+)/mBD2 was identified by endonuclease digestion, PCR, and sequencing analysis. The SiHa cells were transfected with pcDNA3ª±1(+)/mBD2 plasmid and screened by G418 of 100 mg/L over 20 days. Steady expression of mBDª²2 was confirmed by immunofluorescent staining and RTª²PCR. RESULTS: About 250 bp DNA fragment was amplified by RTª²PCR from lung total RNA of the mice injected with LPS. The eukaryotic expression vector, pcDNA3.1(+)/mBD2, was successfully constructed after inserting the mBDª²2 fragment into pcDNA3ª±1(+). Most of SiHa cells transfected with pcDNA3ª±1(+)/mBD2 and screened by G418 could express the mBD2 protein, confirmed by immunofluorescent staining and RTª²PCR. CONCLUSION: The eukaryotic vector of pcDNA3ª±1(+)/mBD2 was successfully constructed and transfected into SiHa cells, which established a solid foundation for further study on the biological characteristics and antiª²tumor mechanisms of the mBD2 protein. ......