[Cloning and eukaryotic expression of murine beta-defensin-2(mBD-2)].

Abstract

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.

• Authors: Xiao-li Wei, Qiao-fa Shi, Hong Li, Wan-yi Li, Zhong-hua Jiang, Ming-yuan Li
• Journal: Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology (Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi) Vol. 23 Issue 1 Pg. 28-31 (Jan 2007) ISSN: 1007-8738 [Print] China
• PMID: 17210101 (Publication Type: English Abstract, Journal Article)

Chemical References

• beta-Defensins

Topics

• Animals
• Cell Line, Tumor
• Cloning, Molecular
• Eukaryotic Cells
• Genetic Vectors
• Humans
• Mice
• Mice, Inbred BALB C
• Plasmids
• Polymerase Chain Reaction
• Reverse Transcriptase Polymerase Chain Reaction
• Transfection
• beta-Defensins (genetics)