Cloning and prokaryotic expression of FnBP ligand binding gene of Staphylococcus aureus
2008
Yin Ronglan | Yang Zhengtao | Zhang Yanjing
[Objective] The study aimed to clone the FnBP ligand binding gene of Staphylococcus aureus and run prokaryotic expression by constructing a prokaryotic expression vector. [Method] The gene encoding FnBP ligand binding gene was amplified from S. aureus chromosomal DNA by PCR technique. After T-A cloning, plasmid pMD18- FnBP was constructed. pMD18- FnBP and pET28a (+) were digested by BamH I and EcoR I double enzymes, then the purified FnBP ligand binding gene was subcloned into the expression vector pET28a (+) , and the prokaryotic expression vector pET28a-FnBP was thus constructed. The constructed plasmid pET28a-FnBP was transformed into Escherichia coli BL21 (DE3) competent cells. The bacterium was induced by IPTG and the expressed products were analyzed by SDS-PAGE and Western blot. [Result] The gene fragment with the length of 370 bp was amplified by PCR approach. One approximately 30 kD exogenous protein was observed in SDS-PAGE analysis. Western blot analysis indicates the protein has antigenicity of S. aureus. [Conclusion] The FnBP ligand binding gene of S. aureus was successfully cloned and expressed in prokaryotic cells.
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