Staphylococcus Aureus Membrane Protein Nebps Prokaryotic Expression System And Antibody Preparation

Staphylococcus aureus is a common bacteria . Mainly through its own elastic fiber surface protein binding protein (Elastin Binding Proteins of S. aureus, nEbpS) infected host. nEbpS owns 1458 bp gene ,which encodes a protein of 486 AA, molecular weight of 25 kDa.The N-terminal 59 AA are the binding sites between Staphylococcus aureus and the main elastic protein.A pair of primers were designed and synthesized according to the sequence of nEbpS gene on the basis of the sequence of Staphylococcus aureus genome from Genbank. And the whole of N-terminal of Staphylococcus aureus gene was amplified by high-fidelity PCR technique from the Staphylococcus aureus genome. Add adenosine to the terminal. Then the recombinant clone plasmids pMD19-T(+)/nebps was constructed after double restriction endonucleases digestion and the host strain DH5αwas transformed. The selected clones were verified by PCR,double restriction endonucleases and DNA sequencing. DNA sequencing results showed that the sequence of DNA fragments inserted into the vector was identical to the one that had been reported before from Genebank. Then the recombinant express plasmids pQE30(+)/nebps was constructed in the similar way as the clone vector. After verifying by PCR and double restriction endonucleases digestion, the plasmid was transformed into expression strain M15 to express nEbpS protein with 37℃and 1mM IPTG induced. The expressed protein with molecular weight of 53kDa was detected by SDS-PAGE electrophoresis and Western blotting. The protein nEbps fused with Trx-His tag. The molecular weight was identical to prospective one(53.33 kDa), and most protein was soluble.The expressed fuse protein was purified by the method of affinity chromatography according to the manual of the producer. Then the purified protein nEbps fused with His tag was detected by Western blotting. The purified product showed good reactivity to anti-His tag antibody. The purified protein was concentrated by ultrafiltration and quantitated by Coomassie brilliant blue to 100 mg / ml concentration.Then misced antigen with complete Freund's adjuvant emulsion to immune animals, tnen harvest antiserum.The antibody was detected by agglutination reaction whih Staphylococcus aureus. The experimental results show that, the fragment of 684bp nEbpS gene was successfully expressed in M15 contained the recombined plasmid pQE30(+)/nebps. The molecular weight of the expressed protein was identical to the anticipant one. Then the expressed nEbpS protein was purified by affinity chromatography and prepared as antigen for animal immunization. And the antiserum was confirmed to own antibody by agglutination reaction.