Prokaryotic Expression Of Lcn2 Gene From Murine Macrophage And The Preliminary Application Of The Recombinant Lcn2

Objective: To construct prokaryotic expression plasmids carrying murine macrophage lcn2 gene, express the recombinant proteins in E.coli BL21(DE3)plysS and observe its biological activity.Methods: The gene from genome of murine macrophage lcn2 was amplified by RT-PCR and inserted into prokaryotic expression vector PET32a(+) to obtain recombinant plasmid pET32a(+)-lcn2. After identified by restriction digestion and DNA sequencing, the constructed recombinant plasmids was transformed into E.coli BL21(DE3)plysS. Then E.coli was induced with IPTG.and the product was analyzed by SDS-PAGE and His-tag in-gel Stain.Then the expressed protein was purified.The recominant protein Lcn2 was added into the LB medium ,and E.coliBL21(DE3) was subcultured on the medium by pouring.A common LB medium without Lcn2 as the control was operated by the same way. Finally , E.coliBL21(DE3),Streptococcus hemolyticus, Staphylococcu saureus were respecrively subcultured on the corresponding medium, the bacteriostatic activity of recombinat Lcn2 was observed by disk diffusion antimicrobial suceptibility test.Result: Restriction endonuclease digestion and DNA sequence analysis indicated that the prokaryotic expression vector was constracted successful.The expressed protein induced by IPTG in E.coliBL21(DE3)plysS had relative molecular mass of about 21 kDa.Bacteria growth test demonstrated that the colonise in medium with recominant protein was smaller than that in control LB medium,but the bacterial numbers is not different.Except for the medium with Staphylococcus aureus,bacteriostatic circles was observed on medium with E.coli and Streptococcus respecrively.Conclusion: The recominant expression plasmid pET32a(+)-lcn2 was constracted successful, and the recominant protein Lcn2 was successfully prepared.Bacteria growth test demonstrated that the recombinant Lcn2 could inhibit the growth of gram-positive bacterias and gram-negative bacteria , but it had no effect for Staphylococcus aureus.These results indicated Lcn2 might be a useful bacteriostatic agent.