[The cloning and expression of rhlR, a quorum sensing gene in Pseudomonas aeruginosa PAO1, and a study of its effect on bacterial clearance rate in mouse lung].

ABSTRACT

OBJECTIVE: To inquire about the molecular characteristics of rhlR, a Quorum Sensing gene in Pseudomonas aeruginosa (P. aeruginosa) PAO1, and to explore the immunogenicity of RhlR protein in mouse. METHODS: The rhlR gene of PAO1 was amplified by PCR and cloned into pGEX4T-1 plasmid. The recombination was expressed in E. coli BL21 (DE3) and analyzed by SDS-PAGE and Western blotting. The fusion protein (GST-RhlR) was purified by GST purification Kit and the purified protein was used to immunize mice. P. aeruginosa PA0315 was injected into mouse lung to explore the immuno-protection of the protein. RESULTS: The 726 bp DNA fragment of rhlR was amplified from PAO1 general DNA. The restriction enzyme map showed that the inserted part of rhlR-pGEX4T-1 was successfully constructed and the gene was 100% homologous to rhlR in GenBank. The recombinant plasmid expressed a 54 kDa fusion protein (rhlR-GST) in E. coli BL21 (DE3) after induction by IPTG. The fusion protein could be recognized by mouse polyvalent antiserum against P. aeruginosa. The results showed that the bacterial clearance rate in mouse lung was 86. 92% in rhlR groups and 49.44% in the control group. CONCLUSION: A 54 kDa protein (RhlR-GST) has been successfully expressed in E. coli BL21 (DE3). The RhlR could increase the bacterial clearance rate in mouse lung and may serve as immunoprotective antigen to develop the genetic engineering vaccine.