Expression And Purification Of PLB Protein Of Candida Albicans And Verification Of Its Function

BackgroundCandida albicans is an important human fungal pathogen, causing a wide variety of infections ranging from mucocutaneous infections to life-threatening systemic infections in individuals.The virulence factors of Candida albicans include adhesion molecules, enzymes for invasion, factors responsible for morphogenesis and phenotypic switching, etc. Phospholipase B is critical among the virulence factors, which is responsible for pathogenicity of Candida albicans directly. Phospholipase B is likely to be involved in the early steps of host invasion (adherence, penetration, and damage). Currently, there is an intense renaissance in the study of the effects of phospholipase B focused on potential role of phospholipases in virulence and fungal pathogenesis.ObjectiveTo construct recombinant vectors containting the gene encoding phospholipase B1 and phospholipase B2 of Candida albicans, verificate the bioactivity of recombinant proteins after they were expressed in E. coli and purified. The study will provide foundation for further investigation in the pathogenic and drug resistance mechanism of phospholipase B1 and phospholipase B2 and developing anti-fungal medicine, diagnostic reagent and DNA vaccine in Candida albicans.MethodsThe target gene fragments PLB1 and PLB2 were obtained by standard PCR amplification while genome DNA of C.albicans SC5314 strain was used as template. PLB1, PLB2 gene fragments and plasmids pEGX-4T-1 were cleaved by two restriction endonucleases EcoR I and Xho I, and the digested products were separated and purified in low melting temperature agarose gels. The purified PLB1, PLB2 gene fragments and plasmid pEGX-4T-1 were recombined by T4DNA ligase, and ligation products were transformed into competent cell, E.coli TOP10. Transformed colonies were screened by Ampr LB plate, and then recombined plasmids were isolated and identified by restricted endonuclease cutting and DNA sequencing. After pEGX-4T-1/PLB1 and pEGX-4T-1/PLB2 were transformed into E.coli strain BL21 (DE3), they were induced to inclusion bodies by IPTG at 37℃. The fusion protein were dissolved and renatured with gradient concentration of urea. The renatured inclusion bodies liquid were purified by GST-Sepharose 4B affinity chromatography column, Mono P 5/50 GL ion exchange prepacked column and reverse phase HPLC in turn, and the fusion proteins were cleaved by thrombin at the same time. Finally, the purified recombinant proteins were verificated the bioactivity with gel diffusion method.ResultsIdentified by agarose gel electrophoresis, the target gene PLB1 and PLB2 were obtained by PCR amplification had the same molecular size as predicted. They were indicated that recombined plasmids pEGX-4T-1/PLB1 and pEGX-4T-1/PLB2 contained inserted PLB1 and PLB2 gene fragment by restricted endonuclease cut analysis, the sequencing data also indicated that inserted PLB1 and PLB2 gene had correct DNA sequence and orientation according to DNA sequence of C.albicans SC5314. After transferred into engineered strain BL21 (DE3), both procaryotic expression plasmid pEGX-4T-1/PLB1 and pEGX-4T-1/PLB2 can express inclusion bodies when induced by IPTG at 37℃. The target proteins were obtained after fusion proteins were dissolved, renatured, purified and cleaved. The recombinant proteins were verificated that they had powerful extracellular activity of the candidal phospholipase.Conclusion1. Procaryotic expression vector, pEGX-4T-1/PLB1 and pEGX-4T-1/PLB2 which can express inclusion bodies proteins were constructed successfully.2. The target proteins, PLB1 and PLB2 were obtained after fusion proteins were dissolved, renatured, purified and cleaved.3. The recombinant proteins had powerful extracellular activity of the candidal phospholipase.