Prokaryotic Expression And Purification And Curative Effect On VVC Of Human Catheliedin LL-37

Objective:Construct the prokaryotic expression vector for LL-37and transformed into Ecoli M15[pREP4]cells. Optimize the expression condition and purify the recombinant protein with the aim of obtaining the bioactive recombinant LL-37protein.To discuss the curative effect and mechanism of LL-37on VVC through the cell and animal experiment.Method:1)Construct the prokaryotic expression vector of pET-Duet/LL-37by genetic engineering, and transformed into Ecoli M15[pREP4]cells,and induce expression by IPTG.Optimize the expression conditions by regulating the concentration of inducer and the induction time.The recombinant protein was purified through Ni-IDA affinity purification system.The antifungal activity was tested by disk diffusion method.2)The vaginal epithelial cells from female vagina were cultured primarily by fractional step digestion method and subcultured4passages for experiment.The epithelial cells and candida were cocultured according to1:1,in order to construct the VVC cell model.Divided into the experimental and control group.Join the recombinant protein to experimental group for treatment, and the control group was joined the same volume PBS. The supernatant of every group wan collected at12h,24h,48h.At each time point,the growth density of Candida albicans was observed by inverted microscope.Glucose consumption testing was used to calculate the growth inhibition rate.(Denoted by absorbancy A). ELISA was applied to detect the level of IFN-γ.IL-10of each time point,and compare the changes of IFN-y/IL-10.3)The estrogen was subcutaneously injected regularly to build the VVC model in mice.30mice was divided into experimental and control group randomly.The experimental group was poured the LL-37into vagina everyday.After five days,observe the microbial load and smear of vaginal lavage fluid.ELISA was applied to detect the concentration of IL-10and IFN-y in homogenate of vaginal wall tissue,and compared the difference of IFN-γ/IL-10.Result:1)Prokaryotic expression vector of LL-37was built successfully.Recombinant protein was mainly expressed in the form of inclusion body.Optimization conditions: IPTG0.5mmol/1,appropriate time6h. The purified recombinant protein which the concentration was433.92ug/ml, purity could reach96%.The disk diffusion test confirmed that the recombinant protein has certain inhibitory of Candida.2)VVC cell model was built successfully. As time progress,the mycelial of the control group grew vigorously, and the tend of absorbancy was notable decline.Both the survival of epithelial cell and the growth of candida of the experimental group were better than the control. The absorbancy are higher and the tend of decline are slower than the control(p<0.05).The result of ELISA prompted that the secretion level of IFN-γ,IL-10reached the max level at24h,and then decreasing.The concentration of IFN-γ of the experimental was higher than the control at the point of24h(p<0.05),and had no significant difference at other time points. The concentration of IL-10at each time point were higher than control,and the ratio of IFN-γ/IL-10were significant higher than the control.3)Model in mice were built successfully.The smear of vaginal lavage fluid prompt that the contention of the hyphae and spores was also significantly lower than control.and the microbial load had the same tend(p<0.05).The experimental concentration of IL-10was obviously lower than the control, while the concentration of IFN-y had no significant difference. The ratio of IFN-γ/IL-10in experimental group was greater than that of control, the difference had statistically significant(p<0.05).Conclusion:1)Successful build the LL-37prokaryotic expression vector, and purified the recombinant proteins with antifungal activity.2) Cells and animal experiments have confirmed that the recombinant protein has obvious therapeutic effect of VVC.3)The possible mechanism of LL-37on the treatment of VVC:a. inhibit the adhesion effect of candida, and kill pathogenic bacteria directly, b.influence the secretion level of IFN-γ,IL-10, and adjust vaginal local immune.LL-37maybe can restore the imbalance of vaginal local immune which caused by VVC and recover the balance between TH1and TH2.By this ways, LL-37can enhance the vaginal local immune to resist candidas.