| Objective The aim of this study was to construct the eukaryotic expression vector of ratβ-defensin-2(rBD-2)gene by gene engineering technique.After transfection of rat corneal epithelial cells by overexpressing lentivirus packaging,the recombinant rBD-2 And to study the protective effect of recombinant rBD-2 on antifungal infection in order to further study the effect of human β-defensin-2 gene therapy on fungal cornea Inflammation to provide basis.Methods The target gene rBD-2 lentiviral expression vector and 293T cells were co-transfected into 293T cells by gene engineering technique.The lentiviral packaging of the target gene was inoculated in vitro.Rat corneal epithelial cell line.The expression of rBD-2 gene in the cells was detected by RT-PCR and Western blotting.The expression of rBD-2 gene in the transfected cells was detected by real-time quantitative RT-PCR.Finally,the animal model of fungal(Aspergillus fumigatus)keratitis was established.The rats were divided into control group and experimental group.The rats in the experimental group were treated with RSB-S16111411/rBD-2 overexpressing lentiviral packaging cell suspension with rBD-2 gene.The rats in control group were treated with empty vector transfected positive cell suspension.After 3,6,9,12,and 15 days,the eyes were observed by slit lamp microscope on the clinical score.The effects of rBD-2 overexpressing lentivirus packaging plasmid were evaluated by clinical manifestation,fungal keratitis score and corneal scraper.Results 1.The recombinant plasmids of pHS-GS034-rBD-2 were identified by electrophoresis and sequencing of BamHI enzyme and Nhel enzyme.The inserted nucleotide sequence was rBD-2,which proved that the recombinant vector was successfully constructed.2.The corneal epithelial cells transfected with RSB-S16111411/rBD-2 were cultured in inverted fluorescent microscope and the corneal epithelial cells infected with the virus were stained with green fluorescence.The untransfected empty cell group had no fluorescence.3.The expression of rBD-2 gene in corneal epithelial cells of RSB-S16111411/rBD-2 transfected group was significantly higher than that of the other two groups by using qPCR method.4.The model of rat fungal keratitis was established by corneal scoring method and corneal stromal injection.The clinical model,corneal scraping and culture confirmed that the model was successfully established.5.The cornea of rats was titrated by cell suspension.The effects of rBD-2 overexpressing lentivirus packaging plasmid were evaluated by clinical manifestation,fungal keratitis score and corneal scraper.Conclusion The rBD-2 eukaryotic expression vector was transfected into rat corneal epithelial cells by using the recombinant plasmid RSB-S16111411/rBD-2,which was able to make the exogenous rBD-2 gene in rat cornea Epithelial cells are transcribed into mRNA.The use of this cell line suspension eye drops,the fungal keratitis in rats have a certain effect. |
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