In Vitro Experimental Study Of RNA Interference Mediated Gene Silencing Of Pneumocystis Carinii BCK1 Gene

The present study was initiated to assess the effect of RNAi technical against Pneumocystis cainii, a PC BCK1 miRNA expression vector was constructed and transferred into pneumocystis carinii cultured in vitro.The effect was evaluated by the count of cyst, the ultrastructural change and the RT-TCR result.Method: Pneumocystis Carinii Pneumonia rat models were established by hypodermic injection of dexamethasone twice a week for 6 weeks. According to algorithm for selection of functional siRNA sequences, two pieces of siRNAs targeting the PC BCK1 gene were designed. hairpin siRNA templates were synthesized and cloned into PTZU6+1 vector. Pneumocystis Carinii was purified and cultured in vitro.then the siRNA of BCK1 gene was transferred into Pneumocystis Carinii by Lipofectamine 2000,three group included green fluorescent protein vector control group, the blank control group without any disposal was also established. Sample of supernatant were withdrawn every day and evaluated for PC cyst counts by GMS stain , the ultrastructural change was observed by transmission and scanning electron microscope after 72h of transfection. the inhibitor of BCK1gene was evaluated by RT-PCR.Result:The Pneumocystis Carinii Pneumonia rat model was established successfully.Two BCK1 gene siRNA was constructed successfully, named BCK1-shRNA1 and BCK1-shRNA2 .then transfected BCK1-shRNA1 and BCK1-shRNA2 into PC in vitro. Compared with the blank control group,the inhibitory rate of BCK1-shRNA1 and BCK1-shRNA2 was 35.4% and 42.5% respectively after 72h of transfection. the cell wall defect was found by scanning electron microscope. the inhibitory effect on PC BCK1 mRNA was detected by RT-PCR, the inhibitory rate was 57.38% and 64.45% respectively.Conclusion: There was a significant effect of interference against the replication and expression of PC BCK1 gene with PC BCK1 siRNA eukaryotic expression vector. The PC proliferation was inhibited and PC cell wall was destructed by PC BCK1 siRNA eukaryotic expression vector...