Empirical Study Of Abaissement Of RNAi To Thymidylate Synthetase Gene Of Rat With Pneumocystis Carinii

In all eukaryotic and prokaryotic organism, thymidylate synthetase is a key enzyme in synthetic route of dTMP (Deoxyadenosine Triphosphate), dTMP is the product of dUMP methylation, in the synthetic procedure of dTMP, thymidylate synthetase which catalyze the production of dTMP methylation is the essential enzyme of DNA composition and recovery.RNA interference is a kind of conservation response of conservative from low organism to mammal existed. Double-stranded RNA was cut abo -ut 21- 23nt siRNA by endonuclease RNaseâ…¢in cell, and formatted RNA- induced silencing complex, RISC identified and degraded mRNA of homol -ogical rank, it lead to ultimately specific silence of gene expression. To co -nstract carrier that it can transcript functional siRNA in pneumocystis cari -nii, and it was advantaged to confuse expression of thymidylate synthetase, to establish background for further investigation of gene function and thera -peutic tool.This study included two parts.The first part: Construction and identification of plasmid vector of short interfering RNA on pneumocystis carinii thymidylate synthase gene's upstream conserved sequence Methods: Hairpin shRNA oligonucleotides of thymidylate synthetase were chemically synthesized and inserted into plasmid vector pTZU6+1 after annealing, then transformed into E.coil Top10. The recombinant plasmid was identified by restriction endonuclease Hindâ…¢and EcoRâ… and DNA sequencing. Results: Gel electrophoresis and DNA sequencing showed that the products was the same as the expected gene(shRNA).Conclusion: Eukaryotic expression plasmid of shRNA on TS was constructed successfully.The second part: Extraorgan empirical study of Abaissement of RNAi to thymidylate synthetase gene of rat with Pneumocystis carinii: Methods: PCP rat models were established by hypodemic injection of dexamethasone twice a week for 6 weeks. Extraorgan abstraction and depuration of pneumocystis carinii, taking the transfection of plasmid pTZU6+1 which contain GFP and Lipofectamine 2000 to pneumocystis carinii respectively according to quality and volume ratio 2 : 2, 2 : 4, 2 : 6, 2: 8, and then express the quantum of the epipolic trophozoite under the fluorescence microscope counting after 24, 48, and 72 hours. Taking the transfection of plasmid to pneumocystis carinii, then respectively count the mean cyst number per visual field after 24, 48, and 72 hours. 48 hours after transfection the mRNA level of thymidylate synthetase gene should be determined by RT-PCR, and take pneumocystis carinii sample to observe the difference in ultrastructure between recombinant plasmid transfection group and control group with a scanning electron microscope. Results: The most transfection green fluorescent protein expression were observed after 48 hours, but mean cyst number per field of Recombination plasmid is at the least, and compared with vacant group or vacant plasmid group, the remarkably difference could be observed. shRNA eukaryotic expression vector can actively suppress the pneumocystis carinii thymidylate synthetase genetic expression, compared with the transfected gene with empty vector group or vacant group, thymidylate synthetase gene mRNA expression decreased to 45.07%, fundament damage on pneumocystis carinii surface is the microvilli shedding, survival microvilli submit to bulb form for edema. Some pneumocystis carinii encyst surface emerge caverns inequality in size to the damage degree. Conclusion: PCP rat models were established by hypodemic injection with dexamethasone for 6 weeks. Lipofecttamine2000 has comparatively strong transfection efficiency to the recombinant plasmid. shRNA eukaryotic expression vector can effectively suppress thymidylate synthetase gene mRNA expression, and has a certain degree of destroy to the pneumocystis carinii ultrastructure.