Silencing Pneumocystis Carinii MSG-UCS Gene By RNA Interference

Objective:In this research,we constructed and identificated plasmid vector of short interfering RNA on Pneumocystis carinii major surface glycoprotein gene's upstream conserved sequence, then we researched its effect after the recombinant plasmids were transfected into PC and were expressed.Methods:Short hairpin RNA (shRNA) oligonucleotide targeting PC MSG UCS gene was chemically synthesized and was inserted into RNAi-Ready plasmid vector pTZU6+1 . The recombinant plasmid, pPC-UCS, were dentified by gel electrophoresis and DNA sequencing. pPC-UCS were transfected into PC by liposome in vitro. The UCS mRNA expression was detected by RT-PCR and the fine structure of PC was observed by electronmicroscope.The rats were injected with pPC-UCS by hydrodynamics transfection after to be infected with PC. After 48 hours, the impression slides of the lung of rat were stained and we counted cysts, slices of lung tissue were stained by HE stain and the alteration of pathology of lung tissue was observed, the fine structure of PC were observed by electronmicroscope. Results: Gel electrophoresis and DNA sequencing showed that the recombinant plasmid containing the correct and full shRNA oligonucleotide.The expression of MSG UCS mRNA was lower in PC whith were transfected with pPC-UCS then that of were transfected with pTZU6+1,and the difference was statistically significant (P<0.05).The inhibition rate of UCS mRNA expression was 77.47%.48 hours after transfected pPC-UCS into PC , PC were observed by scanning electronmicroscope, we discovered PC were swollen and largen, some showing microvilli fall off,some showing membrane broken. The rats were injected with pPC-UCS by hydrodynamics transfection after to be infected with PC. After 48 hours,we found the number of cysts was lower than control group,the slip is 67.88%, the difference was statistically significant (P<0.05). Airsacculitis of rats was lighter than control group, the difference was statistically significant (P<0.05). PC were observed by scanning electronmicroscope, we discovered PC were swollen and largen, some showing microvilli fall off,some showing membrane broken.Conclusion: The siRNA plasmid, pPC-UCS, was constructed successfully. It could knock down the expression of UCS gene in PC in vitro , it could destroy and kill PC. It could destroy and kill PC in vivo, relieve inflammation of lung.