Study On Microsporum Canis PQ-LRP Gene Silencing By RNA Interference Technology&1Case Of Facial Linear Lupus Erythematosus

Objective:We had got the the full sequence of membrane protein PQ-LRP gene by rapidamplification of cDNA ends (RACE).In this study,the gene RNA interference vectorof Microsporum canis PQ-LRP was constructed, in order to research the effects of thisRNA interference vector on the expression of Microsporum canis PQ-LRP gene. Wealso constructed the PQ-LRP gene RNA interference lines, to provide theory for laterto investigate the role of this gene in the pathogenesis, and to provide design target fordevelopment of new medicine.Method：1, Extracted the total RNA,and reverse transcript this to cDNA as the PCRtemplate. Amplified interference sequence of LPR gene with primers LPR-F and LPR-R,to get the length of600bp positive interference fragment, and the fragment wasconnected between the PUC-PUT vector Ptrpc promoter and interval sequence, so thatwe got obtain the intermediate vector PUC-PLUT.Also we used the primer LPR-F andLPR-R to amplify interference the sequence of LPR gene, to get the length of600bpreverse interference sequence, and it was connected between the PUC-PLUT vectorinterval sequence and Ttrpc terminator, to obtain the intermediate vectorPUC-PLULT,and it was connected to the PCB309, and finally we constructed aPCB309-PLULT interference vector.2, According to hygromycin concentration control method, to determine theoptimum concentration of selection of Microsporum canis transformants hygromycin. 3, Transformed the plasmid which containing the vector PCB309-PLULT intoAgrobacterium by electroporation and used the transformation system ofAgrobacterium to mediate the PQ-LRP gene silencing on Microsporum canis.4, Detected the effects of PCB309-PLULT vector on Microsporum canis PQ-LRPgene expression by the application of Real Time PCR method.Result:The intermediate vector PUC-PLUT, PUC-PLULT were constructed, and wereidentified by PCR, gene sequencing; the8825bp interference vector PCB309-PLULThad been built, and the objective vector was identified by enzyme assay; screening ofMicrosporum canis transformants hygromycin optimum concentration was300μ g/ml;identification of Microsporum canis interference by using real-time quantitative PCRmethod: with undisturbed PQ-LRP relative expression level of1.0standard,interference PQ-LRP relative expression level of0.39, interference by61%.Conclusion:The expression of PQ-LRP gene in Microsporum canis was inhibited byPCB309-PLULT interference vector,this contribute to the study on pathogenesis ofmembran protein PQ-LRP gene in Microsporum canis.