Study On Construction, Identification And Function Of RNA Interference Lentivirus Of Rat UCHL1 Gene

ObjectiveThis study was to design,construct and screen RNAi lentiviral expression vector of UCHL1,and to investigate its influence on the expression level of UCHL1 and the survival and proliferation of neuron.Materials and Methods1.Plasmid,pWPXL-MOD,was used to construct the recombinate expression vector of UCHL1 and to acquire identified positive clone.2.UCHL1 was designed to be consist of three specific siRNA target sequence by miRNA devising means,and Negative sequence as controls to synthesize oligonucleotides.3.Using muco-terminal linear plasmid pRNAT-U6.2 the siRNA vector of UCHL1 was constructed.Respectively,three DNA fragments of duo-muco UCHL1 target clips and one of independent control clips were combined with pRNAT-U6.2 to transform competent cells.Positive recons were acquired by PCR,naming pRNAT-U6.2-shRNA1,pRNAT-U6.2-shRNA2, pRNAT-U6.2-shRNA3 and pRNAT-U6.2-NEG correspondingly.4.293FT cells were cultured,and after the number of those researching 5×10⁶ transfected and cotransfected with pWPXL-MOD-UCHL1, pRNAT-U6.2-shRNA1,pRNAT-U6.2-shRNA2,pRNAT-U6.2-shRNA3, pRNAT-U6.2-NEG and plasmid mixture,VIRAPOWER^TMPACKAGING MIX.And naming Lv-UCHL1,Lv-shRNA1,Lv-shRNA2,Lv-shRNA3, Lv-shNEG correspondingly.Blasticidin assayed the concentration of purified viruses.5.The protein expression of UCHL1 in 293FT cell was analyzed by Western-blot after transfected with Lv-UCHL1,Lv-shRNA1,Lv-shRNA2, Lv-shRNA3,Lv-shNEG.The most inhibited lentivirus was chose.6.The experimental model was standardized by the assessment of morphology, NSE and Hochest33258 fluorescence dyeing of cerebral neuron in rats7.The infection rate of lentivirus was determined by fluorescence,and interfering effection by the expression of UCHL1 from RT-PCR and Western-blot.8.Biological behavior of neuron transinfected was observed,and cell survival and proliferation were assessed by MTT,apoptosis was assessed by flow cytometer and Annexin V.Result1.The recombinate plasmid of UCHL1 was constructed successfully,and positive clone acquired and identified.2.The vector of lentivirus was designed and constructed effectively.3.UCHL1 recombinate lentivirus producing from 293FT cells was assayed.4.The most inhibited virus was screen out by UCHL1 RNAi recombinate lentivirus transfecting 293FT cells.5.Stable experimental model was established on the basis of separation and culture of cerebral neuron in rat.6.The expression of UCHL1 mRNA and protein decreased significantly after RNAi lentivirus transinfecting neuron in cerebral cortex.7.The apoptotic proportion increased significantly as soon as RNAi lentivirus inhibited.ConclusionLentivirus not only silent specific gene expression but also exert its influence on gene function research.The established stable experimental model of cortex neuron culture in vitro provided basic information for further investigation of gene regulation by RNA interference.UCHL1,highly specifically distributing in neuron,is a pivot enzyme in the degradation of ubiquitn protein,and RNAi lentivirus transfection shows a stable,effective,especial inhibition of its expression in both mRNA and protein leve.post transcription gene silencing of UCHL1 could influence neuron survival or apoptosis.