Construction Of The Recombinant Plasmid PAAV-ShRNA(BACE1)-NGF And Expression In BT325Cell

ObjectivesOn the basis of pre-construction of the recombinant expression vector of AAV-NGF-IRES-hrGFP,in this experiment we plan to use RNAi principle to construct the multi-functional plasmid AAV-U6-shRNA-CMV-NGF-IRES-hrGFP, which mediated NGF enhanced expression while specific targeting β-secretase (BACE1) expression of silence. And then the effective interference plasmid is screened in vitro to provide a theoretical basis for further in vivo study of NGF and BACE1as a target for gene therapy for Alzheimer’s disease (AD).Methods1. Construction of pAAV-U6-shRNA-CMV-IRES-hrGFP interference vectorAccording to the BACE1mRNA sequence which was found in NCBI and different interferences, three pairs of the siRNA oligonucleotide sequence were designed for the synthesis of the corresponding hairpin DNA fragment., with a pair of unrelated sequence as a negative control. Using gene cloning technology, we inserted it into the RNAi expression plasmids(pSilencer) to build the plasmid pSilencer-U6-shRNA. The U6-shRNA fragments was amplified by PCR and then cloned into pAAV- MCS-IRES-hrGFP to construct a recombinant pAAV-U6-shRNA-CMV-IRES-hrGFP, which was identified by restriction enzyme digestion and sequencing.2. Construction of pAAV-U6-shRNA-CMV-NGF-IRES-hrGFP multi-functional vectorThe NGF gene was amplified by PCR from the vector containing NGF, and inserted into pAAV-U6-shRNA-CMV-IRES-hrGFP using multiple cloning sites, to build pAAV-U6-shRNA-CMV-NGF-IRES-hrGFP recombinant plasmid, which was identified by restriction enzyme digestion and sequencing.3. Screening of cell line with endogenous expression of NFG and BACE1gene in vitroBT325and several other cells of neuron and pancreatic tumor were cultivated, followed by total RNA extraction and reverse transcriptase synthesis of cDNA, NGF of BACE1gene was amplified by PCR method to screen out the cell lines with endogenous expression of the NFG and the BACE1gene. pAAV-GFP plasmids were transfected into positive cells by calcium phosphate co-precipitation method, then cell lines with high transfection efficiency was screened measured by the expression efficiency of green fluorescence protein under fluorescence microscope.4. Screening of most effective and specific plasmids by the analysis of NFG and BACE1expression in transfected BT325cell The plasmids of different experimental groups are transfected into BT325cells by calcium phosphate coprecipitation method. Transfection efficiency of the cells were determined by observation of GFP expression under an inverted fluorescence microscope,followed by Real-time PCR, Western-Blot, and ELISA methods to analyse the suppressed expression of BACE1and the enhanced expression of NGF in gene and protein level to screen out the most effective and specific plasmids.Results1. The adeno-associated virus vector plasmid with shRNA effectively targeting BACE1gene was construted.2.The multi-functional recombinant adeno-associated virus vector plasmid which mediated enhanced expression of NGF and specifically targeted BACE1expression of silence. The BT325, SK-N-SH and SW1990cells were filter out to express pairs of NFG and BACE1genes endogenously. And pAAV could be transfected into BT325cells by calcium phosphate coprecipitation method efficiently.4. The interference effeciency of each group oh shRNA targeting BACE1gene was found not the same. The inhibitory effect of shRNA1and shRNA2was significantly better than shRNA3. Multi-functional plasmids were successfully transfected into BT325cells,resulting in shRNA synthesis by the method of expression in vivo, which could effectively inhibit the BACEl gene expression in cells, and not affect the enhanced NGF gene expression in these cells in the mean time.ConclusionsThe multi-functional recombinant adeno-associated virus vector plasmids were successfully constructed which were mediated NGF enhanced expression,suppressing endogenous BACEl expression specifically and GFP expression for showing efficiency of the transfection.The plasmids were transiently transfected into BT325cells by calcium phosphate precipitation method. And then the optimal plasmids were screened out, which mediated NGF enhanced expression in the BT325cell, and targeted BACE1silencing effectively and specifically in the mean time.