The Building Of Tandem GFP-mRFP Fluorescent Reporter Labeled Lysosome Membrane Protein Vector-PLamp2c-EGFP-mRFP

Object Lysosomal cell death is triggered by lysosomal membrane permeabilization(LMP)and subsequent release of lysosomal hydrolases from the lysosomal lumen into the cytosol,at same time,various protease and signaling pathways are involved into the process.However,the mutation of lysosomes ensure cancer cell having the ability to maintain the lysosome membrane stability to avoid the occurrence of LMP.If there existed some drugs inducing LMP,it may be effective drugs for the treatment of tumor.Because the dissolve of acid enzyme in lysosome,p H is about 5,the LMP can result in acidification around the lysosome membrane.As we all known,the difference of p Ka exists between enhanced green fluorescent protein(EGFP)and monomeric red fluorescent protein(m RFP),and the m RFP is more suitable for p H value in the lysosomal cavities.EGFP fluorescence was attenuated by acidic conditions and degraded by lysosomal hydrolases,whereas m RFP fluorescence was relatively stable.We utilized this finding to assess the ability of novel drugs to induce tumor cell apoptosis by the way of LMP.Thus,the targeted LMP could avoid the disadvantages of single targeted treatment therapy and may be a new future research direction of the tumor treatment.For the purpose of construction and identification of eukaryotic expression vector of Lysosomal membrane proteins(Lamp2c)which have be marked by tandem EGFP-m RFP fluorescent reporter and assessment of vector expression in prostate carcinoma PC3 cells,meanwhile,we investigate the positioning of fluorescent carrier p Lamp2c-EGFP-m RFP in the lysosomes.All of those would contribute to subsequent research about targeted drug therapy of LMP.Methods This research includes two parts: 1.Design and build the eukaryotic expression vector of Lysosomal membrane proteins(Lamp2c)which have be marked by tandem GFP-m RFP fluorescent reporter.Additionally,we appended Kozak sequence in front of the Lamp2 c and applied PCR,enzyme and sequencing technique to verify the Lamp2 c gene construction to enhance the expression of fluorescent proteins and targeted proteins.2.Assess the colocalization of fluorescent vector p Lamp2cEGFP-m RFP with the lysosomes by using Lyso Tracker? Blue DND-22 under a fluorescence laser scanning confocal microscope.Results 1.After application of PCR,enzyme and sequencing technique,the Lamp2 c gene construction was verified successful.2.By X-treme GENE HP DNA Transfection Reagent Transfection system,p Lamp2c-EGFP-m RFP was transfected into PC3 cell.EGFP and m RFP all expressed normally,while the red fluorescence was slightly weaker than green fluorescence.3.The positioning of blue fluorescence,red fluorescence and green fluorescent in the p Lamp2c-EGFP-m RFP-PC3 cells were colocalization after Using Lyso Tracker? Blue DND-22 under a fluorescence laser scanning confocal microscope.Conclusion The p Lamp2c-EGFP-m RFP eukaryotic expression vectors were constructed successfully.The expression of Lamp2 c,EGFP and m RFP proteins was stable in transfected PC3 cell line.The p Lamp2c-EGFP-m RFP displayed lysosome colocalization.Above all,the vector can be used to detecting the lysosome.Laying the foundation of researching the relationship between LMP and cancer treatment.