| Cell signal transduction networks control various basic physiological processes in cells,regulate cell behavior,and determine cell fate.The development o f regulatory strategies for cell signal transduction is an important entry point for realizing user-defined cell behavior.In current research,the regulation strategies rely on the modification of cell receptors,and most of them are based on recoding or modifying of cell surface receptors.When there are signal molecules in the environment,the modified receptor undergoes a ligand-mediated activation response,which generates downstream signals through specific cell signal transduction pathways,and ultimately regulates and controls cell behavior.There are two types of methods for engineering cell receptors.Genetic modification can expand the functions of receptor proteins.However,there are some problems which cannot be ignored:genetic engineering is cumbersome and complicated,and it is difficult to precisely control the advanced structure of proteins,and to construct a stable responding module.DNA-based non-genetic modification of cells is relative simple and powerful with high biologically safety.However,the current methods all rely on the specific binding between aptamers and receptors,which are limited by binding affinity,types,expression levels of receptors,and so on.Hence,it is necessary to develop noval non-genetic methods for regulation of cell signal transduction,and further cell behaviors.In this paper,a lipid vesicle based on protein-nucleic acid chimera（protein-DNA chimera vesicle,PDV）was constructed,which performed non-genetic modification of cell through fusion,and anchored functional molecules on cell membrane.A preliminary expl oration of the artificial cell signal transduction regulation was carried out.The fusion between PDV and cells is cell type un-limited.The protein-nucleic acid chimera was formed through covalent connection,which not only contains the multifunctional characteristics of protein but also the advantages of DNA programming.Therefore,it can expand the function of PDV through rational design.Dynamic DNA strand displacement was introduced as the signal recognition,and the fluorescence signal translocation f rom cell membrane to cytoplasm or nucleus was used as the signal readout.The specific research content of the thesis is as follows:1.In vitro expression and property analysis of the fusion proteins EGFP-DCVm and NLS-EGFP-DCVm.Based on the preliminary work of our group,fusion protein EGFP-DCVm was constructed,expressed,and purified in vitro to provide a tool basis for the subsequent research.Duck circovirus protein（duck circovirus,DCV）has unique activities as both restriction endonuclease and ligase.DNA with specific sequence can be covalently connected with DCV to form a stable protein nucleic acid chimera.EGFP（enhanced green fluorescent protein）is widely used as a fluorescent reporter.NLS（nuclear localization sequence）is a functional peptide that can mediate protein transport into nucleus.In order to expand the protein function,we constructed the fusion protein NLS-EGFP-DCVm.To improve the expression level and purify the fusion protein,the conditions for inducing expression of NLS-EGFP-DCVm in vitro were explored.The two fusion proteins both（1）have the optical properties of EGFP,and（2）retain the activity of covalent connecting to the specific DNA sequence.Under the optimized conditions,we found that the connecting efficiency was over 80%.2.Construction of PDV based on protein nucleic acid chimera and performance optimization.We first verified the assembly of the protein nucleic acid chimera A-D-protein EGFP-DCVm by gel electrophoresis analysis.Two ss DNA A and D formed a partially complementary ds DNA,where D strand contains the DCVm recognition sequence and can covalently connected with the protein EGFP-DCVm to form a chimera,the 5’end of A strand was modified with a lipid-affinity cholesterol,and then the chimera could be inserted into the lipid membrane during the synthesis process.PDV was constructed by classical hydration method.The diameter of PDV was about200 nm,as detected by DLS analysis.Using EGFP as the fluorescent label,fluorescence imaging was carried out,and it was found that green fluorescence was evenly dispersed on the cell membrane,suggesting that the vesicle can fuse with the cell,and the protein-nucleic acid chimera could be anchored in the cell membrane.Subsequently,we optimized the PDV concentration and reaction time during the fusion process,and verified that PDV could fuse with diff erent types of cells.The protein-nucleic acid chimera remained stable on the cell membrane for a long time（10 hours）.There was no crosstalk between fusion cells.High biocompatibility of PDV was proven by cytotoxicity verification.3.Preliminary exploration of the function of the intracellular artificial signal responding module.Here,DNA strand displacement strategy was introduced as the signal recognition strategy to preliminarily explore the function of the signal response module.PDV1 was prepared based on the chimera A-D-protein EGFP-DCVm,and PDV2was prepared based on the chimera A-D-protein NLS-EGFP-DCVm.The artificial signal chain Target was input into the fusion cell by Lipo8000,which underwent a toehold-mediated strand displacement with the nucleic acid part of the chimera,and released chimera from the membrane.Therefore,a visualized intracellular translocation of the fluorescent signal was observed,as the cell responded to the input artificial signal.We first verified that the DNA stran d displacement could happen in vitro by gel electrophoresis analysis,and then optimized the concentration and the time of the Target chain delivery.Finally,two kinds of PDV transformed cells responding to the signal were observed through cell imaging.D epending on the protein,the signal response of the cell could be shown as the translocation of the fluorescence from cell membrane to cytoplasm（regulated by PDV1）,or from cell membrane to nucleus（regulated by PDV2）. |
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