| The epidemic of COVID-19(Corona Virus Disease2019,COVID-19)has been causing serious loss of life,health and economy all over the world.So far,it has not been effectively controlled,the vaccine has become the last hope in the global fight against the COVID-19 epidemic.Since the outbreak of COVID-19,hundreds of vaccine research and development projects that can be divided into five main categories have been launched around the world,including inactivated vaccine,recombinant protein subunit vaccine,virus vector vaccine,DNA vector vaccine and mRNA vaccine.As of March2021,a total of 9 varieties have been urgently licensed in many countries.Remarkably,the mRNA vaccine was used afficially for the first time in the outbreak of COVID-19..Compared with other types of vaccines,mRNA vaccine has unique advantages of security and flexibility,especially it is easy to develop new vaccines against mutated virus strains.In addition,mRNA itself can be used as a natural immune adjuvant to improve the effectiveness of vaccination,the production process of mRNA vaccines does not rely on high-grade biosafety production facilities.The fast production speed and low production cost of the mRNA vaccine means it can better meet the urgent needs of the global epidemic for vaccine dose.In view of all these advantages,mRNA vaccine has attracted much attention.In November 2020,Moderna released clinical data showing that it has shown great anticancer potential in a variety of solid tumors in the development of tumor mRNA vaccine(mRNA-4157).The preparation of nano-nucleic acid carrier and the synthesis of mRNA in vitro are two key technologies for the research and development of mRNA vaccine.In this paper,we carry out the work to construct the preparation of nano-nucleic acid carrier and mRNA synthesis system in vitro.The main contents are as follows:Part one: preparation and characterization of PEI lipid nano-nucleic acid carriers.Based on the cationic polymer polyetherimide(Polyetherimide,PEI),a lipid nano-nucleic acid delivery carrier for DNA and mRNA was developed,and the preparation method of the carrier was optimized by characterization and analysis.The results showed that the prepared PEI lipid nano-nucleic acid vector had small particle size and good uniformity;CCK8 assay proved that the prepared PEI lipid nano-nucleic acid vector had low cytotoxicity;using the green fluorescent protein eukaryotic expression vector as the test gene,confocal fluorescence microscope analysis demontreted that the PEI lipid nano-nucleic acid vector had high transfection efficiency to the plasmid.Part two: in vitro synthesis of mRNA and construction of mRNA nanoparticles.Using green fluorescent protein GFP as the report gene,we successfully prepared and purified GFP-mRNA.Subsequently,PEI-GFP-mRNA lipid nanoparticles was prepared by encapsulating GFP-mRNA with lipid nano-nucleic acid carrier.Characterization analysis showed that the obtained nanoparticles have small particle size and excellent uniformity.After PEI-GFP-mRNA was transfected into Hela cells,green fluorescent protein was successfully expressed and in vivo experiments proved that PEI-GFP-mRNA lipid nanoparticles were low toxic and safe in vivo.Part ?: optimization and characterization of antibody modified liposomes.Firstly,blank liposomes were prepared by ethanol injection combined with extruder extrusion.Then anti-HER2 antibodies were used to screen and optimize the immunization methods mediated by DSPE-PEG2000-NHS or DSPE-PEG2000-Mal.Characterization analysis of prepared immune nanoparticles showed that the average particle size of the immunoliposomes was small and uniform,while the stability was also good.SDS-PAGE electrophoresis showed that the connection efficiency of DSPE-PEG2000-NHS liposome was 44.24% and 36.56%.The results of confocal microscopy and flow cytometry showed that the prepared immunoliposomes had low cytotoxicity.The results of confocal microscopy and flow cytometry showed that Anti-HER2 immunoliposomes had significant targeting specificity to HER2 positive cells. |
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