Fluorescence Labeling,Targeted Modification And Assembly Of The Coat Protein Of Red Clover Necrotic Mosaic Virus

In recent years,with the deepening of precision medicine and precision therapy research,a series of more targeted molecular drugs have been developed.Plant viruses have been reported as novel targeted drug delivery systems and due to its small toxic and side effects,large drug load and it can be combined with CRISPR/Cas9 system to precisely edit relevant pathogenic genes of human beings and related genes of microorganisms so that more accurate and thorough treatment of various human diseases can be achieved.Therefore,the research on shell protein of plant viruses as the carrier of targeted agents has been widely concerned in the medical field,and has become a new hot spot in the research of drug dosage forms for the treatment of various diseases.In this study,the coat protein of RCNMV was used as the research object.The prokaryotic expression vector pET-21 a containing mScarlet/GFP and CP gene sequences was constructed to achieve the purpose of CD46-mScarlet/GFP fluorescent labeling and targeted modification of CP.It is expressed and purified,and assembled with the already expressed CP subunit into a viral coat protein of RCNMV containing CD46-mScarlet/GFP label,which provides a trace basis for targeted drug delivery system.The research content and results are as follows:1.Fluorescent labeling and targeted modification of the coat protein of red clover necrotic mosaic virusThe GFP and mScarlet gene sequences were searched on NCBI,and the primer sequence was designed using Premier 5.0 with GFP and mScarlet gene sequences as a template.And the restriction endonuclease site Eco RI and a homologous arm sequence corresponding to the pET-21a-His-CP-MBP expression vector restriction site were added at the 5’end.The mScarlet,GFP gene and pET-21a-His-CP-MBP vector plasmid were ligated seamlessly to construct a recombinant plasmid expression vector pET-21a-His-CP-mScarlet/GFP-CD46-MBP.2.Expression,purification and identification of CP-mScarlet/GFP-MBP fusion proteinThe pET-21a-His-CP-mScarlet/GFP-CD46-MBP expression vector was transformed into E.coli BL21(DE3)p Lys S,and expressed under the induction of IPTG,and the expression conditions were optimized to obtain a large amount of fusion protein.And it was detected by SDS-PAGE gel electrophoresis.The results indicated that the CP-mScarlet/GFP-CD46-MBP fusion protein was mostly present in a soluble form and was approximately 114 k Da in size.The expressed cells were irradiated with an ultraviolet lamp,and the cells showed green and red fluorescence,which confirmed that the fusion protein was soluble.3.Modification of fluorescence labeling targeted modification vectorsThe mScarlet/GFP-MBP gene and the CP-MBP gene were excised using the cleavage sites on the expression vectors pET-21a-His-CP-mScarlet/GFP-CD46-MBP and pET-21a-His-CP-MBP.The mScarlet/GFP-CD46 and CP genes were ligated into an expression vector by genetic engineering to construct the final recombinant plasmid expression vectors pET-21a-His-CP-mScarlet/GFP-CD46 and pET-21a-His-CP-CD46.4.Assembly,collection and electron microscopic detection of RCNMV coat proteinThe pET-21a-His-CP-mScarlet/GFP-CD46 and pET-21a-His-CP-CD46 recombinant expression vectors were transformed into E.coli BL21(DE3)p Lys S respectively.And CPmScarlet/GFP-CD46 and CP-CD46 were expressed and assembled to RCNMV protein shell under IPTG induction.The cells after induction of expression were collected,subjected to low temperature and high pressure disruption,linear gradient centrifugation of sucrose,and virion shell were assembled by pasteurization.The targeted modified RCNMV virus particles were detected by transmission electron microscopy,which proved that CP-CD46 completed the assembly of virions in E.coli.The targeted modification and fluorescent labeling of CP-mScarlet/GFP-CD46 failed to complete the assembly.