The GFP Fluorescence Expression Of Transfection Of Different Kinds Nanomaterials And The Construction Of Eukaryotic Expression Recombinant Plasmid Of TB Protective Antigens Gene

Objective: With the dramatic development and interdiscipline of nanotechnology andbiomedical technology, the use of the inorganic nanomaterials in medical domain provide a newway for the treatment of disease, vaccine design and early diagnosis. Global TB epidemic isincreasing severe since1980s. A new effective tuberculosis vaccine and diagnostic reagents areneeded urgently. The study of using nanomaterials into tuberculosis vaccine could make somebreakthroughs. In this study, the researchers used three inorganic nanometer materials(such asSiO2, ZnO nanotube and nanorod) to investigate their cytotoxicity and the influence on genetransfection efficiency, besides, the construction of eukaryotic expression recombinant plasmidof TB protective antigens gene(pEGFP-N1-TB10.4) have laid a foundation for the researchwhich combine TB vaccine with nanomaterials.Method: Researchers respectively infected THP-1and RAW267.4with three kinds ofnanometer materials to determine the best transfection concentration which did not affect thecellular activity seriously, and then transfected eukaryotic expression vector GFP intoRAW267.4cells. Then researchers got the transfection cell RAW267.4, observed theirexpression of green fluorescent protein under fluorescence microscope. The influence ofnanometer materials on the GFP gene transfection efficiency was evaluated by comparing withthe transfection efficiency of liposomal and naked plasmid. At last, using molecular biologytechniques cloned the TB10.4gene to connect with the eukaryotic expression vector pEGFP-N1,and got the recombinant plasmid pEGFP-N1-TB10.4.Result:1. Then researchers determine the best transfection concentration which did notaffect the cellular activity seriously, the best effection concentration of nanometer SiO2is50μg/mL. Both the best effection concentration of ZnO nanorods and nanotube are10μg/mL.2. The group of Nanomaterials transfection is observed the expression of green fluorescentprotein after transfected for24h and48h, and expression percentage is5%, significantly lowerthan Liposome transfection group.3. Successfully cloned the gene of mycobacterium tuberculosis protective antigen TB10.4.Got the recombinant plasmid pEGFP-N1-TB10.4successfully.Conclusion:1. The cytotoxicity of inorganic nano-particles is detected to rise as theincreasing of concentration. It is also concerned with some physicochemical properties like size,shape, dispersibility and surface modifications of nano-particles. 2. The three kinds of nanometer materials could mediate gene transfection cells. But thetransfection efficiency is lower than it of the Liposome transfection.3. Successfully get recombinant plasmid pEGFP-N1-TB10.4.