Optimization of gene delivery using laser and nanomaterials : In vitro study /
دراسة معملية لتحسين النقل الجينى باستخدام الليزر والمواد النانوية
Marina Atef George Adeeb , Tareq Youssef , Salwa Farouk Sabet ,Taher Ahmed Salaheldin,
- Cairo : Marina Atef George Adeeb , 2016
- 89 P. : charts , facsimiles ; 25cm
Thesis (M.Sc.) - Cairo University - National Institute of Laser Enhanced Sciences - Department of Laser Applications in Environmental Metrology, Photochemistry and Agriculture
The present work aimed to prepare stable gelatin nanoparticles conjugated with the nonstructural protein 2 (NS2) gene of hepatitis C virus (HCV) genotype 4a as an extra efficient method of vaccine delivery where nucleic acids can be loaded onto GNPs through electrostatic attraction. We synthesized GNPs by two step desolvation method and resulted were found to be of spherical shape, of size around 150±2nm and with zeta potential of +17.6 mv. We amplified NS2 gene form HCV genome (strain ED43), then cloned and expressed it in E.coli M15 using pQE-30 vector. Antigenicity of the recombinant NS2 protein was confirmed by Western blotting to verify the efficiency of NS2 as a possible vaccine before conjugation with GNPs. We successfully conjugated NS2 gene and GNPs and that was confirmed by confocal laser scanning microscope. We then investigated effects of conjugated GNPs/NS2 complex on transformation in E.coli DH5Ü. According to many trials, we found that the conjugated GNPs/NS2 with ratio (2:1) respectively was transformed into E.coli DH5Ü more efficiently as gene delivery system than NS2 transformation alone or other prepared ratios (1:2, 1:1). We confirmed the complex delivery using confocal laser scanning microscope excited by three lines of laser 633nm, 405nm and 514nm.Moreover, we concluded that GNPs didnt affect the properties of NS2 gene verified using gel electrophoresis. This declared that we could succefully deliver NS2 gene using GNPs as first step for Vaccine development against HCV