Construction Of Chicken Complement C3d And Newcastle Disease Virus OptiF Eukaryotic Expression Vector And Preparation Of Chitosan Nanoparticles

Newcastle disease (ND) is highly contagious and lethal disease, economically important viral disease affecting most of avian species worldwide, which is caused by Newcastle disease virus (NDV).It was listed into the group diseases by Office International des Epizooties (OIE). At present, inactivated vaccine and attenuated vaccine are mainly used in the market. Although inactivated vaccine antibody levels persisted for longer periods of time, it need to add adjuvants. The attenuated vaccine may be interfered by maternal antibodies and has reversed its virulence, which suggests a hidden danger in the vaccination. With the development of recombinant DNA technique, DNA vaccine gradually becomes a hotspot about the research of NDV vaccine. DNA vaccines do not only provide the advantage of simple and cost efficient manufacturing, but also the possibility to generate custom vaccines for different regions by creating complex mixtures of plasmids picked from a collection of DNA vaccines encoding various antigens or variants. Because poor cellular uptake and a rapid in vivo degradation,DNA vaccine adoes not induce high titers antibodies.At present, enhancing the efficacy of DNA vaccines is A critical initial step in DNA vaccination.A series of measures, such as vector optimization, molecular adjuvant and gene modification were studied. The interaction of the C3d fragment, part of C3, with its receptor(CR2or CD21) on B cells and/or follicular dendritic cells (FDCs) is key for the induction and maintenance of a normal humoral immune response.The system respiratory tractl and digestive tract infected by NDV. Mucosal immune system plays an important role in the pathogenesis and induce both mucosal immune and humeral cellular immune response. Chitosan is permanently positively charged, imparting the ability to condense negatively charged DNA through electrostatic. So,chitosan is effective delivery systems,which is required to deliver DNA to antigen-presenting cells and to specific organs and increase cellular uptake.It prevents rapid in vivo degradation of DNA. DNA-chitosan complexes are generally stable and are amenable to longterm storage.Additionally, chitosan is biocompatible and biodegradable.In this study, NDV-F of F48E9was coloned into pVAX I. the cDNA of C3d was amplified by RT-PCR (reverse transcription polymerase chain reaction). The C3d fragments were directly inserted into pUC18vector. pUC18-C3d.n (n=1、2、4、6) were constructed using the ligation of Xho I recognition site to the Sal I recognition site.After that, The gene fragment encoding the C3d.n gene, Which were designed by EcoR I and Xba I, were inserted into pVAX I-F (o).In addition, Chitosan microspheres containing pVAX I-F (o)-C3d were prepared by using a complex coacervation process.The experiment results illustrated that pVAX I-F (o)-C3d-NPs were spherical shape with a regular surface, fine dispersibility. The size values of microspheres was237.5nm with polydispersity index0.241.The zeta potential values of microspheres was36.4mv. The encapsulating rate was92.1±0.03)%(n=5) and the loading efficiency was (36.12±0.19)%(n=5).This reseach improve NDV-DN A vaccine application in poultry industry and defense the prevalence of NDV. It possessed significance theoretical and application values and has potential prospect.