Expression In Vitro Of FMDV Vp1 And The Immune Effects Of Vp1 DNA Vaccine

Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofedanimals, including cattles, pigs and sheep. Current FMD vaccines are mainly based oninactivated virus, although effective, but outbreaks of FMD have been directly associated withincomplete inactivation of virus, and even some time contribution to the escape of virus fromvaccine manufacturing facilities. Alternative adjuvants or regimens have been investigated todevelop more safe and effective FMD vaccines. Our studies have been concentrating on utility ofthe FMDV VP1 protein as a model for a new FMDV vaccine approach, specially includes theeukaryotic expression in yeast, chemical adjuvants for DNA vaccines and prime –boost regimensfor enhancing immune effect of FMDV DNA vaccines. To express and identify bovine O type FMDV VP1 in yeast Pichia pastoris. FMDV vp1gene was cloned into secretary Pichia pastoris expression vector-pSuperY. After beinglinearized with enzyme digestion, the construct was transformed into Pichia pastoris SMD1168Hby electroporation and integrated into the genome in yeast. The transformant was screened byzeocin selection. Expressive VP1 protein in yeast were analyzed and identified by SDS-PAGEand Western blot and then were used to immuniz mice for its Immunogenicity. Therecombinant VP1 protein can elicit similar humoral and cellular immune response in mice to thatfrom the traditional FMDV killed vaccine. DNA delivery is an important technology in the study of expression and function of genes.Many laboratories are making great efforts to find a safe and high-efficient delivery vehicle. Theconstruct of pcDNA3/vp1 was transfected into eukaryotic cells in vitro with low molecularweigh of chitosan, a cational polymer designated as chitosan-16. After expressions of HPRT andvp1, the efficiencies of such facilitative transfection were determined by RT-PCR and real-timequantitative PCR technologies compared with transfected using the lipofectin and calciumphosphate methods. The results showed that higher expression of the vp1 at mRNA levelmediated with the chitosan-16/DNA was observed in COS-7 than that from the calciumphosphate and lipfectin. Formation of nano-particles of chitosan-16 with DNA was observedunder a scanning electron microscope and this particle complex may improve the stability ofDNA and the efficiency of transfection. It could be applicable to non-viral based delivery systemfor gene therapy as well as DNA vaccination applications. DNA vaccination is useful for generating immune responses, particularly the cell-mediated - 5 -新疆大学硕士论文immune response, in a wide variety of species. However, DNA vaccination generally inducesonly relatively weak responses; particularly in large animals and human. Although, variousapproaches have been developed recently in order to improve its efficacy or immunopotency,limited success has been achieved in a practical term. We have recently tested several chemicalsfor their usefulness as adjuvants in facilitating DNA vaccination. Of a total of five chemicalstested, levamisole exhibited strongest Th1 stimulatory activity whereas Tween80 showedweakest Th1 activity, as determined by IgG2a production, and saline formulation induced weakT cell proliferation, and DTH, in animals inoculated with a DNA construct expressing thefoot-mouth-disease viral capsule protein VP1. Furthermore, co-inoculation of levamisoleincreased the production of IFN-γ by more than 100-fold as compared to that by DNAinoculation formulated in saline. In contrast, a previously reported chemical adjuvant,bupivacaine, stimulated only modest levels of overall antibody production, with relatively lowlevel of Ig2a. These results demonstrate the usefulness of various chemicals, particularlylevamisole, for modulating the outcome of DNA vaccination, in both the intensity of the immuneresponse and the polarity of such respons...