The Research About Saccharomyces Cerevisiae-vectered APCs-targeted Nucleic Acid Vaccine

The use of Saccharomyces cerevisiae expressing antigen protein intracellularly to inoculate animals and human being through subcutaneous injection gained robust antigen-specific immune responses. The genome sequencing of Saccharomyces cerevisiae had completed. The genetic backgrownd of Saccharomyces cerevisiae is clear, and the the techniques for gene engineering of Saccharomyces cerevisiae is advanced. The safety of Saccharomyces cerevisiae is recognized, and there is no endotoxin. The antigen presentation cells can uptake the anitgen-carrying Saccharomyces cerevisiae exclusively and efficiently because of the particle structure of Saccharomyces cerevisiae. The structure and component of Saccharomyces cerevisiae cell wall can work as immune adjuvant powerfully, to upregulate three signals needed to activate the immune system, of antigen presentation cells. The vaccine system, based on Saccharomyces cerevisiae expressing antigen protein intracellularly, is very potential of application and benefits, because of low production cost, especialy for animal use. Antigen proteins expressed in Saccharomyces cerevisiae, however, because of the nature of the proteins, are often limited in some cases, in the manner of undesirable post-translation modifications and low quantities of production.Saccharomyces cerevisiae has the muticopy episomal plasmid, which has been genetically manipulated normally. A research paper published last year reported that a Saccharomyces cerevisiae muticopy episomal plasmid was engineered to replace the promoter, muticlonal sites and terminator by the relavant units of a mammal expression vector, to integrate a sequence of eGFP, and the cells harbouring the plasmids captured by in vitro macrophage were able to dilivry functional plasmids to express the eGFP proteins. This system, replicating plasmids in Saccharomyces cerevisiae cells and expressing the antigen proteins of interest in mammal antigen presentation cells, is more universive of application, in contrast to expression of antigen of interest in Saccharomyces cerevisiae cells as vaccine, especially when the antigen protein is toxic to the expression host.In this research, the CMV promoter, muticlonal sites and BGH terminator of pcDNA3.1-GFP plasmid is amplified by PCR, and cloned into pYES2/NT A plasmid, the promoter, muticlonal sites and terminator of which is deleted through restriction enzymes. The plasmid pcDNA3.1-HA and the final construct are digested with the same tow restriction enzymes and the HA coding sequence is cloned into the vector.In the next step of the experiment, the immune efficiency will be tested through animal protection experiment.The episomal plasmid and the Saccharomyces cerevisiae strain will be reengineered to make the Saccharomyces cerevisiae to grow with high cell density and high plasmid stability in nutrition-rich medium without itself2micron plasmid.