The Research Of Saccharomyces Cerevisiae As Difunctional Carrier Vaccine

Saccharomyces cerevisiae is a kind of eukaryote, which is safe non-toxic and has no pathogenicity. The condition needed to culture S. cerevisiae is simple. And S. cerevisiae has a clear genetic background, which makes it easy for genetic operation. S. cerevisiae is generally used as a research model to investigate eukaryotes. When the foreign genes expressed by S. cerevisiae, the protein can be processed, which make foreign protein have a certain degree of folding processing and glycosylation modification. Therefore, S. cerevisiae is suitable for the expression of eukaryotic gene and expressing proteins which have specific function.When we use the intracellular antigens expressed by S. cerevisiae as a vaccine to inoculate animals and human being with the method of subcutaneous injection, powerful antigen specific immune response can be observed. S. cerevisiae has a special kind of cytoderm structure, which makes S. cerevisiae have an intense resistance effect against hydrochloric acid in gastric juice from the digestive tract. The cytoderm of S. cerevisiae is a kind of natural biological capsule which can provide a good protection for biological macromolecules and S. cerevisiae cytoderm contains ingredients which can adjust the function of intestine and stomach. The granular structure of S. cerevisiae make it targeted and effective being uptaken by antigen-presenting cell. The structure and composition of S. cerevisiae cytoderm make it have strong immune adjuvant activity and upregulate three signals needed to activate the immune system. The vaccine system, which is based on intracellular antigen protein expressed by S. cerevisiae has a large potential and economic value due to its low production cost. But resulting from the difference in the nature of the purpose antigen, in many cases, the expression of intracellular antigen protein by S. cerevisiae, due to the reason that the modification is non-ideal compared with that they are supposed after translation and that the expression level is low, is limited.Yeast episomal plasmid is a kind of DNA fragments which is independent from the genome. Currently, the gene manipulation technology of yeast episomal plasmid is mature. In order to study yeast as a carrier in the nucleic acid vaccine and the function of the oral gene therapy, we need a shuttle carrier which can be replicated in yeast and expressed in mammalian cells. The CMV promoter can promote transcription in mammalian cells, which make the target protein expression in mammalian cells have proper folding and modification.In this research we do some experiments as follows:1, Use HA and eGFP as the target proteins, clone the coding sequence to CMV control gene expression, and make it exist in yeast episomal plasmid.2, Use the conventional lithium chloride method to translate yeast episomal plasmid into the cells of INVScl and the auxotrophic method to screen the transformants.3, Extract the genome DNA of the transformants, and through the PCR identification we screen the right transformants.4, Recombination yeast episomal plasmid transfect Hela by liposome mediatation, observe and detect its expression, to prove that the plasmid can promote and express the target protein in mammalian cells.5, Expand the culture of the transformants that carry the target gene, collect and broke up the S. cerevisiae cells. And then use western blotting to detect whether the protein is expressed in S. cerevisiae or not to verify whether CMV promoter start express in S. cerevisiae.6, Use the method of cocultivation, in which the phagocytes of mice named RAW1phagocytose S. cerevisiae transformants, to observe and detect the target protein. So that we can verify whether the purpose gene express or not post-phagocytosed by macrophages.7, Inoculate the mice subcutaneously, after a period of time collect the blood from the orbit, use ELISA to detect whether the antibody express or not, to verify its immune effect.8, Expose the mice to lethal doses of the H1N1virus. And then observe and take a record of the number of the mice who are still alive with the passage of time, so that we can verify the immune effect from the number of the mice alive.In this experiment we screen S. cerevisiae transformants which carry the stable episomal plasmid containing target gene, and turn out that episomal plasmid containing purpose gene can be expressed in mammalian cells. By subcutaneous injection of inactivated S. cerevisiae transformants, we confirmed that it has a certain immune function in mice reaserch. The results from the reaserch can be a certain foundation for the nucleic acid vaccine using S. cerevisiae and oral vaccine research, and make a contribution to the oral vaccine research later. But from the point of immune test result, the immune effect is not satisfactory. The next step of the experiment, we will furtherly reconstruct the carrier to make S. cerevisiae can be cultured in a normal medium without missing the episomal plasmid containing target gene. In addition, we will try to raise the number of copies of episomal plasmid containing target gene. And we will also change the way of vaccination to make the immune effect more obvious. And in the end we will do some reaserch in the form of oral vaccine to test whether S. cerevisiae transformants can induce effective immune response or not.