Secretive Expression,Purification And Charactriation Of Toxoplasma Gondii Multi-Antigenic Gene Encoding P30 And CTA2/B In Pichia Pastoris

Toxoplasma gondii (T. gondii) is a harmful intracellar protozoa, which can cause a kind of worldwide , severe parasitic diseases named Toxoplasmosis. T. gondii occurs in almost all mammals, including human beings. The prevalence rate among the population is high range from 25% to 50%. Congenital toxoplasmosis is that toxoplasma crosses the placental barrier from the mother's blood, which can cause abortions, teratosis and stillbirths. Toxoplasma is an opportunistic protozoan. In immunosuppressed and immunodeficient individuals, including patients with organ transplantation, malignant tumor, acquired immune deficiency syndrome (AIDS), toxoplasma can cause them to death. Toxoplasma is also capable of causing severe disease in animals so it could destroy farming badly. So far we do not have any methods effectively to cure the disease, the prevention of toxoplasmosis is very important. The manufacture of Toxoplasma gondii'svaccine becomes urgent and a common direction among the researches.P30 is the main surface antigen of the tachyzoite. It is highly immunogenic andcould induce IgG, IgM, IgA, antibodies and CD8+T cell. It is regarded as one of the diagnostic antigens and candidated molecular vaccine to preventing Toxoplasma gondii infection. In addition to Toxoplasma gondii invades into the body though the mucous membrane, nontoxic CTX A2/B was used as an adjuvant in order to enhance the immunogenicity of p30, which play a important role in Toxoplasma gondii's vaccine. Though both home and aboard have expressed p30 and CTX in various system, the effect of the protein in animals and human beings can not cater to the requirement.It is well known that genes of eukaryotic cells can be transcripted and translated in prokaryotic cells in a simple and high productive way. But the prokaryotic cells cannot produce proteins with natural configuration and activity, due to its incompetence of accurate post-translation processings. Although the cell of mammals can express the protein with activity, the product is low in does, and it can not suit to get a great deal of protein required.Yeast pichia pastores expression system was used in the experiment. It was applied popular for the system both have the characters of prokaryotic cells and eukaryotic cells> simplicity in operation n fast in growth and has competence of accurate post-translation processings. Furthermore it also has the character of productivity. The expressing vector used in experiment is pGAPZ a A has a a signal peptide, which can make the product secrete from the cells simpJicizing the purification.The compound gene P30-CTXA2/B, cloning plasmid pMD]8-P30-CTXA2/B and pichia pastores expression piasmid pGAP-P30-CTXA2/B were successfully constructed by means of molecular biological clone-oriented method and gene combination technique. Then the recombinant pichia pastores expression plasmid pGAP-P30-CTXA2/B transfected Yeast pichia pastores cells GS115. Meanwhile, fusion protein of compound gene P30-CTXA2/B was successfully expressed in Yeast pichia pastores cells, which was detected by running SDS-PAGE, showing a specific protein band and was further confirmed by Western Blotting, showing that the specific fusion protein containing P30 immunological activity. And the protein expressed has the characters of high productiveity and purification.The successful expression of P30-CTX A2/B fusion protein in Yeast pichia pastores expression system plays a basic role in the aspect of diagnosis and the research in vaccine at the level of gene and protein. And it also paves way for further study on animals.