The Construction Of Three Forms Recombinant Plasmid Of Toxoplasma Gondii P30 And Preliminary Estimation On The Mice Immunized Genetically With The P30 Constructs

Background: Toxoplasma gondii is an obligate intracellular protozoan which cause toxoplasmosis of human andlor domestic livestock. It distributes worldly. Almost one third adults in the world are infected with Toxoplasma gondii.If primary infection occurs during pregnancy, transplacental transmission can lead to abortion or neonatal malformations. In immunocompromised hosts such as tumor and AIDS patients, Toxoplasma gondii is recognized as one of major lethal pathogen which serve as a opportunistic pathogen. Thus there is an urgent need for the development of an effective vaccine against Toxoplasma gondii. It has been shown that P30, the maj or surface antigen of Toxoplasma gondii is a highly immunogenic protein and an appropriate antigen for developmerit into a vaccine. Since 1990抯, the DNA-based vaccine technology has been applicated abroadly and promptly due to its several potential advantages compared with the conventional vaccines. Some progresses have been obtained in DNA vaccine of Toxoplasma gondii. Genetic immunization with recombinant plasmid encoding P30 antigen of Toxoplasma gondii has been proven to be able to elicit the immune responses and to protect from the pathogens. However, the efficacy of different forms of P30 constructs is still not clear. Aims: to screen a better nucleic acid vaccine which can induce a protective immune response to Toxoplasma gondii by immunization mice with plasmid DNA encoding three different forms of P30 antigen (secretory, membrane and intracellular. Method: 1 Construction of recombinant plasmid encoding membrane P30 gene: Two primers were designed and synthesized to amplified the whole P30 encoding sequence(include signal peptide and hydrophobic tail) from the Toxoplasma gondii ZS 1 strain genomic DNA. Then the amplified DNA fragment was cloned into eukaryotic espression plasmid pcDNA3 to construct pcDNA3-P3 0Mb. 2 Construction of recombinant plasmid encoding secretory P30 gene: the amplified DNA fragment of PCR was purified and subsequently digested with PstI. The truncated P30 gene without C-terminal hydrophobic tail was cloned into the plasmid pcDNA3 to construct pcDNA3-P3OSe. 6 3 Construction of recombinant plasmid encoding intracellular P30 gene: The recombinant plasmid pBV22O-P30Jn was digested with EcoRI and BamHI to produce the small P30 fragment. Then the recovered P30 fragment was subcloned into pcDNA3 to construct pcDNA3-P301n. 4 The mice was immunized by intramuscular injection with different form recombinant plasmid into the quadriceps(the ratio of DNA:lipofectin was 5:2, Suglmice).Mice were boosted two weeks later.Before the injection and after the boosted injection 2, 4 and 6 weeks , bleed the mice from the tail to detect IgG antibody by ELISA and Western blot. 5 P30 protein was expressed in E.coli and purified to detect IgG antibody.The conditions of purification were optimized. Results: 1 Identification by restrictive enzyme digetion and sequencing showed that the insert was surely the encoding gene of different form of P30 protein. 2 IgG antibody was detected by western blot and ELJSA: After 2 weeks of immunization, a weak active band occurred in membrane and secretory groups while intracellular group not and the colour of the secretory group was darker. After 4 and 6 weeks of immunization, there was a clear band in three immune groups and no remarkable difference wa...