Bioinformatics Analysis、prokaryotic Expression And Histological Localization Of Toxoplasma Gondii ROP7

Toxoplasmosis is a worldwide epidemic diseases suffering from people and animals. Toxoplasmosis can cause serious diseases such as the nervous system disease, eye disease and neonatal systemic disease, especially in people with weakened immune systems. However, in immunocompetent adults, infection is usually latent and has no obvious symptoms. When the immune system was damaged, latent infection with Toxoplasma gondii can transform into acute infections, causing extensive tissue damage and severe disease. Toxoplasmosis can also cause death in patients with AIDS. Toxoplasma is not only harmful to human health, but also seriously damage the development of livestock husbandry, causing serious economic losses. Currently it has no effective drugs and vaccines which can be actually commercialized and applied on toxoplasmosis. Thus, the direction of current research is to develop an effective vaccine for toxoplasmosis.Toxoplasma top complex –rhoptry, plays an important role in the invasion of host cells and the infection of organism. ROP7 is secreted by rhoptry and is related to the invasion of host cells of Toxoplasma gondii. But the topology of the parasitophorous vacuole membrane has not been determined, and the epitope of ROP7 or whether it has the immune protection for host is unclear.(1) In this study, physicochemical properties, soluble properties, secondary structure, epitope and whether it contains a signal peptide and transmembrane domains of Toxoplasma rhoptry protein ROP7 were predicted by using bioinformatics, the results showed that the full length of Toxoplasma rhoptry protein ROP7 gene was 1728 bp, encoding 575 amino acids, Nterminal 1-33 amino acids with a signal peptide sequence, and 12-34 amino acids of a transmembrane domain in ROP7, but it has a plurality of post-translational modification sites, multiple epitopes. These results theoretically supported that Toxoplasma rhoptry protein ROP7 has the potential of immunogenicity, and has a potential of becoming vaccine candidate gene.(2) According to the online prediction results of bioinformatics software, the signal peptide parts of rhoptry protein ROP7 gene of Toxoplasma gondii RH strain were removed, PCR replicated 561 bp which was Intercepted from the remaining sequence, the amplified fragment was recycled and purified, then cloned it into pMD18-T vector. The recombinant plasmid pMD18-T-ROP7 was digested by NcoⅠand NotⅠand inserted into pET-28 a prokaryotic expression vector, sequence analysis results showed the Recombinant plasmids were correctlytransferred into DE3 competent cells. Inducing by IPTG, ROP7 recombinant proteins was analyzed by SDS-PAGE and Western blot. Recombinant protein ROP7 was expressed successfully, sizing about 22 kDa. NI-NTA purified and concentrated a concentration of 0.3mg/mL ROP7 by PEG 20000 assay after dialysis.(3) Use mice immunized with purified protein to prepare murine polyclonal antibody Tg2ROP7. ELISA detective titer was 1:6400,And successfully applied in Toxoplasma gondii tachyzoites Tg2ROP7 partial top immunofluorescence experiment.This study not only proved that Toxoplasma rhoptry ROP7 genes have antigenicity in theory and have feasibility as toxoplasmosis DNA vaccine in technically, but bioinformatics technology was also applied to predict the Toxoplasma protein, laying the foundation for future research in terms of immunogenic antigen of Toxoplasma gondii.