Prokaryotic Expression And Immunogenicity Assay Of Dense Granule Protein GRA12 Of Toxoplasma Gondii

Toxoplasma gondii is an intracellular parasitic protozoan,which causes a wide range host of toxoplasmosis,including mammals and birds.Toxoplasmosis was reported all around the world,and about one third of people were infected with T.gondii.Although most of them are asymptomatic,some of them may suffer from the symptomatic pathological changes in the lymph nodes,eyes,and central nervous system.In addition,pregnant women with primary infection with T.gondii may transmit to the fetus leading to congenital disease.The immunocompromised individuals infected with T.gondii may develop a life-threatening disease.It is very common that the livestock infected with T.gondii,such as dogs,cats,pigs,cattle,sheep and others,and the infection rate is from 10% to 47.3%.The toxoplasmosis not only causes livestock abortion but also inhibites the development of animal husbandry.T.gondii is the main infection of human toxoplasmosis.Toxoplasmosis has become one of the important public health problems that need to be solved urgently in our country.However,there is still no ideal preventive vaccine and drugs against toxoplasmosis.Therefore,looking for effective vaccine candidate antigens or drug targets is key to the prevention and control toxoplasmosis.Dense granule protein GRA is the main components of excretory secretory antigens(ESA)in T.gondii,and it plays an important role not only in the protective immune response induced by the host,but also in modifying the parasitophorous vacuole membrane structure.It participated in the parasitophorous vacuole membrane network formation,and protected the survival and proliferation of T.gondii in host cells.GRA12 is a dense granule protein discovered recently.In this study,GRA12 was cloned and expressed and analyzed its immunogenicity,and the results showed that GRA12 is an important secretory protein and has potential immunogenicity.The details are as follows:The bioinformatics analysis of GRA12 gene By using bioinformatics softwares including GENE Runner,DNASTAR,DNAMAN and Expasy the sequence coding regions of GRA12 were analyzed.The physical and chemical properties,signal peptide,transmembrane domain,secondary structure,hydrophilicity/hydrophobicity and antigen epitopes of the gene encoding protein were predicted by those softwares.The results showed that the full-length of GRA12 was 1 311 bp,and it encoded 436 amino acids.The theoretical molecular weight was about 47.8 ku,no signal peptide,the protein was stable to hydrophilic amino acid,and there were 6 transmembrane regions and 15 potential antigenic tables.It is predicted that GRA12 has a good immunogenicity.The cloning,expression and purification of T.gondii GRA12 A pair of specific primers were designed by using the GRA12 sequence of Toxoplasma gondii RH strain published in GenBank,and the Sac?and Hind III cleavage sites were added to the upstream and downstream primer respectively.The total RNA of T.gondii was extracted and amplified by RT-PCR.The gene was connected to pMD18-T vector,and then identified by enzyme digestion and sequencing.The positive clone was obtained,the plasmid was extracted,and the target fragment was connected to pET-28a(+)after the enzyme digestion.Then the prokaryotic expression plasmid named pET-28a-GRA12 was constructed and transformed into E.coli(DE3).The expression was induced by IPTG,and the recombinant GRA12 protein was purified by Ni column affinity chromatography.The recombinant protin was determined by SDS-PAGE and Western blotting.The immunogenicity of GRA12 antigen in T.gondii Kunming mice were randomly divided into three groups: GRA12 recombinant protein immunized group,adjuvant control group and PBS control group.The blood samples and serum were collected at different time intervals after immunization,and then the content of immunoglobulin in serum was determined by ELISA.Collecting the spleen of mouse,and then The value of CD3,CD4 and CD8 in spleen lymphocytes of mice were detected by flow cytometry;The levels of cytokines IL-12 and TNF-? were detected by indirect ELISA.Finally,the immunogenicity of recombinant protein were tested in mice after challenging with T.gondii RH.The results showed that GRA12 could induce high level of IL-12 and TNF-? production,the values of CD4 and CD4/CD8 in the immunized mice were significantly higher than those in the control group,and GRA12 protein could prolong the survival time of mice.The results suggested that the GRA12 is an immune related protein.This study will provide theoretical basis on looking for potential drug targets and vaccine candidate antigens of toxoplasmosis.