Bioinformatics-based Vaccine Design And Its In Vivo Verification Of Toxoplasma Gondii Dense Granule Protein GRA3

Toxoplasma gondii is an obligate intracellular protozoan parasite with a high pathogenic potential. Toxoplasmosis affects a wide range of hosts, such as mammals and birds, and humans as well. Toxoplasmosis, transmitted to human commonly through ingestion of raw or undercooked meat, or of the oocyst expelled by pets. For the course of its zoonotic nature, this disease has endangered public health worldwide. However, T. gondii strains differ both in genotypic and phenotypic characteristics. Recently, the pathogenicity and the molecular mechanisms of toxoplasmosis have been brought into their genetic signature. Most isolates of T. gondii from Europe and North America fall into three genetically distinct clonal lineages recognized as types â… , â…¡ and â…¢, which have been extensively studied abroad. Our laboratory has long engaged in the study of T. gondii, and a number of Toxoplasma strains have been isolated and characterized from animals, such as Type I, ToxoDB#204, ToxoDB#205, Chinesel (ToxoDB#9) etc. ToxoDB#9is the most common T.gondii genotype in China. Whatsoever the pathogenic mechanisms and the virulence factor functions of Chinese1, which is vital for the diagnosis and prevention of toxoplasmosis, has not received much investigation.Objective:Through investigating the expression and amino acid sequence polymorphism of GRA3among Chinese1strains, we initiated the approach for pathogenic mechanisms of Chinese1. The potential value of GRA3as a candidate molecule for Toxoplasma DNA vaccine was studied. Additionally, our work would provide a theoretical and experimental basis for diagnosis and treatment of toxoplasmosis.Meterials:In the study, we first used qRT-PCR to detect the value of expression of GRA3in Chinese1strains. Subsequently, PCR technique was used to amplify the virulence factor fragments. Next, we built GRA3eukaryotic expression plasmid for TgCtwh3and TgCtwh6strains. The recombinant plasmid was then injected into mice from day zero (First day) and subsequently at two weeks intervals for four weeks (A total of3times). The mice were then challenged by tachyzoites (Wh3) and cysts (Wh6). At six weeks post-inoculation, the specific antibodies (IgG, IgGl and IgG2a) and cytokines (IL-2, IL -4, IL-10and IFN-y) were investigated by using ELISA. The number of deaths and brain cysts of mice were determined and statistically analyzed. Also, the physicochemical properties, secondary structure prediction, antigen epitope, and3D model construction of dense granular proteins of GRA3were determined by using bioinformatics to predict the correlation of the molecular structure with its in vivo protection.Results:(1) The expression level of GRA3in TgCtwh6was significantly (P<0.01) higher compared to GRA3expression levels in other Chinese1strains.(2) Sequence alignment:in terms of sequence alignment,22amino acids of GRA3were different in both TgCtwh6and TgCtwh3strains.(3) The successful building and express GRA3-GFP eukaryotic plasmid of TgCtwh6and TgCtwh3.(4) Results from the immune protection experiments did not support the candidature of GRA3as a nucleic acid vaccine against the Toxoplasma strains studied. Both the levels of the specific antibodies (IgG, IgGland IgG2a) and cytokines (IL-2IL-4, IL-10and IFN-y) and the number of deaths and brain cyst in mice were statistically insignificant (P>0.05)(5) The bioinformatics analysis established the physico-chemical properties, antigen epitope and preliminary3D model of GRA3in Chinese1strains.Conclusions:The present results show that TgCtwh3and TgCtwh6exhibited different expression levels and sequence polymorphisms in GRA3. Dense granular proteins of GRA3were not specifically enough to serve as Toxoplasma vaccine candidate molecule, for that the GRA3prediction protein was not stable and had less antigen epitope coupled with a relatively simple3D structure. Analyses of bioinformatics of GRA3protein coincides well with the in vivo protective experiments, suggesting that bioinformatic approach is a usefull tool for prediction of the candidate in the development of T. gondii molecular vaccine.