Identification of Toxoplasma Gondii Tyrosine Hydroxylase (TH) Activity and Molecular Immunoprotection against Toxoplasmosis

Abstract

The neurotropic parasite Toxoplasma gondii (T. gondii) infection can change the behavior of rodents and cause neuropsychological symptoms in humans, which may be related to the change in neurotransmitter dopamine in the host brain caused by T. gondii infection. T. gondii tyrosine hydroxylase (TgTH) is an important factor in increasing the neurotransmitter dopamine in the host brain. In this study, the enzyme activity of TgTH catalytic substrate for dopamine production and the molecular characteristics of TgTH were identified. In order to amplify the open reading frame (ORF), the designing of the specific primers for polymerase chain reaction (PCR) was on the basis of the TgTH sequence (GenBank Accession No. EU481510.1), which was inserted into pET-32a (+) for the expression of recombined TgTH (rTgTH). The sequence analysis indicated that the gene of TgTH directed the encoding of a 62.4-kDa protein consisting of 565 amino acid residues, which was predicted to have a high antigen index. The enzyme activity test showed that rTgTH and the soluble proteins extracted separately from T. gondii RH strain and PRU strain could catalyze the substrate to produce dopamine in a dose-dependent manner, and the optimum catalytic temperature was 37 °C. The result of the Western Blotting assay revealed that the rTgTH and the native TgTH extracted from somatic of T. gondii RH tachyzoite were successfully detected by the sera of mice infected with T. gondii and the rat serum after rTgTH immune, respectively. Immunofluorescence analysis using antibody against rTgTH demonstrated that the protein was expressed and located on the surface of T. gondii RH tachyzoite. Freund’s adjuvant was used to emulsify the rTgTH, which was subsequently applied to BALB/c mouse immune thrice on week 0, week 2, and week 4, respectively. The result of the animal challenge experiments showed an integral increase in IgG, IgG2a, IgG1, and IFN-γ, IL-4, and IL17 were as well significantly increased, and that the rTgTH vaccinated animals apparently had a prolonged survival time (14.30 ± 2.41) after infection with the RH strain of T. gondii compared with that of the non-vaccinated control animals, which died within 11 days. Additionally, in the rTgTH vaccination group, the number of brain cysts (1275 ± 224) significantly decreased (p < 0.05) compared to the blank control group (2375 ± 883), and the size of the brain cysts in the animals immunized with rTgTH vaccination was remarkably smaller than that of the control mice. All the findings prove that TgTH played an important role in increasing the neurotransmitter dopamine in the host brain and could be used as a vaccine candidate antigen to mediate cell-mediated and humoral immunity.

The Molecular Characterization and Immunity Identification of Trichomonas vaginalis Adhesion Protein 33 (AP33)

“…According to the method described in the reported literature ( Engbring and Alderete, 1998b ; Zhang et al, 2020 ), the protein of TvAP33 was expressed and purified. The Bradford procedure ( Bradford, 1976 ) was applied to determine the TvAP33 protein concentration with the standard of bovine serum albumin (BSA) used as a standard.…”

Advances in Toxoplasma gondii Vaccines: Current Strategies and Challenges for Vaccine Development

“…The recombinant T. gondii CDPK3 vaccine elicited partial protection against RH tachyzoite and a PRU challenge infection in mice, characterized by high antibody titers and a Th1-dominant immune response [ 54 ]. Protective efficacy of tyrosine hydroxylase-based vaccines has been explored, but the protection induced was mild in spite of the humoral and cellular immunity induced through immunization [ 55 ]. In one study, T. gondii vaccines expressing extracellular signal-related kinases were used to immunize the mice on a weekly basis for 5 consecutive weeks.…”

Immune responses induced by a combined vaccination with a recombinant chimera of Mycoplasma hyopneumoniae antigens and capsid virus-like particles of porcine circovirus type 2

“…The protein and complete Freund's adjuvant (CFA) were mixed at 1:1 ration. According to the instruction of CFA provided by Sigma Company and Guidelines for the Use of Adjuvants in Research: Special Emphasis on Freund's Adjuvant (Animal Research Advisory Committee Guidelines; NIH Office of Instramural Research Office of Animal Care and Use), and usages in other reports [16,48,49], the concentration of Mycobacterium tuberculosis is 1 mg/mL, and the final concentration of Mycobacterium tuberculosis is 0.5 mg/mL in the emulsion. Serum samples were obtained from the retro-orbital sinus 0, 14, 28, 35, and 42 days after the first immunization (DAI).…”