title: Induction of specific humoral immune response in mice immunized with ROP18 nanospheres from Toxoplasma gondii.
abstract: Toxoplasmosis is one of the most common zoonotic protozoal diseases. Recent advances in biotechnology have produced recombinant protein, which are immunogenic, and progress in nano-pharmaceutics has generated encapsulated protein in nanospheres, which are suitable for vaccine delivery. DNA was extracted from Toxoplasma gondii oocysts and was confirmed through nested PCR and sequencing. The 1665 bp of ROP18 was cloned into the easy vector system: pGEM-T by the T-A cloning method. DH5α bacteria were transfected with pGEM-ROP18. ROP18 was subcloned from pGEM-ROP18 into pET28-ROP18. BL21 bacteria were transfected with pET28-ROP18. Thus, rROP18 protein was expressed in BL21 bacteria by induction at different concentrations of isopropyl β-D-1-thiogalactopyranoside. Protein expression was confirmed through SDS-PAGE and Western blotting. The immunoblot of rROP18 was recognized by anti-HIS antibodies and sera from infected mice at 67 kDa. Recombinant ROP18 protein was encapsulated in nanoparticles with PLGA and was characterized through scanning electron microscopy. Intraperitoneal immunizations with rROP18 protein and intranasal immunization of nanospheres were carried out in mice, and the immune response was detected by ELISA. Results showed that rROP18 in nanospheres administered intra-nasally elicited elevated responses of specific IgA and IgG2a as compared to groups inoculated intra-nasally with rROP18 alone, or injected subcutaneously with rROP18 in montanide adjuvant. It was concluded that nanospheres of ROP18 would be a non-invasive approach to develop vaccination against T. gondii. Further experiments are needed to determine the cellular response to these nanospheres in a mouse model for chronic toxoplasmosis.
authors: Department of Parasitology, University of Veterinary and Animal Sciences, Lahore, Pakistan.
authors: Habibun
authors: H
authors: Nabi
authors: Department of Parasitology, University of Veterinary and Animal Sciences, Lahore, Pakistan. imran.rashid@uvas.edu.pk.
authors: Imran
authors: http://orcid.org/0000-0002-0280-9335
authors: I
authors: Rashid
authors: Department of Parasitology, University of Veterinary and Animal Sciences, Lahore, Pakistan.
authors: Nisar
authors: N
authors: Ahmad
authors: Department of Clinical Medicine and Surgery, University of Veterinary and Animal Sciences, Lahore, Pakistan.
authors: Aneela
authors: A
authors: Durrani
authors: Department of Parasitology, University of Veterinary and Animal Sciences, Lahore, Pakistan.
authors: Haroon
authors: H
authors: Akbar
authors: Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Lahore, Pakistan.
authors: Saher
authors: S
authors: Islam
authors: Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Lahore, Pakistan.
authors: Amna Arshad
authors: AA
authors: Bajwa
authors: Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Lahore, Pakistan.
authors: Wasim
authors: W
authors: Shehzad
authors: Department of Parasitology, University of Veterinary and Animal Sciences, Lahore, Pakistan.
authors: Kamran
authors: K
authors: Ashraf
authors: Government Degree College for Women, Ferozewala, Sheikhupura, Pakistan.
authors: Nyla
authors: N
authors: Imran
mesh_terms: D000276:Adjuvants, Immunologic; D000818:Animals; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D056724:Immunity, Humoral; D051379:Mice; D054118:Nanospheres; D015800:Protozoan Proteins / Q000276:immunology*; D016052:Protozoan Vaccines / Q000276:immunology*; D011994:Recombinant Proteins / Q000235:genetics; D014124:Toxoplasmosis, Animal / Q000517:prevention & control*; D014162:Transfection
chemical_list: D000276:Adjuvants, Immunologic; D015800:Protozoan Proteins; D016052:Protozoan Vaccines; D011994:Recombinant Proteins
keywords: Cloning and expression; Humoral response; Mucosal response; Nanoparticles; ROP18; Toxoplasma gondii