Molecular cloning, sequencing, and biological characterization of GRA4 gene of Toxoplasma gondii

Ram, Hira; Rao, J. R.; Tewari, A. K.; Banerjee, P. S.; Sharma, Ajay Kumar

doi:10.1007/s00436-013-3414-3

Cited by 7 publications

(3 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Electrophoresis of GRA-4 recombinant protein using SDS PAGE (Figure -5) showed the protein recombinant with molecular weight of 48 kDa. This Available at www.veterinaryworld.org/Vol.13/October-2020/6.pdf data differed from data observed by Ram et al [23] and Meng et al [24]. The difference in molecular weight is related to the histidine-tagged application [18].…”

Section: T Gondii=toxoplasma Gondiimentioning

confidence: 78%

Cloning and expression of Toxoplasma gondii GRA-4 recombinant protein as a toxoplasmosis diagnostic kit candidate

et al. 2020

View full text Add to dashboard Cite

Aim: The objective of this study was to produce recombinant protein GRA-4 (rGRA-4) of a local Toxoplasma gondii isolate as a candidate for a toxoplasmosis diagnosis kit in Escherichia coli BL21 (DE3) competent cells using pET SUMO plasmid. Materials and Methods: Samples used were stock T. gondii tachyzoites DNA from the Parasitology Laboratory, Faculty of Veterinary Medicine, Gadjah Mada University, Yogyakarta. Amplified GRA-4 polymerase chain reaction product of T. gondii tachyzoite DNA was cloned in the pET-SUMO TAR cloning vector. The GRA-4 gene from T. gondii local isolate was sequenced, followed by plasmid transformation, recombinant plasmid DNA isolation, and recombinant protein expression in DE3 competent cells. Results: The amplification product of GRA-4 T. gondii gene was 1036 bp, with 48 kDa molecular weight after expression in DE3 competent cells. An alignment of the amino acid sequence of GRA-4 from the local isolate which was cloned with GRA-4 was obtained from NCBI database and showed 99.61% homology to the predicted GRA-4 from the T. gondii Izatnagar isolate. Amino acid sequence of the predicted GRA-4 protein from local isolate was different at positions 19 and 304. Conclusion: This research cloned rGRA-4 in pET SUMO plasmid.

show abstract

Section: T Gondii=toxoplasma Gondiimentioning

confidence: 78%

Cloning and expression of Toxoplasma gondii GRA-4 recombinant protein as a toxoplasmosis diagnostic kit candidate

et al. 2020

View full text Add to dashboard Cite

show abstract

“…The GRA proteins, which are highly expressed by the parasite, constitute the circulating antigens in the acute and chronic phases of infection and are of primary relevance to host immunity. Studies demonstrated the ability of several GRA antigens to confer protective immunity in mice infected with T. gondii [ 32 , 33 ], in particular GRA4 [ 17 , 34 - 38 ]. Reports demonstrated that GRA4 might be used to design novel and alternative diagnostic methods for toxoplasmosis [ 39 , 40 ].…”

Section: Introductionmentioning

confidence: 99%

Analyzing and identifying novel B cell epitopes within Toxoplasma gondii GRA4

Wang

et al. 2014

Parasites Vectors

View full text Add to dashboard Cite

BackgroundThe identification of specific epitopes targeted by the host antibody response is important for understanding the natural response to infection and for the development of epitope-based marker vaccines and diagnostic tools for toxoplasmosis. In this study, Toxoplasma gondii GRA4 epitopes were identified using software-based prediction and a synthetic peptide technique.MethodsThe complete GRA4 gene sequence was obtained from T. gondii of the Gansu Jingtai strain of tachyzoites. The potential B cell epitopes of GRA4 was predicted using the PROTEAN subroutine in the DNASTAR software package. The peptides with good hydrophilicity, high accessibility, high flexibility and strong antigenicity were chemically synthesized and assessed by ELISA using pig sera from different time points after infection.ResultsThe potential B cell epitopes of GRA4 predicted by bioinformatics tools focused on six regions of GRA4, 52–77 aa, 93–112 aa, 127–157 aa, 178–201 aa, 223–252 aa and 314–333 aa. Eleven shorter peptides from the six regions were synthesized and assessed by ELISA using pig sera from different time points after infection. Three of the eleven peptides (amino acids 62–77, 233–252 and 314–333) tested were recognized by all sera.ConclusionsWe precisely located the T. gondii GRA4 epitopes using pig sera collected at different time points after infection. The identified epitopes may be useful for additional studies of epitope-based vaccines and diagnostic reagents.

show abstract

“…The GRA proteins, which are highly expressed by the parasite, constitute the circulating antigens in the acute and chronic phases of infection and are of primary relevance to host immunity. Studies demonstrated the ability of several GRA antigens to confer protective immunity in mice infected with T. gondii [32,33], in particular GRA4 [17,[34][35][36][37][38]. Reports demonstrated that GRA4 might be used to design novel and alternative diagnostic methods for toxoplasmosis [39,40].…”

Section: Introductionmentioning

confidence: 99%