التعرف على المُعَلَمات السطحية لطفليل التوكسوبلازما من خلال استخدام امصال المصابين ذو التركيز العالي بالمضادات المناسبة للتشخيص المناعي
“Identification of immunoreactive Toxoplasma gondii epitopes selected by high antibody titer positive human sera suitable for immunodiagnostic techniques”
دينا نبيل خليل زيغان
Dina Nabil Khalil Zighan
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Toxoplasma gondii is a coccidian obligatory intracellular protozoan parasite that utilizes cats as a definitive host and it is the causative agent of the toxoplasmosis (Abu-Madi, Behnke et al. 2008). The parasite affect about 30% of human population and it is considered a major zoonotic disease. (Chunlei Sua, Zhou et al. 2012; R. Edelhofer 2010) The disease is endemic in many countries in the world including Europe, United States and Mediterranean regions. The present study was conducted to identify immunoreactive Toxoplasma gondii epitopes using M13 phage display library for identifying peptides that mimic Toxoplasma gondii antigens. Phage display was done by plaque assay in 12 amino acid peptide phage library. For phage screening purposes; sera were collected from Toxoplasma gondii infected pregnant women, high antibody titer sera were pooled from different patients after screening for anti-Toxoplasma crude antigens by Modified Agglutination Test (MAT). By the aid of the high antibody titer Toxoplasma gondii pooled sera, three screening cycles of M13 phage display library, (12 amino acids peptides) were achieved. In the third screening cycle; 329 reactive plaques were obtained, 138 plaques that gave the strongest signals with pooled sera were selected for further analysis. Reactive peptides were isolated and sub-cloned in recombinant expression plasmid and were used in dot-ELISA to screen the selected M13 phage clones with pooled sera. ELISA test was also carried out to detect Toxoplasma infected individuals were identified. Phage Dot-Blot assay standardized for the confirmation of reactivity selected M13 phage clones with Toxoplasma gondii high antibody titer pooled sera and ELISA Assay revealed reactivity between selected amplified phages and some collected sera of Toxoplasma gondii infected individuals. The DNA coding the 12 amino acid peptide fused with the PIII M13 phage gene was amplified using specific primers flanking the peptide designed based on the PIII gene. DNA sequence was determined for 24 different clones, followed by bioinformatics DNA analysis by means of DNA alignment and BLAST search analysis. From the 24 sequenced peptides, 19 selected peptide clones represent the selected M13 phages showed no direct similarity to Toxoplasma gondii parasite DNA genomic sequences. So, due to that we was searching on amino acids sequence for the selected M13 phages through BLAST program. Data analysis showed a cross-reactivity between the selected M13 phages and anti-Toxoplasma pooled sera; which the sequences represent the selected M13 phages related to protozoan parasites such as Plasmodium knowlesi and Plasmodium fragile. These protozoan parasites are closely related to Toxoplasma family (Sarcocystidae). The selected M13 phage clones were pooled and tested for their reactivity against pooled anti-Toxoplasma sera in dot-blot and ELISA format. The obtained results showed specific interaction that need further studies and evaluation for the use of these phages in future Toxoplasma diagnosis.
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