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      Molecular characterization and protective efficacy of a new conserved hypothetical protein of Eimeria tenella Translated title: Caractérisation moléculaire et efficacité protectrice d’une nouvelle protéine hypothétique conservée d’ Eimeria tenella

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          Abstract

          Eimeria tenella is an obligate intracellular parasite that actively invades cecal epithelial cells of chickens. This parasite encodes a genome of more than 8000 genes. However, more than 70% of the gene models for this species are currently annotated as hypothetical proteins. In this study, a conserved hypothetical protein gene of E. tenella, designated EtCHP18905, was cloned and identified, and its immune protective effects were evaluated. The open reading frame of EtCHP18905 was 1053bp and encoded a protein of 350 amino acids with a molecular weight of 38.7kDa. The recombinant EtCHP18905 protein (r EtCHP18905) was expressed in E. coli. Using western blot, the recombinant protein was successfully recognized by anti GST-Tag monoclonal antibody and anti-sporozoites protein rabbit serum. Real-time quantitative PCR analysis revealed that the EtCHP18905 mRNA levels were higher in sporozoites than in unsporulated oocysts, sporulated oocysts and second-generation merozoites. Western blot analysis showed that EtCHP18905 protein expression levels were lower in sporozoites than in other stages. Immunofluorescence analysis indicated that the EtCHP18905 protein was located on the surface of sporozoites and second-generation merozoites. Inhibition experiments showed that the ability of sporozoites to invade host cells was significantly decreased after treatment with the anti-r EtCHP18905 polyclonal antibody. Vaccination with r EtCHP18905 protein was able to significantly decrease mean lesion scores and oocyst outputs as compared to non-vaccinated controls. The results suggest that the r EtCHP18905 protein can induce partial immune protection against infection with E. tenella and could be an effective candidate for the development of new vaccines.

          Translated abstract

          Eimeria tenella est un parasite intracellulaire obligatoire qui envahit activement les cellules épithéliales du caecum des poulets. Ce parasite code un génome de plus de 8000gènes. Cependant, plus de 70% des modèles de gènes de cette espèce sont actuellement annotés en tant que protéines hypothétiques. Dans cette étude, un gène de protéine hypothétique conservé d’ E. tenella, désigné par EtCHP18905, a été cloné et identifié, et ses effets immuno-protecteurs ont été évalués. Le cadre de lecture ouvert d’ EtCHP18905 était de 1053 pb et codait pour une protéine de 350 acides aminés avec un poids moléculaire de 38,7kDa. La protéine recombinante EtCHP18905 (r EtCHP18905) a été exprimée dans E. coli. En utilisant le Western blot, la protéine recombinante a été reconnue avec succès par un anticorps monoclonal anti-GST-Tag et un sérum de lapin anti-protéines de sporozoïtes. Une analyse PCR quantitative en temps réel a révélé que les niveaux d’ARNm d’ EtCHP18905 étaient plus élevés dans les sporozoïtes que dans les oocystes non sporulés, les oocystes sporulés et les mérozoïtes de deuxième génération. L’analyse par Western blot a montré que les niveaux d’expression de la protéine EtCHP18905 étaient plus faibles dans les sporozoïtes que dans les autres stades. L’analyse par immunofluorescence a indiqué que la protéine EtCHP18905 était localisée à la surface des sporozoïtes et des mérozoïtes de deuxième génération. Des expériences d’inhibition ont montré que la capacité des sporozoïtes à envahir les cellules hôtes était significativement diminuée après le traitement par l’anticorps polyclonal anti-r EtCHP18905. La vaccination avec la protéine r EtCHP18905 a permis de réduire significativement les scores moyens des lésions et les sorties d’oocystes par rapport aux témoins non vaccinés. Les résultats suggèrent que la protéine r EtCHP18905 peut induire une protection immunitaire partielle contre l’infection par E. tenella et pourrait être un candidat efficace pour le développement de nouveaux vaccins.

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          Most cited references 54

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          Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.

          The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data. Copyright 2001 Elsevier Science (USA).
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            We have determined the relationship between mRNA and protein expression levels for selected genes expressed in the yeast Saccharomyces cerevisiae growing at mid-log phase. The proteins contained in total yeast cell lysate were separated by high-resolution two-dimensional (2D) gel electrophoresis. Over 150 protein spots were excised and identified by capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS). Protein spots were quantified by metabolic labeling and scintillation counting. Corresponding mRNA levels were calculated from serial analysis of gene expression (SAGE) frequency tables (V. E. Velculescu, L. Zhang, W. Zhou, J. Vogelstein, M. A. Basrai, D. E. Bassett, Jr., P. Hieter, B. Vogelstein, and K. W. Kinzler, Cell 88:243-251, 1997). We found that the correlation between mRNA and protein levels was insufficient to predict protein expression levels from quantitative mRNA data. Indeed, for some genes, while the mRNA levels were of the same value the protein levels varied by more than 20-fold. Conversely, invariant steady-state levels of certain proteins were observed with respective mRNA transcript levels that varied by as much as 30-fold. Another interesting observation is that codon bias is not a predictor of either protein or mRNA levels. Our results clearly delineate the technical boundaries of current approaches for quantitative analysis of protein expression and reveal that simple deduction from mRNA transcript analysis is insufficient.
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              Correlation between Protein and mRNA Abundance in Yeast

              We have determined the relationship between mRNA and protein expression levels for selected genes expressed in the yeastSaccharomyces cerevisiaegrowing at mid-log phase. The proteins contained in total yeast cell lysate were separated by high-resolution two-dimensional (2D) gel electrophoresis. Over 150 protein spots were excised and identified by capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS). Protein spots were quantified by metabolic labeling and scintillation counting. Corresponding mRNA levels were calculated from serial analysis of gene expression (SAGE) frequency tables (V. E. Velculescu, L. Zhang, W. Zhou, J. Vogelstein, M. A. Basrai, D. E. Bassett, Jr., P. Hieter, B. Vogelstein, and K. W. Kinzler, Cell 88:243–251, 1997). We found that the correlation between mRNA and protein levels was insufficient to predict protein expression levels from quantitative mRNA data. Indeed, for some genes, while the mRNA levels were of the same value the protein levels varied by more than 20-fold. Conversely, invariant steady-state levels of certain proteins were observed with respective mRNA transcript levels that varied by as much as 30-fold. Another interesting observation is that codon bias is not a predictor of either protein or mRNA levels. Our results clearly delineate the technical boundaries of current approaches for quantitative analysis of protein expression and reveal that simple deduction from mRNA transcript analysis is insufficient.
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                Author and article information

                Journal
                Parasite
                Parasite
                parasite
                Parasite
                EDP Sciences
                1252-607X
                1776-1042
                2021
                03 May 2021
                : 28
                : ( publisher-idID: parasite/2021/01 )
                Affiliations
                [1 ] Key Laboratory of Animal Parasitology of Ministry of Agriculture, Shanghai Veterinary Research Institute, CAAS 200241 Shanghai PR China
                [2 ] College of Life and Environment Sciences, Shanghai Normal University 200234 Shanghai PR China
                Author notes
                [a]

                Equal contributors.

                [* ]Corresponding author: hhysh@ 123456shvri.ac.cn
                Article
                parasite200156 10.1051/parasite/2021037
                10.1051/parasite/2021037
                8095096
                33944773
                © H. Zhao et al., published by EDP Sciences, 2021

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                Page count
                Figures: 8, Tables: 1, Equations: 0, References: 49, Pages: 12
                Categories
                Research Article

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