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A Novel Polyclonal Antiserum against Toxoplasma gondii Sodium Hydrogen Exchanger 1.

ABSTRACT: The sodium hydrogen exchanger 1 (NHE1), which functions in maintaining the ratio of Na(+) and H(+) ions, is widely distributed in cell plasma membranes. It plays a prominent role in pH balancing, cell proliferation, differentiation, adhesion, and migration. However, its exact subcellular location and biological functions in Toxoplasma gondii are largely unclear. In this study, we cloned the C-terminal sequence of T. gondii NHE1 (TgNHE1) incorporating the C-terminal peptide of NHE1 (C-NHE1) into the pGEX4T-1 expression plasmid. The peptide sequence was predicted to have good antigenicity based on the information obtained from an immune epitope database. After induction of heterologous gene expression with isopropyl-b-D-thiogalactoside, the recombinant C-NHE1 protein successfully expressed in a soluble form was purified by glutathione sepharose beads as an immunogen for production of a rabbit polyclonal antiserum. The specificity of this antiserum was confirmed by western blotting and immunofluorescence. The antiserum could reduce T. gondii invasion into host cells, indicated by the decreased TgNHE1 expression in T. gondii parasites that were pre-incubated with antiserum in the process of cell entry. Furthermore, the antiserum reduced the virulence of T. gondii parasites to host cells in vitro, possibly by blocking the release of Ca(2+). In this regard, this antiserum has potential to be a valuable tool for further studies of TgNHE1.

SUBMITTER: Xiao B

PROVIDER: S-EPMC4792324 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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A Novel Polyclonal Antiserum against Toxoplasma gondii Sodium Hydrogen Exchanger 1.

Xiao Bin B Kuang Zhenzhan Z Zhan Yanli Y Chen Daxiang D Gao Yang Y Li Ming M Luo Shuhong S Hao Wenbo W

The Korean journal of parasitology 20160226 1

The sodium hydrogen exchanger 1 (NHE1), which functions in maintaining the ratio of Na(+) and H(+) ions, is widely distributed in cell plasma membranes. It plays a prominent role in pH balancing, cell proliferation, differentiation, adhesion, and migration. However, its exact subcellular location and biological functions in Toxoplasma gondii are largely unclear. In this study, we cloned the C-terminal sequence of T. gondii NHE1 (TgNHE1) incorporating the C-terminal peptide of NHE1 (C-NHE1) into ...[more]

PMID: 26951975

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Project description:BackgroundIn 2007, Park et al. identified a novel serotype among Streptococcus pneumoniae serogroup 6 which they named serotype 6C. The aim of this study was to evaluate with the Neufeld test a novel S. pneumoniae serotype 6C type specific polyclonal antiserum. In addition, serotype 6C isolates found in Denmark in 2007 and 2008 as well as eight old original serotype 6A isolates were characterised.MethodsIn this study, 181 clinical Streptococcus pneumoniae isolates from Denmark 2007 and 2008 were examined; 96 isolates had previously been typed as serotype 6A and 85 as serotype 6B. In addition, eight older isolates from 1952 to 1987, earlier serotyped as 6A, were examined. Serotype 6C isolates were identified by PCR and serotyping with the Neufeld test using the novel type specific polyclonal antiserum, factor antiserum 6 d, in addition to factor antisera 6b, 6b* (absorbed free for cross-reactions to serotype 6C) and 6c. All antisera are commercially available and antiserum 6b obtained from the supplier after 1 January 2009 is antiserum 6b*. All serotype 6C isolates were further characterised using multi-locus sequence typing.ResultsWhen retesting all 96 original serotype 6A isolates by PCR and the Neufeld test, 29.6% (24 of 81) of the invasive isolates in Denmark from 2007 and 2008 were recognised as serotype 6C. In addition, three of eight old isolates originally serotyped as 6A were identified to be serotype 6C. The oldest serotype 6C isolate was from 1962. The serotype 6C isolates belonged to eleven different sequence types (ST) and nine clonal complexes (CC), ST1692 (CC395), ST386 (CC386) and ST481 (CC460) were the predominant types.ConclusionsWe tested a novel polyclonal antiserum 6 d, as well as modified antiserum 6b*, provided a scheme for the serotyping of S. pneumoniae serogroup 6 using the Neufeld test and compared the serotyping method with PCR based methods. The two types of methods provided the same results. In future, it will, therefore, be possible to test also serotype 6C in accordance to the standard method for serotyping of S. pneumoniae recommended by WHO.Among all invasive isolates from Denmark 2007 and 2008, serotype 6C constituted 29.6% of the original serotype 6A isolates. The serotype 6C isolates were found to be diverse belonging to a number of different STs and CCs of which most have been observed in other countries previously. Serotype 6C is regarded as an "old" serotype being present among S. pneumoniae isolates in Denmark for at least 48 years. The genetic diversity of serotype 6C isolates and their genetic relationship to other serotypes suggested that serotype 6C strains may have arisen from several different independent recombination events involving different parental strains such as serotypes 6A, 6B, 23F and 4.

Dataset Information

A Novel Polyclonal Antiserum against Toxoplasma gondii Sodium Hydrogen Exchanger 1.

Publications

A Novel Polyclonal Antiserum against Toxoplasma gondii Sodium Hydrogen Exchanger 1.

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