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Molecular cloning and characterization of a novel immunoreactive ATPase/RNA helicase in human filarial parasite Brugia malayi

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Abstract

DEAD box proteins are putative RNA unwinding proteins found in organisms ranging from mammals to bacteria. We have identified a novel immunodominant cDNA clone, BmL3-helicase, encoding DEAD box RNA helicase by immunoscreening of a larval stage cDNA library of Brugia malayi. The cDNA sequence exhibited strong sequence homology to Caenorhabditis elegans and C. briggsae RNA helicase, a prototypic member of the DEAD (Asp-Glu-Ala-Asp) box protein family. The clone also showed similarity with RNA helicase of Wolbachia, an endosymbiotic bacterium of filarial parasite. It was overexpressed as ∼50 kDa His-tag fusion protein, and ATP hydrolysis assay of recombinant enzyme showed that either ATP or dATP was required for the unwinding activity, indicating BmL3-helicase as an ATP/dATP-dependent RNA helicase. The recombinant protein also demonstrated cross-seroreactivity with human bancroftian sera. The presence of BmL3-helicase in various life stages of B. malayi was confirmed by immunoblotting of parasite-life-cycle extracts with polyclonal sera against the BmL3-helicase, which showed high levels of expression in microfilaria, L3, and adult (both male and female) stages. In the absence of an effective macrofilaricidal agent and validated anti-filarial drug targets, RNA helicases could be utilized as a rational drug target for developing agents against the human filarial parasite.

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Acknowledgments

The author acknowledges University Grants Commission, New Delhi, India, for financial assistance in the form of Senior Research Fellowships to M.S. We are grateful to Mr. A. K. Roy and R. N. Lal for their excellent technical assistance in maintenance of B. malayi infection in laboratory.

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Authors and Affiliations

  1. Division of Parasitology, Central Drug Research Institute, P.O. Box-173, Lucknow, 226001, India

    Meghna Singh & Shailja M. Bhattacharya

  2. Division of Microbiology, Central Drug Research Institute, P.O. Box-173, Lucknow, 226001, India

    K. K. Srivastava

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  1. Meghna Singh
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  2. K. K. Srivastava
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  3. Shailja M. Bhattacharya
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Corresponding author

Correspondence to Shailja M. Bhattacharya.

Additional information

Nucleotide sequence reported in this paper is available in the GenBank™, EMBL, and DDBJ databases under the accession number EF409381.

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Singh, M., Srivastava, K.K. & Bhattacharya, S.M. Molecular cloning and characterization of a novel immunoreactive ATPase/RNA helicase in human filarial parasite Brugia malayi . Parasitol Res 104, 753–761 (2009). https://doi.org/10.1007/s00436-008-1251-6

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  • DOI: https://doi.org/10.1007/s00436-008-1251-6

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