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Cloning, Expression and Characterization of Spore Wall Protein 5 (SWP5) of Indian Isolate NIK-1S of Nosema bombycis

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Abstract

SWPs are the major virulence component of microsporidian spores. In microsporidia, SWPs can be found either in exospore or endospore to serve as a putative virulence factor for host cell invasion. SWP5 is a vital protein that involves in exospore localization and supports the structural integrity of the spore wall and this action potentially modulates the course of infection in N. bombycis. Here we report recombinant SWP5 purification using Ni-NTA IMAC and SEC. GFC analysis reveals SWP5 to be a monomer which correlates with the predicted theoretical weight and overlaps with ovalbumin peak in the chromatogram. The raised polyclonal anti-SWP5 antibodies was confirmed using blotting and enterokinase cleavage experiments. The resultant fusion SWP5 and SWP5 in infected silkworm samples positively reacts to anti-SWP5 antibodies is shown in ELISA. Immunoassays and Bioinformatic analysis reveal SWP5 is found to be localized on exospore and this action could indicate the probable role of SWP5 in host pathogen interactions during spore germination and its contribution to microsporidian pathogenesis. This study will support development of a field-based diagnostic kit for the detection N. bombycis NIK-1S infecting silkworms. The analysis will also be useful for the formulation of drugs against microsporidia and pebrine disease.

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Abbreviations

SWPs:

Spore wall proteins

PTPs:

Polar tube proteins

ELISA:

Enzyme-linked immunosorbent assay

CD:

Circular dichroism

GFC:

Gel Filtration Chromatography

Ni-NTA IMAC:

Nickel- Trinitilo acetic acid Immobilized metal affinity chromatography

SEC:

Size exclusion chromatography

Trx:

Thioredoxin

ALP:

Alkaline phosphatase

BCIP:

5-bromo-4-chloro-3-indolyl phosphate

NBT:

Niro blue tetrazolium

ECL:

Enhanced Chemiluminescence

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Acknowledgements

KMP thanks the Central Silk Board (CSB), Government of India for the financial support in the form of a research grant (Project No. AIT-5872).

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Authors and Affiliations

  1. Genomic Division, Seribiotech Research Laboratory, Carmelaram Post, Kodathi, Bengaluru, 560035, Karnataka, India

    Vijaya Gowri Esvaran, Anupama Jagadish & Kangayam M. Ponnuvel

  2. Institute of Bioinformatics and Applied Biotechnology, Electronic City Phase I, Biotech Park, Bengaluru, 560100, Karnataka, India

    Shobana Ponnuvel & Hosahalli S. Subramanya

  3. Department of Biochemistry, Biological Sciences Building, Indian Institute of Science, C.V. Raman Road, Bengaluru, 560012, Karnataka, India

    Shobana Ponnuvel & Handanahal S. Savithri

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  1. Vijaya Gowri Esvaran
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  2. Shobana Ponnuvel
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  3. Anupama Jagadish
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  4. Handanahal S. Savithri
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Contributions

VGE: Concept/ Experimentation; SP: Experimentation/ Manuscript writing; AJ: Initial Manuscript writing and editing; HSS: Scientific Inputs/ Infrastructural support; HSS: Scientific Inputs/ Infrastructural support; KMP: Concept/ Technical advice/ Formal analysis/ Scientific inputs;

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Correspondence to Kangayam M. Ponnuvel.

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Esvaran, V.G., Ponnuvel, S., Jagadish, A. et al. Cloning, Expression and Characterization of Spore Wall Protein 5 (SWP5) of Indian Isolate NIK-1S of Nosema bombycis. Protein J 41, 596–612 (2022). https://doi.org/10.1007/s10930-022-10078-1

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