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Molecular Cloning, Expression, and In Silico Structural Analysis of Guinea Pig IL-17

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Abstract

Interleukin-17A (IL-17A) is a potent proinflammatory cytokine and the signature cytokine of Th17 cells, a subset which is involved in cytokine and chemokine production, neutrophil recruitment, promotion of T cell priming, and antibody production. IL-17 may play an important role in tuberculosis and other infectious diseases. In preparation for investigating its role in the highly relevant guinea pig model of pulmonary tuberculosis, we cloned guinea pig IL-17A for the first time. The complete coding sequence of the guinea pig IL-17A gene (477 nucleotides; 159 amino acids) was subcloned into a prokaryotic expression vector (pET-30a) resulting in the expression of a 17 kDa recombinant guinea pig IL-17A protein which was confirmed by mass spectrometry analysis. Homology modeling of guinea pig IL-17A revealed that the three-dimensional structure resembles that of human IL-17A. The secondary structure predicted for this protein showed the presence of one extra helix in the N-terminal region. The expression profile of IL-17A was analyzed quantitatively in spleen, lymph node, and lung cells from BCG-vaccinated guinea pigs by real-time PCR. The guinea pig IL-17A cDNA and its recombinant protein will serve as valuable tools for molecular and immunological studies in the guinea pig model of pulmonary TB and other human diseases.

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Acknowledgments

This work was supported by a subcontract from Colorado State University under NIH contract HHSN 266200400091c. The authors thank Dr. Larry Dangott of the Protein Chemistry Lab at Texas A&M University for mass spectrometry analysis.

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Author notes

  1. Vijaya R. Dirisala

    Present address: Division of Hematology, Department of Internal Medicine, Ohio State University Medical Center, Columbus, OH, 43210, USA

Authors and Affiliations

  1. Department of Microbial and Molecular Pathogenesis, College of Medicine, Texas A&M Health Science Center, College Station, TX, 77843-1114, USA

    Vijaya R. Dirisala, Amminikutty Jeevan & David N. McMurray

  2. Division of Chemistry and Chemical Engineering, California Institute of Technology (Caltech), Pasadena, CA, 91125, USA

    Suresh K. Ramasamy

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  1. Vijaya R. Dirisala
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  2. Amminikutty Jeevan
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Corresponding author

Correspondence to Vijaya R. Dirisala.

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Supplementary Fig. 1

The superposition of the structure of IL-17A from Cavia porcellus predicted by modeler (blue), by Expasy server (green) and the human IL-17 which are used as templates in predicting the Cavia porcellus IL-17A structure (red) (PPTX 153 kb)

Supplementary Fig. 2

The front and back view of superimposition of guinea pig IL-17A from modeller (blue), Swiss-Prot (green), and the Human IL-17A crystal structure (red). The loop region has been highlighted in box marked in red for viewing the difference between the predicted models (PPTX 316 kb)

Supplementary Fig. 3

The front and back view of superimposition of guinea pig IL-17A and the human IL-17A crystal structure (PPTX 291 kb)

Supplementary Fig. 4

The front and back view of electrostatic surface charges for IL-17A from Cavia porcellus. Surface charges are calculated based on electrostatic potential which is color-ramped from negative (−)10kB/e (red) to positive (+)10 kB/e (Blue) (PPTX 906 kb)

Supplementary Fig. 5

The closer look of disulfide bond in the modeled IL-17A from Cavia porcellus (PPTX 189 kb)

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Dirisala, V.R., Jeevan, A., Ramasamy, S.K. et al. Molecular Cloning, Expression, and In Silico Structural Analysis of Guinea Pig IL-17. Mol Biotechnol 55, 277–287 (2013). https://doi.org/10.1007/s12033-013-9679-z

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