Molecular cloning, sequencing, and characterization of bovine transporter associated with antigen processing 2 (BoTAP2)

Aruna P N Ambagala, Zhengyu Feng, Raul G Barletta, Subramaniam Srikumaran

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

The transporter associated with antigen processing (TAP) 1/2 heterodimer is essential for the transport of antigenic peptides from the cytosol into the lumen of the endoplasmic reticulum. Bovine herpesvirus 1 (BHV-1) inhibits bovine TAP (BoTAP) activity, as a means of down-regulating MHC class I expression on the cell surface, and hence evasion of the cytotoxic T-lymphocyte response of the host. Identification of BHV-1 protein(s) responsible for TAP inhibition, and elucidation of the mechanism of TAP inhibition necessitate cloning and high-level expression of BoTAP1 and 2. In this study, we cloned and sequenced BoTAP2. Cytoplasmic RNA isolated from bovine peripheral blood mononuclear cells was used for cDNA synthesis. Rapid amplification of cDNA ends was used to amplify the 5′ and the 3′ ends of BoTAP2 cDNA. Based on the 5′ and 3′ sequences, primers were designed and the full-length BoTAP2 cDNA was PCR-amplified and sequenced. The full-length cDNA encodes a 719-amino acid polypeptide with a predicted molecular weight of Mr 79,000. BoTAP2 has ∼80% homology, at the amino acid level, to its mammalian counterparts. Similar to human TAP2, BoTAP2 consists of seven putative transmembrane segments followed by an ATP-binding cassette. As expected, the level of BoTAP2 mRNA expression was up-regulated by treatment with recombinant bovine interferon-γ. In Northern blot analysis, BoTAP2 transcripts were detected in several bovine tissues with the highest level observed in jejunum. BoTAP2, when expressed as a green fluorescent fusion protein, exhibited a typical endoplasmic reticulum localization pattern.

Original languageEnglish (US)
Pages (from-to)30-38
Number of pages9
JournalImmunogenetics
Volume54
Issue number1
DOIs
StatePublished - May 20 2002

Fingerprint

Antigen Presentation
Molecular Cloning
Complementary DNA
Bovine Herpesvirus 1
Endoplasmic Reticulum
Amino Acids
Peptides
Cytotoxic T-Lymphocytes
Jejunum
Green Fluorescent Proteins
Northern Blotting
Cytosol
Interferons
Organism Cloning
Blood Cells
Adenosine Triphosphate
Molecular Weight
RNA

Keywords

  • Bovine
  • Cloning
  • Expression
  • Sequencing
  • TAP2

ASJC Scopus subject areas

  • Immunology
  • Genetics

Cite this

Molecular cloning, sequencing, and characterization of bovine transporter associated with antigen processing 2 (BoTAP2). / Ambagala, Aruna P N; Feng, Zhengyu; Barletta, Raul G; Srikumaran, Subramaniam.

In: Immunogenetics, Vol. 54, No. 1, 20.05.2002, p. 30-38.

Research output: Contribution to journalArticle

Ambagala, Aruna P N ; Feng, Zhengyu ; Barletta, Raul G ; Srikumaran, Subramaniam. / Molecular cloning, sequencing, and characterization of bovine transporter associated with antigen processing 2 (BoTAP2). In: Immunogenetics. 2002 ; Vol. 54, No. 1. pp. 30-38.
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