Cloning, Expression and Characterization of NAD+-dependent glyceraldehyde-3-phosphate Dehydrogenase of Adult Haemonchus Contortus

J Helminthol. 2011 Dec;85(4):421-9. doi: 10.1017/S0022149X10000763. Epub 2010 Dec 21.

Abstract

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) regulates a wide range of biological processes, including pathogen evasion. In the present research, the GAPDH gene of Haemonchus contortus (HcGAPDH) was cloned and characterized. Specific primers for the rapid amplification of cDNA ends (RACE) were designed based on the expressed sequence tag (EST, AW670737) to amplify the 3' and 5' ends of HcGAPDH. The full length of cDNA from this gene was obtained by overlapping the sequences of 3' and 5' extremities and amplification by reverse transcription polymerase chain reaction (RT-PCR). The biochemical activities of the recombinant protein HcGAPDH, which was expressed in prokaryotic cells and purified by affinity chromatography, were analysed by assays of enzymatic activity, thermal stability and pH. The results showed that the cloned full-length cDNA comprised 1303 bp and encoded a peptide with 341 amino acid residues which showed sequence similarity to several known GAPDHs. The biochemical assay showed that the protein encoded by the HcGAPDH exhibited enzymatic activity with NAD+ as a cofactor. HcGAPDH was stable between pH 5 and 9 and maintained activity at high temperatures of up to 75°C. The natural GAPDH of Haemonchus contortus detected by immunoblot assay was approximately 38 kDa in size, and the recombinant HcGAPDH was recognized strongly by serum from naturally infected goats.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Cloning, Molecular*
  • Enzyme Stability
  • Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)* / chemistry
  • Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)* / genetics
  • Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)* / immunology
  • Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)* / metabolism
  • Goat Diseases / immunology
  • Goat Diseases / parasitology
  • Goats
  • Haemonchiasis / immunology
  • Haemonchiasis / parasitology
  • Haemonchiasis / veterinary
  • Haemonchus / classification
  • Haemonchus / enzymology*
  • Haemonchus / genetics
  • Haemonchus / immunology
  • Helminth Proteins / chemistry
  • Helminth Proteins / genetics
  • Helminth Proteins / immunology
  • Helminth Proteins / metabolism
  • Hydrogen-Ion Concentration
  • Molecular Sequence Data
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / immunology
  • Recombinant Proteins / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sequence Analysis, DNA
  • Sequence Homology, Amino Acid

Substances

  • Helminth Proteins
  • Recombinant Proteins
  • Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)

Associated data

  • GENBANK/HM145749