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Cloning, expression and characterization of NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase of adult Haemonchus contortus†

Published online by Cambridge University Press:  21 December 2010

K. Han ,
L. Xu ,
R. Yan ,
X. Song  and
X. Li
K. Han
Affiliation:
College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, 210095, P.R. China
L. Xu
Affiliation:
College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, 210095, P.R. China
R. Yan
Affiliation:
College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, 210095, P.R. China
X. Song
Affiliation:
College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, 210095, P.R. China
X. Li*
Affiliation:
College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, 210095, P.R. China
*
*Fax: +86 25 8439 8669 E-mail: [email protected]
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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.

Type
Research Papers
Information
Journal of Helminthology , Volume 85 , Issue 4 , December 2011 , pp. 421 - 429
Copyright
Copyright © Cambridge University Press 2010

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