Molecular and biochemical characterisation and recognition by the immune host of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) of the abomasal nematode parasite Teladorsagia circumcincta

作者：Umair S^*; Bouchet C L G; Knight J S; Pernthaner A; Simpson H V

来源：Experimental Parasitology, 2017, 181: 40-46.

DOI：10.1016/j.exppara.2017.07.006

• Glyceraldehyde 3-phosphate dehydrogenase
• GAPDH
• Teladorsagia circumcincta
• Cloning
• Expression
• Kinetic properties
• ELISA

A 1023 bp full length cDNA encoding Teladorsagia circumcincta GAPDH (TeciGAPDH) was cloned, expressed in Escherichia coli and the recombinant protein purified and its kinetic properties determined. A phylogenetic tree was constructed using helminth GAPDH sequences. The predicted protein consisted of 341 amino acids and was present as a single band of about 38 kDa on SDS-PAGE. Multiple alignments of the protein sequence of TeciGAPDH with homologues from other helminths showed that the greatest similarity (93%) to the GAPDH of Haemonchus contortus and Dictyocaulus viviparus, 82-86% similarity to the other nematode sequences and 68-71% similarity to cestode and trematode enzymes. Substrate binding sites and conserved regions were identified and were completely conserved in other homologues. At 25 degrees C, the optimum pH for TeciGAPDH activity was pH 8, the V-max was 1052 +/- 23 nmol min(-1) mg(-1) protein and the apparent Km for the substrate glyceraldehyde-3-phosphate was 0.02 +/- 0.01 mM (both mean +/- SD, n = 2). Antibodies in both serum and saliva from field-immune, but not nematode-naive, sheep recognised recombinant TeciGAPDH in enzyme-linked immunosorbent assays. The recognition of the recombinant protein by antibodies generated by exposure of sheep to native GAPDH indicates similar antigenicity of the two proteins.

• 出版日期2017-10