Immunoproteomic And Comparative Proteomic Analysis Of Adult Haemonchus Contortus

Haemonchus contortus is an important gastrointestinal nematode parasite of ruminants (notably goats and sheep) in many parts of the world. The nematode is a blood-feeding parasite that infects the abomasum and small intestine of ruminants. Infection with this parasite can lead to anaemia, weight loss and death of the host (especially in lambs), causing significant economic losses to the industry. Current control of this parasite is through periodic use of anthelmintic drugs and pasture management. However, the growing emergence of resistant strains of this parasite has resulted in the need to search for alternative control strategies, such as vaccination.It is necessary to find new immunogenic proteins to develop effective vaccines. In this study, whole proteins and excretory-secretory products from male and female adult H. contortus were analysed by immunoproteomic techniques. Some shared and different immunogenic proteins were reported.1. Immunoproteomic analysis of whole proteins of adult Haemonchus contortusWhole proteins of male and female adult Haemonchus contortus were analysed by immunoproteomic techniques. Approximately 662 and 680 spots were detected on proteome maps of male and female nematodes, respectively, stained by Coomassie brilliant blue G-250. There were 609 shared spots. Approximately 193 and 196 spots, respectively, were recognised on western blot maps of male and female nematodes using the naturally infected antiserum as primary antibody. The gender-specific spot numbers were 129 in male and 132 in female nematodes, respectively. Twenty-three shared immunogenic spots were analysed and identified by database searching. These proteins included glutamate dehydrogenase (GDH), homologues of dim-1, actin, globin-like ES protein F6, glutathione S-transferase (GST), ATPase and glyceraldehyde-3-phosphate dehydrogenase. Except for GDH and GST, the other proteins were not previously reported in similar researches. These proteins as new findings enrich the data set of immunogenic proteins of H. contortus.2. Comparative proteome analysis of whole proteins of male and female adult Haemonchus contortusWhole proteins of male and female adult Haemonchus contortus were analyzed by two-dimensional gel electrophoresis (2-DE) and mass spectrometry (matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS)). Twenty different spots which were gender-specific or had significant changes in abundance were identified by MS. The search results showed that homologues of Major Sperm Protein family member (msp-49) and Kinesin-Like Protein family member (klp-17) were male-specific proteins. Heat shock protein 20, homologues of flavoprotein subunit of succinate dehydrogenase, F32A5.2b protein and VEGF (vascular endothelial growth factor) Receptor family member (ver-4) were female-specific proteins. Glutamate dehydrogenase (GDH) and homologue of ACTin family member (act-4) were expressed at higher level in male nematodes. Western blot using the naturally infected antiserum as primary antibody indicated that GDH, heat shock protein 20, homologues of klp-17 and act-4 could be recognized by the antiserum.3. An immunoproteomic approach to the identification of shared immunogenic proteins of male and female Haemonchus contortus excretory-secretory products (ESPs)To find new potential vaccine candidates against Haemonchus contortus, excretory-secretory products (ESPs) of male and female nematodes were analyzed with immunoproteomic approach. Approximately 176 and 183 spots were detected respectively on proteome maps of male and female nematodes and the shared spots were 158. About 149 and 156 spots were recognised on western blot maps using naturally infected antiserum as primary antibody. There were 121 shared spots. Twenty shared immunogenic spots were analyzed by mass spectrometry. These proteins included 24 kDa excretory/secretory protein, serine/threonine protein kinase (HcSTK), P100-GA, zinc metallopeptidase and glutamate dehydrogenase (GDH) of H. contortus, homologues of ZK287.1 and ENOLase family member (enol-1) of Caenorhabditis elegans, homologues of hypothetical proteins CBG13342, CBG07031, CBG03816 and CBG24211 of Caenorhabditis briggsae and homologue of arginine kinase of Procambarus clarkia. HcSTK, homologue of arginine kinase and homologues of Caenorhabditis proteins were not previously reported and enriched the data of immunogenic proteins of H. contortus.4. Cloning and expression of Haemonchus contortus actin geneA pair of gene-specific degenerate primers were designed according to the sequences of actin open reading frames (ORF) of other nematodes accepted in GenBank. The 1100 bp product was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) with total RNA extracted from adult H. contortus as template. Nucleotide sequencing showed that the gene consists of an ORF of 1131 bp and encodes a protein of 376 amino acids. Through BLAST in NCBI base, the nucleotide and amino acid sequences of the gene had high degree of homology to other nematodes such as Caenorhabditis elegans, Caenorhabditis briggsae and Cooperia oncophora. The gene was cloned into pET-28a(+) and transformed into BL21 (DE3). The expressed product was 46 kDa. Western blot analysis showed that the product was recognized by naturally infected anti-H. contortus goat serum. Natural actin expression protein in H. contortus was analysed by western blotting, using mice serum immunized with purified recombinant protein. The result showed there was a display strip at 43 kDa.5. Cloning and expression of Haemonchus contortus glutamate dehydrogenase geneA pair of gene-specific primers were designed according to the sequences of glutamate dehydrogenase open reading frames (ORF) of H. contortus accepted in GenBank. The 1600 bp product was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) with total RNA extracted from adult H. contortus as template. Nucleotide sequencing showed that the gene consists of an ORF of 1617 bp and encodes a protein of 538 amino acids. The gene was cloned into pET-28a(+) and transformed into BL21 (DE3). The expressed product was 63 kDa. Western blot analysis showed that the product was recognized by naturally infected anti-H. contortus goat serum. Natural glutamate dehydrogenase expression protein in H. contortus was analysed by western blotting, using mice serum immunized with purified recombinant protein. The result showed there was a display strip at 59 kDa.