Haemonchus Contortus: Immunoprotection Of Glyceraldehyde-3-Phosphate Dehydrogenase & Characterization Of Enolase

Haemonchus contortus is a blood-sucking nematode that primarily infects the abomasum of sheep and goats worldwide. Infections with this parasite can cause anaemia, weight loss and death. The current methods for the control of gastrointestinal nematodes rely heavily on the use of chemicals, but this has led to an increase of parasite anthelmintic-resistant and environmental pollution. So we must develop alternative or supplementary means of control, such as molecular vaccines or new anthelmintic chemicals that leave little residue.This research mainly focuses on the characteration of glyceraldehyde-3-phosphate dehydrogenase and its immunoprotection on goats, other research includes the characteration of H. contortus enolase, the immunolocalization of GAPDH in H.contortus, the bioinformatics analysis of H.contortus phosphoglycerate kinase.1. Characterization of adult Haemonchus contortus GAPDH (HcGAPDH)Specific primers for the rapid amplification of cDNA ends (RACE) were designed based on the expression sequence tag (EST, GenBank accession no.AF670737) 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 PCR, then this gene was send to NCBI(accession no.HM145749). The results showed that the cloned full length cDNA comprised 1303bp, a 1026 bp open reading frame (ORF) was found that terminated with the TAA stop codon. The biochemical activities of the recombinant protein HcGAPDH, which was expressed in prokaryotic cells and purified by affinity chromatography, were analyzed by assays of enzymatic activity, thermal stability and pH. 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℃. 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.2. Histochemistry of the GAPDH in Haemonchus contortus In order to study the localization of the glyceraldehyde-3-phosphate dehydrogenase of Haemonchus contortus (HcGAPDH) in the parasite, the whole paraffin sections of adult worm of H. contortus were studied by immunohistochemical staining with anti-rHcGAPDH serum as the primary antibody. The results showed that GAPDH was expressed in the intestinal tracts of adult female and male worms.3. Construction and identification of DNA Vaccine pVAXl-HcGAPDHFirstly, pVAX1-HcGAPDH DNA vectors were constructed, and then the recombinant DNA plasmids were injected into leg muscle of mice. RT-PCR and Western blot were utilized to analyze the expression of vaccines in mice. The results indicated that DNA vaccines could be well transcripted and expressed in injected tissues.4. Immunoprotective effect of DNA vaccine pVAX1-HcGAPDHFifteen locally bred goats (9-10 months) were raised under nematode-free conditions. The goats were allocated into 3 experimental groups randomly. The H. contortus DNA vaccine was administered 100μg each of pVAX1-HcGAPDH dissolved in lml of PBS (pH7.4). After that, the lml injection volume was equally divided between two injections sites, alternating between the shoulder and thigh muscle. Unchallenged group were not challenged with L3, but vaccinated with lml of PBS of instead, served as the unvaccinated control. Vaccinated animals received two vaccinations at 2 week intervals.5000 infective H. contortus L3 were given two weeks after the final injection. Faecal egg counts were undergone every two days from day 22 after challenge until the end of the experiment. All of the goats were killed 35 days after challenge L3 larvae. Meanwhile, the abomasum was opened immediately, and worm counts were performed. In all goats, serum HcGAPDH specific immunoglobulin IgG titres, IgA titres, mucosal surface (MS) IgA and mucosal homogenate (MH) IgA titres were determined with ELISA. Meanwhile, the peripheral CD4+, CD8+T and B lymphocytes were determined by the method of flow cytometry. The effects of pVAX1-HcGAPDH on cytokine levels of interferon-γ(IFN-γ), IL-4, IL-22 and transforming growth factor-β(TGF-β) of goat peripheral blood were also examined in this study. At last, the peripheral blood eosinophil, basophil, neutrophil, monocyte counts and hemoglobin concentration were determined, respectively. The results showed:After a single L3 larvae challenge of group 1 (pVAX1-HcGAPDH) immunized goats, cumulative mean EPG (eggs per gram of feces) and worm burdens were reduced by 34.9% and 37.73%, respectively. Before L3 challenge infection, the vaccinated groups had significantly higher HcGAPDH-specific IgG titres and IgA titres than the unchallenged and challenged control goats. After L3 challenge, the vaccinated and challenged control groups had significantly higher serum HcGAPDH-specific IgG titres and IgA titre compared to the unchallenged controls. These findings suggested that the HcGAPDH-specific IgG and MH IgA could play a role in protection against H. contortus parasite. Meanwhile, prior to L3 challenge infection, the CD4+ T lymphocytes and B lymphocytes were significantly higher in the vaccinated group as compared to the unchallenged control. After L3 challenge infection, the vaccinated and challenged control groups had higher CD4+ T lymphocytes than the unchallenged control group. Also, we found that the challenged control group had significantly higher B lymphocytes than the vaccinated group. By the determination of cytokine levels, we found the concentration of IL-4 was significantly higher after DNA vaccination. The mean number of blood eosinophils, neutrophil and monocyte increased after immunization coincident with low total worm burdens in the pVAX1-HcGAPDH protected goats.5. Characterization of adult Haemonchus contortus enolase (HcENO)Specific primers for the rapid amplification of cDNA ends (RACE) were designed based on the expression sequence tag (EST, GenBank accession no.BF422728) to amplify the 3’-and 5’-ends of HcENO. The full length of this gene was obtained by overlapping the sequences of 3’-and 5’-extremities and amplification by RT-PCR. The results, showed that the cloned full length cDNA comprised 1583bp. a 23 bp 5’-untranslated region (5’-UTR) was detected before the ATG initiation codon and a 1305 bp open reading frame (ORF) was found that terminated with the TAA stop codon. On the 3’-end, the cDNA had a 216 bp 3’-UTR with a potential functional polyadenylation signals (AGTAAA) in frame followed by a 13 bp poly-A tail. The biochemical assay showed that the protein encoded exhibited enzymatic activity, the optimal pH for enzymatic activity of recombinant enolases were about pH 7.0. Also, the recombinant HcENO was recognized strongly by serum from naturally infected goats. These results suggested that the enzyme might play potential roles in the infection of H, contortus.6. Bioinformatics analysis of HcPGKThe phosphoglycerate kinase gene of Haemonchus contortus (HcPGK) was cloned and characterized. Specific primers for the rapid amplification of cDNA ends (RACE) were designed based on the expression sequence tag (EST, GenBank accession no.CB 192372) to amplify the 3’-and 5’-ends of HcPGK. After this, some bioinformatics analysis were performed, for example, amino acid and atomic compositions, isoelectric point, extinction coefficient, secondary structure predictions, signal peptide cleavage sites, et al. The results showed that the cloned full length cDNA comprised 1584bp. With bioinformatics analysis, we found that the HcPGK encodes 417 amino acids, molecular weight 44,594 Da, also found that it has a few active domains and motifs.