Cloning And Immunoprotection Of HC29, DIM-1 And Lipase From Haemonchus Contortus

Haemonchus contortus is an important gastrointestinal nematode parasite of ruminants (notably goats and sheep) in many parts of the world, which belongs to Haemonchus genus of Trichostrongylidae family. It is a blood-feeding nematode parasite that infects the abomasums (fourth or true stomach) and small intestine of ruminants. Infection can lead to anaemia, loss of condition and the death of the host (especially lambs) in severe cases, which cause significant economic loss of husbandry 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 present study, the complete coding sequence of Haemonchus contortus HC29, Dim-1 and Lipase was generated by the rapid amplification of cDNA ends (RACE) and PCR. Then, the analysis of nucleic acid and amino acid was carried out and the characteristics of recombinant protein were evaluated. It was reported that GPX and Lipase were involved in developmental regulation of H. contortus, and Dim-1 was a strongly immunogenic protein. Recombinant DNA vaccines encoding GPX and Dim-1 was tested for protection against experimental H. contortus infections in goats separately and the immunologic mechanisms of protective effect generated by both DNA vaccines were researched.1. Cloning and characterization of HC29 from adult Haemonchus contortusIn this study, the complete coding sequence of Haemonchus contortus HC29 cDNA was generated by the rapid amplification of cDNA ends (RACE) and PCR using primers based on the 5'and 3'ends of the partial mRNA published. The cloned HC29 cDNA, was 1113 bp in size with an open-reading frame of 507 bp assumed to encode a protein of 168 aa with a calculated molecular mass of 18.9 kDa. Analysis of amino acid sequence revealed that cloned HC29 had conserved residues of catalytic triad and dimer interface of selenium-independent glutathione peroxidase (GPX). Alignment of the predicted amino acid sequences demonstrated that the protein shared 44.7-80.4% similarity to GPX homologues in thioredoxin_like family. Phylogenetic analysis revealed close evolutionary proximity of GPX sequence to counterpart sequences. These results suggested that HC29cDNA was a GPX, a member of the thioredoxin_like family. Alignment of nucleic acid and amino acid sequences of HC29 with the reported selenium-independent GPX of Haemonchus contortus showed HC29 had different types of spliced leader sequences and dimer interface sites. However, the active sites of both were the same. Enzymatic analysis of recombined HC29 protein of prokaryotic expression showed activity to hydrolyze H2O2. Those indicated that HC29 was a selenium-independent GPX of Haemonchus contortus.2. Immunoprotection of HC29 DNA vaccine from adult Haemonchus contortusRecombinant HC29 DNA vaccine was tested for protection against experimental H. contortus infections in goats. Fifteen animals were allocated into three trial groups. The animals of HC29 group was vaccinated with a recombinant HC29 DNA vaccine twice on days 0 and 14, and challenged with 5000 infective H. contortus L3 (third larval stage) on days 28. An unvaccinated positive control group was challenged with L3 at the same time. An unvaccinated negative control group was not challenged with L3. The results indicated HC29 DNA vaccines were transcribed at local injection sites and expressed in vivo post immunizations respectively. Following L3 challenge the mean eggs per gram feces and worm burdens of HC29 group, were reduced by 36.1% and 35.6% respectively. After immunization with the DNA vaccine, significantly high levels of serum IgG, serum IgA, mucosal IgA, CD4+ T lymphocytes and B lymphocytes were produced. Increased numbers of blood eosinophils and lymphocytes and declined haemoglobin level after immunization were observed in the vaccinated group. These data suggest that recombinant H. contortus HC29 glutathione peroxidase DNA vaccine induced partial immune response and has protective potential against caprine hemonchosis.3. Cloning and immunoprotection of a Disorganized Muscle family member (Dim-1) from adult Haemonchus contortusDue to the strong immunogenicity to host's immune system, Dim-1 in Haemonchus contortus is potential candidate for vaccine to control haemonchosis. However, information on the complete coding sequence and protection potential of this molecule is lacking. In this study, full length of Dim-1 cDNA was cloned using a rapid amplification of cDNA ends (RACE) strategy and the DNA vaccine encoding Dim-1 was tested for protection against experimental H. contortus infections in goats. The grouping of experimental animals and procedures of animal experiment were following the methods described previously. The results indicated Dim-1 DNA vaccines were transcribed at local injection sites and expressed in vivo post immunizations respectively. Following L3 challenge the mean eggs per gram feces and worm burdens of Dim-1 group, were reduced by 45.7% and 51.1% respectively. Significantly high levels of serum IgG, serum IgA, mucosal IgA, CD4+ T lymphocytes and B lymphocytes were produced. Increased numbers of blood eosinophils and lymphocytes and declined haemoglobin level after L3 challenge were observed in the Dim-1 group. These data suggest that recombinant H. contortus Dim-1 DNA vaccine induced partial immune response and has protective potential against caprine hemonchosis.4. Cloning and characterization of a lipase from adult Haemonchus contortusThe proteins in parasites involved in live stages were thought to possess potential of developing alternative strategies for the control of this parasite. In this study, the complete coding sequence of H, contortus Lipase cDNA was generated by the rapid amplification of cDNA ends (RACE) and PCR using primers based on the 5'and 3'ends of the partial mRNA cloned previously. The cloned Lipase cDNA, was 1121 bp in size with an open-reading frame of 918 bp assumed to encode a protein of 305 aa. Analysis of amino acid sequence revealed that cloned Lipase had conserved residues of catalytic triad, lid structure, signal peptide and nucleophilic elbow of this enzyme. These results suggested that this cloned cDNA was a lipase, a member of the lipase-3 family.