Isolation and immunolocalization of a putative protective antigen (p26/23) from adult Haemonchus contortus

Abstract

The putative protective antigen, p26/23, from adult Haemonchus contortus was isolated. A soluble extract from adult helminths obtained from the abomasa of hyperinfected (12,000 infective larvae) female Manchego lambs and treated with a mixture of protease inhibitors was subjected to affinity chromatography (hexylgluthatione) to eliminate the enzyme gluthatione S-transferase. The eluate was analyzed by electrophoresis under denaturing and reducing conditions (sodium dodecyl sulfate polyacrylamide gel electrophoresis), electrotransferred to nylon membranes, and assayed by Western blot with sera from immunized lambs. The bands recognized by the lambs' sera corresponding to proteins with a molecular weight of 23-26 kDa (p26/23) were excised, eluted, and separated by reverse-phase chromatography. This allowed the isolation of a single protein, which was expressed in both infective larvae (L3) and the adult stage of the parasite. The first 20 amino acids of the N-terminal end of the purified protein were determined. The partial amino acid sequence revealed a 100% identity with the N terminus of one of the peptides present in the p26/23 immunoprotective fraction previously tested by us against sheep haemonchosis. No significant homology with any reported sequence was found except for the deduced sequence of a hypothetic H.contortus protein (HCC00515). Immunolocalization studies showed that the protein was expressed in the hypodermic chords of the nematode.

Intranasal Immunization of Lambs with Serine/Threonine Phosphatase 2A against Gastrointestinal Nematodes

“…Greatest efficacy has been achieved with vaccines that use irradiated larvae (5), but the logistical problems involved in their production and administration (6) have led to a search for native antigens or recombinant ones (7). The latter are easier to produce and distribute, and many of them are proteases, cysteine proteases, metalloproteases, aspartyl proteases (PEPs), all proteins from the helminth intestine, and some with unknown biological functions (8)(9)(10)(11). Due to the homology in their sequences, some of these antigens have proved to be effective against different nematode species (3,12,13).…”

Immunization against Lamb Haemonchosis with a Recombinant Somatic Antigen ofHaemonchus contortus(rHcp26/23)

“…Purification of p26/23 involved the sequential use of affinity chromatography with S-Hexyl Glutathione and reverse-phase chromatography (Vydac 214TP5415 column) [14, 15]. Proteins were analyzed by 15% polyacrylamide gel electrophoresis with 1% sodium dodecyl sulphate (Merck) and 5% mercaptoethanol (Merck) (SDS-PAGE).…”

“…RT-PCR was carried out in two independent steps: synthesis of cDNA (1st Strand cDNA Synthesis Kit, AMV, Roche) using hexanucleotide random primers in 40  μ L final volume and amplification by PCR following the procedures described below. The partial N-terminal amino acid sequence of the protein p26/23 purified from adult H. contortus [14] showed 85% identity with the hypothetical protein HCC00515 from H. contortus (NEMBASE), deduced on the nucleotide sequence from a cDNA library. Therefore, the available N-terminus sequence of this protein and the nucleotide sequence of HCC00515 were used as template to design two primers with BamHI and HindIII restriction targets: FBamHI (5′  GGA TCC GCA GGA CTG TTC GCA CAT  3′) and RHindIII (5′  AAG CTT TCA GTC TTT CGC GGA CTT G  3′).…”

“…Immunolocalization of p26/23 in adult H. contortus was carried out following the methods described in [14]. Sections were washed with PBS, blocked with 5% foetal bovine serum (Sigma) in PBS for 1 h at room temperature, and incubated with rabbit anti-p26/23 and pooled anti-rHcp26/23 sera (1 : 1000 in PBS).…”

“…Our group has already demonstrated that a protein fraction obtained from adult worms, p26/23, was immunoprotective in 3.5–5-month-old lambs challenged with H. contortus [4, 13]. Recently, the content of this fraction has been analyzed, and the major protein present has been purified, immunolocalized, and partially sequenced [14]. In the present paper we present the cloning and expression of the recombinant protein, rHcp26/23.…”