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Immunization against Lamb Haemonchosis with a Recombinant Somatic Antigen of Haemonchus contortus (rHcp26/23).

García-Coiradas L, Angulo-Cubillán F, Valladares B, Martínez E, de la Fuente C, Alunda JM, Cuquerella M - Vet Med Int (2010)

Bottom Line: Our previous results showed that a soluble fraction from adult stages of the nematode (p26/23) induced partial protection against challenge.Recombinant DNA technology was applied to obtain a synthetic protein (rHcp26/23).Immunization did not induce any significant protection after challenge with 16000 infective larvae of H. contortus, and comparable values for parasite faecal egg output, packed cell volume, and abomasal parasite burdens were found in vaccinated and control animals.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain.

ABSTRACT
Haemonchosis, caused by the abomasal nematode Haemonchus contortus, is a common parasitic disease of sheep. Our previous results showed that a soluble fraction from adult stages of the nematode (p26/23) induced partial protection against challenge. Recombinant DNA technology was applied to obtain a synthetic protein (rHcp26/23). Immunological assays (ELISA, Western blotting, and immunolocalization), using sera from lambs immunized with p26/23, confirmed the identity of the recombinant protein and demonstrated that the synthetic protein is equivalent to the purified protein employed in the previous immunoprophylaxis studies. Vaccination of lambs with 300 mug of rHcp26/23 and Freund's adjuvant elicited a notable specific antibody response. Immunization did not induce any significant protection after challenge with 16000 infective larvae of H. contortus, and comparable values for parasite faecal egg output, packed cell volume, and abomasal parasite burdens were found in vaccinated and control animals.

No MeSH data available.


Related in: MedlinePlus

SDS-polyacrylamide gel  of Coomassie blue stained fractions during purification under nondenaturing conditions of rHcp26/23. Lane 1: MW markers in kDa; 2: uninduced E. coli culture; 3: induced E. coli culture;  4: starting sample; 5: unbound fraction; 6: eluate with buffer I (without imidazole); 7: eluate with  20 mM imidazole; 8: eluate with 250 mM imidazole.(*) purified rHcp26/23.
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fig1: SDS-polyacrylamide gel of Coomassie blue stained fractions during purification under nondenaturing conditions of rHcp26/23. Lane 1: MW markers in kDa; 2: uninduced E. coli culture; 3: induced E. coli culture; 4: starting sample; 5: unbound fraction; 6: eluate with buffer I (without imidazole); 7: eluate with 20 mM imidazole; 8: eluate with 250 mM imidazole.(*) purified rHcp26/23.

Mentions: The optimal overexpression of rHcp26/23 in the pQE30/E. coli M15 system was achieved after induction of bacterial cultures with 0.02 mM IPTG for 2 h (not shown). Electrophoretic analysis of supernatants and sediments obtained from the cultures of transformed E. coli showed that most of the protein was solubilized in the first washing with 8 M urea. Affinity chromatography allowed the purification of the recombinant protein between pH 5.9 and 4.5. Similarly, extraction under nondenaturing conditions rendered, after affinity chromatography, a single band, as assessed by SDS-PAGE (Figure 1, lane 8).


Immunization against Lamb Haemonchosis with a Recombinant Somatic Antigen of Haemonchus contortus (rHcp26/23).

García-Coiradas L, Angulo-Cubillán F, Valladares B, Martínez E, de la Fuente C, Alunda JM, Cuquerella M - Vet Med Int (2010)

SDS-polyacrylamide gel  of Coomassie blue stained fractions during purification under nondenaturing conditions of rHcp26/23. Lane 1: MW markers in kDa; 2: uninduced E. coli culture; 3: induced E. coli culture;  4: starting sample; 5: unbound fraction; 6: eluate with buffer I (without imidazole); 7: eluate with  20 mM imidazole; 8: eluate with 250 mM imidazole.(*) purified rHcp26/23.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2902039&req=5

fig1: SDS-polyacrylamide gel of Coomassie blue stained fractions during purification under nondenaturing conditions of rHcp26/23. Lane 1: MW markers in kDa; 2: uninduced E. coli culture; 3: induced E. coli culture; 4: starting sample; 5: unbound fraction; 6: eluate with buffer I (without imidazole); 7: eluate with 20 mM imidazole; 8: eluate with 250 mM imidazole.(*) purified rHcp26/23.
Mentions: The optimal overexpression of rHcp26/23 in the pQE30/E. coli M15 system was achieved after induction of bacterial cultures with 0.02 mM IPTG for 2 h (not shown). Electrophoretic analysis of supernatants and sediments obtained from the cultures of transformed E. coli showed that most of the protein was solubilized in the first washing with 8 M urea. Affinity chromatography allowed the purification of the recombinant protein between pH 5.9 and 4.5. Similarly, extraction under nondenaturing conditions rendered, after affinity chromatography, a single band, as assessed by SDS-PAGE (Figure 1, lane 8).

Bottom Line: Our previous results showed that a soluble fraction from adult stages of the nematode (p26/23) induced partial protection against challenge.Recombinant DNA technology was applied to obtain a synthetic protein (rHcp26/23).Immunization did not induce any significant protection after challenge with 16000 infective larvae of H. contortus, and comparable values for parasite faecal egg output, packed cell volume, and abomasal parasite burdens were found in vaccinated and control animals.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain.

ABSTRACT
Haemonchosis, caused by the abomasal nematode Haemonchus contortus, is a common parasitic disease of sheep. Our previous results showed that a soluble fraction from adult stages of the nematode (p26/23) induced partial protection against challenge. Recombinant DNA technology was applied to obtain a synthetic protein (rHcp26/23). Immunological assays (ELISA, Western blotting, and immunolocalization), using sera from lambs immunized with p26/23, confirmed the identity of the recombinant protein and demonstrated that the synthetic protein is equivalent to the purified protein employed in the previous immunoprophylaxis studies. Vaccination of lambs with 300 mug of rHcp26/23 and Freund's adjuvant elicited a notable specific antibody response. Immunization did not induce any significant protection after challenge with 16000 infective larvae of H. contortus, and comparable values for parasite faecal egg output, packed cell volume, and abomasal parasite burdens were found in vaccinated and control animals.

No MeSH data available.


Related in: MedlinePlus