Evaluation of a recombinant excretory secretory Haemonchus contortus protein for use in a diagnostic enzyme-linked immunosorbent assay.
The nematode Haemonchus contortus (H. contortus) is one of the most pathogenic and economically important parasites of sheep. A 24 kDa protein is one of the important components in H. contortus excretory/secretory (ES), which was shown to have important biological function. In our research, the cDNA of its open reading frame (ES24) was obtained and analyzed. Then the ES24 was sub-cloned into pET-30a expression vector. The recombinant vector that codes hexahistidyl peptide fusion protein (His-ES24) was transformed into Escherichia coli BL21 (DE3) strain. After induction, a high expression level of His-ES24 was found at 6h taking about 26% of the total bacterial protein analyzed by gel thin-layer scanning. The expressed His-ES24 was purified and then used in an enzyme-linked immunosorbent assay (ELISA) to detect specific antibodies in serum samples. The ELISA was able to differentiate between H. contortus-infected sheep serum and Fasciola hepatica-infected sheep serum or non-infected sheep serum. No cross-reaction was observed in sheep sera that have been experimentally infected with F. hepatica. A total of 153 field sheep serum samples conserved in our laboratory were examined using the His-ES24 ELISA, and 82 (53.6%) of them were found seropositive to H. contortus. Our results demonstrate that the prokaryotic-expressed His-ES24 might be a useful diagnostic reagent for epidemiological studies of H. contortus in sheep.
Li
Xiaojun
X
Institute of Preventive Veterinary Medicine, Zhejiang University, Hangzhou, Zhejiang, China.
Du
Aifang
A
Cai
Weiming
W
Hou
Yuhui
Y
Pang
Linhai
L
Gao
Xiang
X
D000818:Animals; D000909:Antibodies, Helminth / Q000097:blood; D000947:Antigens, Helminth / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000592:standards; D006188:Haemonchiasis / Q000175:diagnosis / Q000662:veterinary*; D006189:Haemonchus / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D015801:Helminth Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D012756:Sheep; D012757:Sheep Diseases / Q000175:diagnosis*
D000909:Antibodies, Helminth; D000947:Antigens, Helminth; D015801:Helminth Proteins; D011994:Recombinant Proteins