[
2008]
"H11 was best characterized as "Hidden antigen", inducing very high levels of antibody-mediated protective immunity, on average a greater than 90% reduction in faecal egg counts (FECs) and >75% reduction in worm burdens. However, recombinant forms of H11, expressed in E. coli and insect cells using recombinant baculovirus, have so far failed to induce protection level similar to that achieved using native antigen. Studies indicate that conformational epitopes and/or glycosylation may be involved in protection.In our study, cDNA of H11 of Haemonchus contortus was cloned and identified via RT-PCR. 2960 bp of cDNA sequence of H11 were amplified from Haemonchus contortus ZJ strain, containing 2919 bp of the integrated open reading frame sharing 99.5% identity with that in database (Smith, 1997). The predicted protein possessed 972 amino acids containing five significant amino acid substitutions of Phe-184 to Leu, Lys-526 to Arg, Glu-561 to Lys, Ser-815 to Phe and Gly-881 to Glu.Due to the probable role of conformational epitopes and glycosylation in protection, a typical region with 347 amino acids from Y224 to E570 was chosen to further study, containing two N-glycosylation sites and one zinc-binding region, corresponding to partial cDNA of H11 from T670 to G1710 expressed in BL21 (DE3).By the alignment analysis between H11 and its homolog in C. elegans and C. briggsae, a partial genomic DNA sequence spanning 4806 bp was amplified using specific primers (HC-GSF and HC-GSR), referring to the patterns of introns and exons of its homologous genes in C. elegans and C. briggsae.Sequence analysis demonstrated typically GT-AG consensus sequences at each intron-exon splice junction and 11 exons were separated by 10 introns in this gemomic DNA sequence, corresponding to 1162 bp cDNA of H11. The 2810 bp 5' flanking region was amplified using the "genome walker" protocol. Except normal transcriptional factors such as TATA box, CCAAT box, more than nine GATA boxes were present in this region, which probably played a certain role in transcription of H11 gene. The antecedent characterizations of H11 may interpret why recombinant antigen failed to induce active protection. "