The gene structure and promoter region of the vaccine target aminopeptidase H11 from the blood-sucking nematode parasite of ruminants, Haemonchus contortus.
Aminopeptidase H11, an integral membrane glycoprotein present only in the gut of Haemonchus contortus, could provide substantial protection as shown by 90% reduction in fecal egg counts, while its recombinant version expressed in E. coli induced little. To investigate the characteristics further, we amplified mRNA of H11 gene via reverse transcriptase polymerase chain reaction, followed by isolation of its 1,517-bp 5'-flanking region and determination of its genomic organization. The H11 gene contained 25 exons separated by 24 introns and spans 14,959 bp of genomic DNA. Analysis of the 1,517 bp 5'-flanking region of the H11 gene revealed a putative "TATA-less" promoter. Partial sequences of the last exon and its 3'-UTR of H11 isoform H11-4 were also identified upstream to the H11 gene with the same transcription orientation. The 1,517-bp 5'-flanking region and part of the first exon of the H11 gene were subcloned into the vector upstream of green fluorescence protein reporter gene and microinjected into the gonads of Caenorhabditis elegans. The transformed animals exhibited fluorescence in the distal intestine in the L4 larvae stage and adult worms. This study characterized gene structure of aminopeptidase H11, demonstrated different transcriptional pattern of its promoter region between free-living and blood-sucking nematode species, and highlights the utility of C. elegans as a heterologous system to study the biology roles of H11 isoforms.
Zhou
Qian-Jin
QJ
Key Laboratory of Animal Epidemic Etiology & Immunological Prevention of Ministry of Agriculture, Institution of Preventive Veterinary Medicine, Zhejiang University, Hangzhou 310029, China.
Zhang
Hong-Li
HL
Jiang
Xiao-Lei
XL
Du
Ai-Fang
AF
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D016386:Chromosome Walking; D017238:Genes, Helminth; D006189:Haemonchus / Q000235:genetics* / Q000472:pathogenicity; D015801:Helminth Proteins / Q000235:genetics*; D007527:Isoenzymes / Q000235:genetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012418:Ruminants / Q000469:parasitology; D016415:Sequence Alignment; D014612:Vaccines
D015801:Helminth Proteins; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D014612:Vaccines; D000626:Aminopeptidases