Characterization of two cDNAs encoding serine proteinases from the hard tick Haemaphysalis longicornis.
Host vaccination against tick infestation is at present the most practical and sustainable alternative tick control method to the current acaricide use which has serious limitations. However the success of this approach to control ticks depends upon the identification of target vaccine antigens. Members of the serine proteinase gene family may represent an interesting group of proteins to target as candidate antigens because of their involvement in regulation of many physiological functions and development processes in a wide range of organisms. We used RT-PCR with the 3' and 5' RACE to clone two cDNAs encoding full-length serine proteinases from the hard tick, Haemaphysalis longicornis. RT-PCR degenerate primers were designed from amino acid sequences surrounding active sites, His(57) and Ser(195) conserved among most known serine proteinase. Gene specific primers designed from nucleotide sequences of the RT-PCR products were used to prime the 3' and 5' RACE. Southern blotting analysis showed that both HLSG-1 and -2 are single copy. The 2 cDNAs, HLSG-1 and -2 are 1.2 and 1.0 kb long in size with open reading frames encoding polypeptides with 37.7 and 31.2 kDa predicted molecular mass respectively. Northern blotting analysis of total RNA from unfed and partially fed whole ticks showed that the expression of mRNAs for both HLSG-1 and -2 was induced by blood feeding. Expression analysis by RT-PCR showed that both HLSG-1 and -2 are expressed in other tick organs in addition to salivary glands and midguts. The 6 serine proteinase consensus cyteine residues are well conserved in both HLSG-1 and -2. We have discussed our findings with respect to tick vaccine development research.
Mulenga
A
A
Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan.
Sugimoto
C
C
Ingram
G
G
Ohashi
K
K
Misao
O
O
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002417:Cattle; D003001:Cloning, Molecular; D018076:DNA, Complementary; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000235:genetics*; D013987:Ticks / Q000201:enzymology* / Q000235:genetics
D018076:DNA, Complementary; C432950:HLSG-1 protein, Haemaphysalis longicornis; C432951:HLSG-2 protein, Haemaphysalis longicornis; D012697:Serine Endopeptidases