Cloning And Sequence Analysis Of Antimicrobial Peptide Genes Of The Tick Rhipicephalus Haemaphysaloide

Ticks are obligate haematophagous ectoparasites, they distribute widely and are the significant vectors transmitting a great number of pathogens of human and animals, such as arboriruses, rickettsiae, spirochetes and parasitic protozoa. At present, there are about 110 kinds of ticks in China, and more than 10 kinds of ticks have evident medical significance. The research of relationship between ticks and pathogens is important for preventing and curing the sick of ticks. The antimicrobial peptide is one kind of effective molecules in nonspecific innate immunity.Firstly, we obtained the M200 and M50 full-length sequences from rapid amplification of cDNA ends protocol, the M200 gene had 542bp edcoding 100 amino acid residues with 9.85kDa predicted molecular weight and showed a rather high homology (46.2% cDNA identities) with an antimicrobial peptide Ixodidin from the tick Boophilus microplus. The M50 gene had 410bp edcoding 100 amino acid residues with 9.0kDa predicted molecular weight and showed a low homology with Protease inhibitor of Anisakis simiplex. The sequence analysis of M200 and M50 also showed a special cysteine-stabilizedαβmotif (C…CXXXC…C…CXC), which is a common character in most cysteine-rich antimicrobial peptides。Secondly, the expression patterns in the different organs and developmental stages were analysised by the reverse transcription polymerase chain reaction method (RT-PCR), the results suggested the expression of M200 gene were expressed in the salivary,gut, shell, eggs, unfed larva, unfed nymphae and adult. the expression of M50 gene were expressed in the salivary,gut, shell, unfed larva, adult. But no in the eggs, and unfed nymphae.The expression of M50 and M200 genes were both up-regulated by blood feeding, which indicated that their expression were related with hemophagia. Thirdly, the recombinant proteins of GST-M200 and GST-M50 were expressed by pGEX-4T-1 vector in E.coli and the fused protein were purified by GST Sepharose. The result indicated that it can be expressed in high level in E.coli.In a word, this study reported cloning and expression of two new genes from the tick Rhipicephalus haemaphysaloides. Bioinformative analysis showed that they are two genes coding two antimicrobial peptides. This research will be an important foundation for the biological functional studies.