Cloning And Expression Of Troponin I Gene From Rhipicephalus Haemaphysaloides

Ticks are obligate ectoparasites infest many hosts and they are the significant vectors transmitting a great number of pathogens of human and animals, such as arboviruses, rickettsiae, spirochetes and parasitic protozoa. To find a new protective antigen for anti-tick vaccine, a expressed sequence tag (EST) was selected from the salivary gland cDNA library of partially fed female Rhipicephalus haemaphysaloides(R.haemaphysaloides),and its full-length sequence was obtained from rapid amplification of cDNA ends(RACE) protocol. Meanwhile we analyzed its distribution in the tick body by the reverse transcription polymerase chain reaction(RT-PCR) ,and checked whether this gene contained intron. Finally, the gene was cloned into vector pET-32a(+)and expressed in E.coli BL21 (DE3) induced by IPTG, and we detected the result of expression by Western blot method. The results revealed that a troponin I(TnI)-like EST was selected from the cDNA library and the full sequence was obtained from 5-RACE protocol. The full length of the gene was 852bp, edcoding 219 amino acid residues with 25kDa predicted molecular weight. The predicted amino acid sequence of the gene show a rather high homology(84.47% amino acid identities)with Haemaphysalis longicornis(H.longicornis) TnI (GenBank,GI:14041807).Furthermore, the actin-binding domains of troponin I in predicted amino acid sequence, just the same as H. longicornis ,at the amino acid residues 147-167, suggesting the gene is K haemaphysaloides TnI gene. RT-PCR analysis revealed that the TnI gene was expressed in eggs, larva, nymphae.adult .shell, salivary gland and gut respectively. The genomic DNA encoding TnI was obtained from PCR protocol using Khaemaphysaloides genomic DNA as complete DNA, and sequence analysis showed that there was no intron. 45 kDa recombine protein of Tn-I was expressed in E.coli with high level .Western blot analysis suggested that the rabbit anti-TnI serum can recognize corresponding protein in Khaemaphysaloides, and there were two bands between 22kDa to 36kDa, suggesting that native TnI may have two isoformsat the same time in tick. To sum up, this study cloned and expressed a TnI gene from Khaemaphysaloides for the first time and researched the function in a pilot study, this will be the foundation to further study.