| Tick is a kind of hematophagous ectoparasite. By feeding on a host, they can obtain a large amount of blood, the weight of which is more than100times that of the unfed ones. Because of its blood meal and the spread of pathogens among the host during the blood-feeding, ticks become a serious threat to animal husbandry development and human health. Tick control is drawing increasing attention of scientists around the world. There are complicated interactions between host and ticks, in which the circumvention of tick from the host immune response system is one of the critical points that researchers are focusing on. With further understanding of ticks, several kinds of proteins in ticks are found to be involved in the regulation of host immune system, and are great significant in protection against tick and tick-borne diseases as well as in medical material development. Immunoglobulin G binding proteins (IGBPs) are such kind of molecules. Researchers have found that they can bind with host IgG and exlude it from tick body, but the detailed mechanism is not clear.Rhipicephalus haemaphysaloides belongs to Ixodidae and is a major kind of ticks in South China. RH-IGBP is one of IGBPs which was cloned from R. haemaphysaloides salivary gland. Previous study have analyzed its expression in tick different developmental stages by RT-PCR, and investigated the anti-tick activity of the recombinant protein rIGBP. As a result, it was expressed in all tick developmental stages, and the recombinant protein showed a protection ability against ticks.For a more detailed description on the bio-function, in this study, we performed works on RH-IGBP from five aspects based on the results we have obtained. First, after expression in E.coli, the recombinant protein was used to immunize mouse and obtained anti-RH-IGBP antibody, then the antibody was used to detect RH-IGBP in different tissues from male and female ticks. The results suggest that RH-IGBP showed a higher expression in male ticks than that in female. And the highest expression was in salivary gland of male ticks. Second, RH-IGBP was expressed in insect cells (His-IGBP) to obtain protein close to native protein, and His-IGBP’s binding activity was evaluated using IgG from seven kinds of host (including rabbit, pig, bovine, goat, mouse, dog and human), The results suggest that it can bind obviously with rabbit and pig IgG, to some extent with dog IgG,, more less with goat, bovine, and mouse IgG, but can not bind with human IgG. Then rabbit IgG fragments F(ab’)2and Fc were employed to locate the binding site, and it was demonstrated that IGBP mainly bound to IgG F(ab’)2fragment, but IGBP adhered to Fc fragment to a certain extent with the increasing of the concentration. Third, rabbits were immunized with Bovine Serum Albumin(BSA). After the third immunization, a high titer of antibody was obtained, then female and male ticks were fed on the rabbits, and ticks were pull down every day and dissected for tissues (Including haemolymph, gut, and salivary gland). By a detection of IgG of rabbit anti-BSA antibody through ELISA and Western blot analysis, the results suggested that IgG from host was transported and migrated in Rhipicephalus haemaphysaloides; and a high concentration of RH-IGBP was detected in salivary gland and haemolymph from4-day-fed male ticks. However, in female ticks, RH-IGBP was only detected in3-day-fed salivary gland. Fourth, RNA interference of RH-IGBP in male ticks was carried out, and the gene was proved to be silenced by Real-time PCR and Western blot analysis, but other parameters including engorgement time, engorged body weight, fecundity, hatchability showed no significant difference between the test and control groups, which suggested that IGBPs had no direct or obvious influence to tick growth and reproduction, or there were other compensatory mechanisms. In the end, a IGBP gene was cloned from Hyalomma asiaticum (HA-IGBP), which showed high homology to RH-IGBP (80%homology in DNA and76%homology in protein sequences). HA-IGBP has a signal peptide and possesses structure related with innate immunity. The expression of HA-IGBP in different developmental stages of tick was investigated by Real-time PCR assay, and the result shows some similarities on the expression between RH-IGBP and HA-IGBP, in another word, HA-IGBP is the most abundant in male ticks. However, HA-IGBP is also highly expressed in female ticks, which is different from that of RH-IGBP. Furthermore, HA-IGBP expression is very low in tick stages except adult ticks, but its expression is relatively high in unfed larve, which have not been verified in other tick species.Overall, considering that IGBP is still poorly studied, our research provided great information for a further explanation on the function of IGBPs. |
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