Cloning and characterization of a cDNA clone encoding calreticulin from Haemaphysalis qinghaiensis (Acari: Ixodidae)

Abstract

The application of anti-tick vaccine has been shown to be the most promising alternative strategy compared to the current use of acaricides that suffer from a number of serious limitations. The success of this method is dependent upon identification and cloning of potential tick vaccine antigens. Previously, we have cloned 21 positive clones (named from Hq02 to Hq22) by immunoscreening complimentary DNA (cDNA) libraries of Haemaphysalis qinghaiensis; however, some of those clones did not contain open reading frames (ORF). In this study, we amplified the entire sequence of Hq07 by using rapid amplification of the cDNA ends. Hq07 contains an ORF of 1,233 bp that encodes for 410 amino acid residues with a coding capacity of 47 kDa. Search of the cloned sequences against GenBank revealed that Hq07 is a calreticulin (CRT)-similar clone and designated HqCRT. Expression analysis by reverse transcription-polymerase chain reaction showed that this gene is ubiquitously expressed at different developmental stages and in different tissues of H. qinghaiensis. The gene was expressed as glutathione S-transferase-fused proteins in a prokaryotic system. Western blot analysis revealed that native HqCRT was secreted into their hosts by ticks during blood sucking. Vaccination of sheep with rHqCRT conferred protective immunity against ticks, resulting in 54.3% mortality in adult ticks, compared to the 38.7% death rate in the control group. These results demonstrated that rHqCRT might be a useful vaccine candidate antigen for biological control of H. qinghaiensis.

Artificial feeding of Rhipicephalus microplus female ticks with anti calreticulin serum do not influence tick and Babesia bigemina acquisition

“…3). As it can be perceived, phylogenetic the immunogenicity of tick CRT (Gao et al, 2008;Jaworski et al, 1995;Parizi et al, 2009;Xu et al, 2004) and, recently, antibodies against tick CRT were used as a biomarker to tick exposure (AlarconChaidez et al, 2006;Vu Hai et al, 2013). Within the present study the rCRT showed to have a weak immunogenicity in mice since compared to control mice antibody titers were no greater than 1:4000 (Fig.…”

“…4). IFA assays revealed that, as expected, CRT is dispersed in salivary glands cells (Gao et al, 2008;Jaworski et al, 1995;Kaewhom et al, 2008). CRT was also found in ovaries and, in this tissue, a predominantly cytoplasmic localization is clear in immature eggs.…”

“…The recombinant CRT protein (rCRT) was obtained with the expected molecular weight of 46 kDa (Fliegel et al, 1989;Gao et al, 2008) and then purified by affinity chromatography (Fig. 2).…”

“…Moreover, being secreted to the host it integrates the group of exposed antigens not requiring re-vaccination; thus suggesting that this protein can be integrated in an anti-tick vaccine (Kaewhom et al, 2008). CRT has been used in different immunization studies (Ferreira et al, 2002;Gao et al, 2008;Parizi et al, 2009;Rachinsky et al, 2008). Rabbits immunized with Amblyomma americanum CRT exhibited necrotic lesions in the tick bite sites, indicating that the immune reaction could disrupt the feeding cycle (Jaworski et al, 1995), vaccination of sheep with Haemaphysalis qinghaiensis rCRT conferred protective immunity against ticks (Gao et al, 2008) or anti-rBmCRT (R. microplus) and anti-rHlCRT (H. longicornis) bovine sera also recognized the native proteins in larvae extracts (Parizi et al, 2009).…”

“…CRT has been used in different immunization studies (Ferreira et al, 2002;Gao et al, 2008;Parizi et al, 2009;Rachinsky et al, 2008). Rabbits immunized with Amblyomma americanum CRT exhibited necrotic lesions in the tick bite sites, indicating that the immune reaction could disrupt the feeding cycle (Jaworski et al, 1995), vaccination of sheep with Haemaphysalis qinghaiensis rCRT conferred protective immunity against ticks (Gao et al, 2008) or anti-rBmCRT (R. microplus) and anti-rHlCRT (H. longicornis) bovine sera also recognized the native proteins in larvae extracts (Parizi et al, 2009). However, in immunization assays with rCRT in cattle against R. microplus, no effect in tick biological parameters was observed mainly due to its low immunogenic capacity in bovines (Ferreira et al, 2002).…”