?(Fig

?(Fig.9d).9d). the host, migrate into the bile duct and then develop into adult worms [6]. has been classified as one of the definite Group 1 carcinogens by the International Agency for Research on Cancer (IARC) [7]. However, it is difficult Fesoterodine fumarate (Toviaz) to dissuade people from eating raw fish which is deeply rooted in the epidemic area and the molecular mechanism by which the causes pathological changes is still poorly understood at present [4]. Hence, effective prevention tactics like vaccines and new antiparasitic drugs are urgently needed. Accumulating evidence showed that immune responses in the intestinal mucosa and bile duct of hosts provoked by infection played important roles in immunotoxicity and growth inhibition on antigen may be a convenient, inexpensive and needle-free strategy to protect humans or animals from infection. Nevertheless, oral immunization suffers from proteolysis, degradation and immune tolerance in the gastrointestinal tract, which may lead to poor immune response [11]. Choosing the ideal vehicles for the delivery of heterologous antigens to extreme environments such as the gastrointestinal tract will overcome these shortcomings. Extensive research demonstrated that spores of are an ideal Fesoterodine fumarate (Toviaz) platform for antigen delivery for oral vaccines [12C14]. First, protected by multiple layers of cortex proteins, the spore can survive in the presence of excessive temperature, desiccation, lytic enzymes and toxic chemicals, UV irradiation and pH [15]. In addition, utilizing the cortex coat proteins of the spore (e.g. CotB, CotC and CotG) as the anchoring protein, heterologous antigen can be stably displayed on the surface of Fesoterodine fumarate (Toviaz) spore [16]. Additionally, the spore is non-pathogenic and noninvasive so that is currently used in probiotics and food additives for humans and other animals [17]. Moreover, spores can be used as an immune adjuvant when administered together with purified antigenic proteins [18, 19]. In our previous studies, antigen display system based on spore was successfully established and was proved to be feasible and effective [6, 20C23]. Paramyosin, a myofibrillar protein present in numerous invertebrates including helminths, is indicated as a multifunctional molecule that related to both muscle physiological contraction and immunoregulation [24, 25]. Vaccine trials with [26, 27], [28], [29] or other parasites [30C32] indicated that paramyosin was a promising vaccine candidate and elicited encouraging protective effect. In our previous study, paramyosin of (CsPmy) was confirmed to be abundantly present in the cyst wall of the metacercariae and tegument of the adult worms [33]. In addition, both the recombinant protein (rCsPmy) obtained from expression system and the eukaryotic plasmid of pcDNA3.1(+)-CsPmy could induce strong immune Fesoterodine fumarate (Toviaz) responses and resulted in significant reduction rates of worm burden and eggs per gram (EPG) in vaccination trials [33]. In the present study, taking advantage of the engineering platform we constructed before, the coding sequence of CsPmy was cloned into a PEB03-CotC plasmid. The expression of fusion protein CotC-CsPmy on the surface of spore was then detected. Both the specific local and systemic immune responses of mice were analyzed after immunized with recombinant spores intraperitoneally and orally. The protective SACS effect was also evaluated after challenging infection and the effects of recombinant spores on hepatic and intestinal functions were investigated. Methods Preparation of rCsPmy protein and antiserum BL21 (DE3) containing the pET-26b(+)-CsPmy plasmid was constructed in our previous study and routinely preserved in our laboratory [33]. It was induced with isopropyl–d-thiogalactoside (IPTG) (Sigma-Aldrich, St Louis, USA) and the inclusion bodies were dissolved in PBS containing 6 M urea. Then rCsPmy was purified with His Bind Purification kit (Novagen, Darmstadt, Germany), eluted with gradient imidazole (Sigma-Aldrich) solution. Purified rCsPmy were renatured, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), as described before [33]. Then rCsPmy was emulsified with complete Freunds adjuvant and subcutaneously injected to SD rats. Each animal was given 200 g recombinant protein for the first injection, and 100 g recombinant protein emulsified with incomplete Freunds adjuvant was given for the next two boosters at a 2-week interval. 2 weeks after the last injection, the rat sera were collected and stored at -80 C after determination of antibody titer by ELISA as described before [33]. Construction of PEB03-CotC-CsPmy To display CsPmy on the surface of (WB600) spores, the full-length coding sequence of CsPmy (GenBank: “type”:”entrez-nucleotide”,”attrs”:”text”:”EF071860.1″,”term_id”:”126116627″,”term_text”:”EF071860.1″EF071860.1) was connected to the 3′ end of the coding sequence of CotC, a component of spore coat proteins, to construct fusion gene of CotC-CsPmy. Briefly, PEB03-CotC, a plasmid previously constructed and routinely stored in our laboratory, was linearized by using adult Fesoterodine fumarate (Toviaz) with specific primers. The sequence of the.