Nevertheless, systemic toxin isn’t indicative of a standard infection scenario [37]. outcomes of the research reveal that recombinant proteins formulated with RBDs of poisons could be useful for vaccine advancement. Additionally, we found that an RBD-TcdA/B vaccine can elicit a stronger humoral immune response and provide better immunoprotection than the univalent vaccines. This RBD vaccine candidate conferred significant protection against disease symptoms and death caused by toxins from a wild-type strain. or [1] is a gram-positive, spore-forming, anaerobic rod bacterium that barely received attention before it was discovered to be associated with pseudomembranous colitis in 1978 [2,3]. It causes more than 25% of the cases of antibiotic-associated diarrhea [1,2]. The current prognosis of infection (CDI) is alarming, with a mortality rate between 3% and 15% and a recurrence rate ranging from 12% to 40% [3]. In the United States, CDI is responsible for 500,000 infections [4], approximately 14,000 deaths, and healthcare costs LXR-623 exceeding $3 billion [5] per year. Most CDIs are caused by a new strain belonging to ribotype 027 (RT027), which spread worldwide in 2003 and resulted in a large number of deaths [6,7]. It has been on the legal surveillance list in many countries, including China, as a pathogen causing infectious disease [8,9]. Besides the use of effective antibiotics and fecal microbiota transplantation (FMT), immunoprophylaxis is generally considered an effective and preferred control strategy [10,11]. Toxin A and toxin B are major virulence determinants of this bacterium. Immunity to these two LXR-623 toxins provides protection by inhibiting the action of the toxins, which can effectively prevent serious illness caused by CDI [6,12]. Toxin A is an enterotoxin and cytotoxin with very high toxicity; toxin B is a potent cytotoxin with a toxicity 100C1000 times as high as that of toxin A [13,14]. In vaccine development, we search for vaccine candidates with high immunogenicity but low toxicity, so we needed to make changes to the toxins for the development of the new vaccine type. Both toxin A and toxin B are single-chain polypeptides consisting of a highly conserved N-terminal catalytic domain that can modify GTPases, a translocation domain, an autoproteolytic domain, and a receptor-binding domain (RBD) [15,16]. The three-dimensional structures of the glucosyltransferase domain and portions of the RBD have been well defined. The only known native receptor of toxin A is the Gal(1,3)Gal(1,4)Glcgly can sequence, which is not found on human intestinal epithelial cells [16,17]. Human receptors of toxin B have been identified [18,19,20]. Crystal structures of toxin fragments indicate that the toxin A RBD possesses seven carbohydrate binding sites [21], and toxin B is predicted to have four sites [16]. Binding to receptors by RBDs is essential for the toxicity of toxins A and B; inhibiting the binding can protect a host from illness caused by the toxins. Usually, the RBDs are known to be nontoxic and immunogenic, and RBDs were used in vaccine candidates against, among others, LXR-623 SARS Coronavirus [22] and [23]. In our study, we constructed recombinant proteins containing the full-length RBD from toxin A or toxin B (named RBD-TcdA and RBD-TcdB, respectively) of reference strain “type”:”entrez-protein”,”attrs”:”text”:”VPI10463″,”term_id”:”1642177071″,”term_text”:”VPI10463″VPI10463 (ribotype RT087). We immunized mice with the recombinant RBD-TcdA and/or RBD-TcdB to test their immunogenicity and protective effect against toxins extracted from the wild-type strain American Rabbit Polyclonal to ACBD6 Type Culture Collection (ATCC) BAA-1870, which belongs to ribotype 027 (RT027). 2. Materials and Methods 2.1. Mice, Cells, and Bacteria All animal studies were conducted in accordance with the Beijing Institute of Microbiology and Epidemiology Animal Care and Use Committee (2012-06-21-02) guidelines. C57BL/6 wild-type mice (6 weeks old, weighing 14C16 g) were obtained from our institute Laboratory Animal Center (Beijing, China). All experimental mice were bred in a specific pathogen-free facility at our institute. The reference strain “type”:”entrez-protein”,”attrs”:”text”:”VPI10463″,”term_id”:”1642177071″,”term_text”:”VPI10463″VPI10463/RT087 and wild-type strain ATCC BAA-1870/RT027 of were purchased from the American Type Culture Collection (ATCC) center. We sequenced the genes encoding toxin A and toxin B of our wild-type strain. A Vero cell line was kept in our lab. 2.2. Protein Expression and Purification The amino acid sequence corresponding to the RBDs of RBD-TcdA (strain “type”:”entrez-protein”,”attrs”:”text”:”VPI10463″,”term_id”:”1642177071″,”term_text”:”VPI10463″VPI10463, residue positions 1867C2708) and RBD-TcdB (strain “type”:”entrez-protein”,”attrs”:”text”:”VPI10463″,”term_id”:”1642177071″,”term_text”:”VPI10463″VPI10463, residue positions 1751C2366) were amplified by PCR from the genome of BL21(DE3) (Transgen, Bejing, China) for subsequent protein expression and purification. The recombinant proteins were expressed in successfully transformed bacteria by induction with isopropyl–D-thiogalactopyranoside (IPTG) in LuriaCBertani medium and then purified with a Ni2+-HiTrap chelating 5 ml prepacked column (GE Healthcare Bio-Sciences Corp, Piscataway, NJ, USA), using imidazole as the elution reagent. The.