These models were used thereafter for mapping conformational epitopes. Structural studies with circular dichroism showed that chimer proteins have slightly more secondary structures than rBoNT/E-HCC. Conclusion The immunological results suggested that the above-mentioned identical region in rBoNT/E-HCC is more exposed. Circular dichroism, computational protein modeling and hydrophobicity predictions indicated a more exposed location for the identical region in rBoNT/E-HCC than the Isosakuranetin chimer protein, which is strongly in agreement with immunological results. strain [3]. Botulism syndrome is classified into three forms: the food-born, wound, and infant (intestinal) botulism [1, 3]. BoNT are initially produced as a stable complex of approximately 900 kDa and then divided into a 150-kDa neurotoxin and non-toxic components [1]. The 150-kDa neurotoxin consists of two polypeptide chains: a light chain (50 kDa) and a heavy chain (100 kDa), which are linked through a disulfide bond [4]. The light chain is organized as an N-terminal catalytic domain, while the heavy chain comprises an internal translocation domain and a C-terminal receptor binding domain. The heavy chain, via receptor-mediated endocytosis, mediates translocation of light chain across the endosomal membrane into the cytosol. BoNT recognize nerve membranes by binding to two components: a group of membrane glycophospho-lipids called gangliosides and specific protein receptors such as synaptotagmin (for BoNT/D and G) or synaptic vesicle membrane protein, SV2 (for BoNT/A and E) [5, 6]. The light chain is a protease that cleaves target proteins in nerve cells such as synaptosomal-associated protein of 25-kDa and vesicle membrane protein synaptobrevin. Cleavage of these proteins causes the blockage of acetylcholine release and finally neuroparalysis [7 , 8]. Vaccination against botulism by toxoids has some limitations, including the need for specific equipments which leads to high cost, the low yield of toxin production by strain, the danger of handling, and the potential side effects and unexpected immunological reactions. Isosakuranetin To prevent botulism, researchers have been recently interested in using recombinant BoNT-based proteins as vaccine [9-11]. These types of vaccine have resolved many previous concerns related to use of toxoids. An example of these recombinant proteins is based on BoNT-binding domains with multivalent and monovalent antigenic properties [12]. Antibodies against these recombinant vaccines are proven to be effective in neutralizing BoNT effects [12]. The multivalent vaccines are more preferable than monovalent vaccines due to their ability to immunize against multiple neurotoxin serotypes. Here, we study two of these binding domain-based recombinant proteins whose BoNT neutralizing ability has been previously reported. These proteins include a multivalent chimer protein (187 amino acid) which is composed of serotypes A, B and E binding subdomains [13] and a monovalent recombinant protein (259 amino acids) which contains 93 amino acid residues of C-terminal heavy chain of BoNT type E (rBoNT/E-HCC) [14]. Both of these proteins have an identical region (48 aa) that contains one of the Isosakuranetin most important BoNT/E epitopes (YLTHMRD sequence) [12]. The protein sequences and their homology have been depicted in Figure 1. In this study, the scale of antibody production against two above-mentioned recombinant proteins in rabbits was compared. Furthermore, we characterized some features of these vaccines as a criterion of multivalent and monovalent vaccine comparison by ELISA. Finally, we further confirmed the results of other studies using circular dichroism and molecular modeling [15]. MATERIALS AND METHODS All molecular biology grade chemicals and bacterial culture media were from Merck (Germany). Chemical agents for nickel nitrilotriacetic acid agarose (Ni-NTA) resin were from Qiagen (USA). LB powder was obtained from Difco Mouse monoclonal to SKP2 (Sparkes, MD, USA). The Isosakuranetin pET-contained Comparison of amino acid sequences of rBoNT/E-HCC and chimer protein (Fig. 1) was carried out using the ClustalW program (http://www.ebi.ac.uk/Tools/clustalw2/index.html) [17]. Open in a separate window Fig. 1 Sequence alignment of recombinant C-terminal heavy chain of BoNT/E (rBoNT/E-HCC) and chimer protein. The amino acid sequences are numbered from the aminoterminal of proteins. Different parts of the proteins have been depicted with different colors. Isosakuranetin Chimer protein is composed of a tag of 58 aa from BoNT/A (BoNT/A subdomain), 61 aa from BoNT/B (BoNT/B subdomain), and 48 aa from BoNT/E (BoNT/E subdomain) from N- to C-terminal, respectively. rBoNT/E-HCC is composed of a tag, and 93-aa of BoNT/E from N- to C-terminal, respectively. The red arrow (48 aa) depicts a region of chimer protein, which is exactly similar to 48-aa sequence of rBoNT/E-HCC (the identical region). The purple box shows the active BoNT/E-epitope (YLTHMRD sequence) of the proteins. Trx tag shows a Trx?Tag? thioredoxin protein in pET 32 [16]. For interpretation of the references to color in this text, the reader is referred to.