Arrows on the blot indicate GAM. == 2.3. structure, plant crossing, vaccine == 1. Introduction == Antigenantibody complexes (AACs) have been shown to regulate immune reactions [1,2]. Immunization using AACs has been used to enhance immunogenicity (R)-P7C3-Ome through the production of specific antibodies against immunogenic epitopes [3]. Although factors that affect the effectiveness of AACs that influence immune responses are not fully understood, several potential mechanisms have been proposed [4]. The Fc region of AAC can increase antigen uptake via the Fc receptor (FcR) that is present on antigen-presenting cells (APCs) and deliver the antigen for cross-presentation [5,6]. For this process, the roles of the Fc region of antibody have been reported, and they include relationships with both inhibitory FcRIIB and activating FcR found on the dendritic cells (DCs) and macrophages [7]. Fc, derived from the immune complex, can identify numerous effector cells of the innate immune system [8]. Focusing on a vaccine to both DCs and antigen-specific B cells for both MHC class I and MHC class II-restricted processing might generate a more diverse and powerful immune response against pathogens and malignancy cells [9]. Antibody-derived immunomodulation induced by the formation of large protein complexes results in the induction of beneficial or protective immune responses [10]. Study concerning the immune complex-mediated enhancement of immunization has been proactively carried out using animal cell systems [11,12]. However, the use of vegetation has garnered attention as an alternative expression system for biopharmaceutical recombinant protein products, such as vaccines and antibodies [13,14,15,16]. The flower expression system, which is a eukaryotic system similar to the animal cell system, is expected to communicate, fold, and assemble restorative glycoproteins like a encouraging alternative with product safety and economic benefit. Furthermore, flower genetic engineering allows for the modification of the protein structure through glycosylation to increase the effectiveness of the vaccine [17]. Therefore, plant-based vaccine candidates have been extensively analyzed for his or her potential to enhance immune reactions [18,19,20]. Recent studies reported that, as compared to the individual antigens, immune complexes via the manifestation of an antigen fused to the C-terminus of the weighty chain (HC) and light chain (LC) in animal expression systems enhance the activation of DCs and the overall immune response [11,21,22]. However, it remains to be determined whether the co-expression of antigens and antibodies in vegetation can induce the assembly of both proteins, resulting in large quaternary AACs that enhance immune responses. In flower, immune complexes have been indicated as an antigen fused to the C-terminus of weighty chain (HC) and light chain (LC) forms [23]. In this study, we shown another way to make immune complex form using the transgenic flower crossing process. Therefore, in the present study, transgenic vegetation expressing the antigen GA733 (an epithelial cell adhesion molecule (EpCAM) highly indicated in colorectal malignancy cells) fused to Fc (Space) [24,25,26], and the anti-colorectal malignancy mAb CO17-1A realizing the antigen GA733 (COP) [27,28,29,30,31,32] were crossed to express both the antigen and antibody in F1 vegetation. The GAPand COPcomplexes indicated in the flower were assessed for his or her expression, structure, and in vitro and in vivo immune functions like a vaccine. == 2. Results == == 2.1. Generation of F1 Vegetation Transporting both GAPand COPGenes == Transgenic vegetation transporting GAPand COPHCK and LC genes were acquired by Agrobacterium-mediated transformation [25,26,27,28,29,30,31,32]. The GAPencoding gene was under the control of the enhanced cauliflower mosaic disease (CaMV) 35S promoter (Ca2p) and the tobacco etch disease 5 leader sequence (TEV). COPHCK and LC were indicated from Ca2p and Pin2p promoters, respectively. F1 seeds were acquired by crossing the vegetation equally expressing GAPand COP(Number HSPB1 1), and were germinated in the presence of kanamycin to obtain (R)-P7C3-Ome the F1 flower line #4 equally expressing both GAPand COPfor AAC formation (Number 1b). == Number 1. == Schematic diagram of generation of F1 vegetation expressing both GAPantigen and COPantibody by crossing transgenic vegetation expressing GAPand COP, and their different complex protein structures. (a) Flower vectors pBI GA733-FcK (remaining) and pBI CO17-1AK (ideal) for manifestation of GAPand COPprotein encoding genes in vegetation. F1 vegetation (Space COP) were generated by cross-fertilization of two different transgenic vegetation expressing GAPand COP. (b) Schematic diagram of different (R)-P7C3-Ome complex constructions of both GAPand COPin F1 vegetation. Dimers created between solid GAPand COP(Top). The linear chain constructions between solid (R)-P7C3-Ome GAPand COP(Middle). Large quaternary circular complex constructions of GAPand COP(Bottom). These constructions are hypothetical. == 2.2. Confirmation of GAPand COPGene Insertion and Protein Manifestation in F1.