Furthermore, an antigen-antibody chimera was reported to provide

Furthermore, an antigen-antibody chimera was reported to provide higher expression levels, better selleck compound yields, and increased stability in plant expression systems [2, 17]. In the present study, 3 different recombinant human colorectal cancer antigen GA733 genes were expressed in a tobacco (Nicotiana tabacum) plant expression system: GA733 fused to the immunoglobulin Fc fragment (GA733-Fc), GA733-Fc with KDEL (GA733-FcK), and GA733 with KDEL (GA733K). The stability and functionality of these colorectal cancer vaccine candidates were confirmed by western blot analysis and ELISA, respectively. In order to understand the fusion of Fc to GA733 and its functionality, the immunogenicity of recombinant GA733-Fc with oligomannose glycosylation was investigated in mice. 2. Materials and Methods 2.1.

Construction of the Plant Expression Vector The synthetic DNA sequence encoding GA733 (Gln38-Lys279, GenBank accession no. “type”:”entrez-nucleotide”,”attrs”:”text”:”AY189981″,”term_id”:”28864237″,”term_text”:”AY189981″AY189981) was modified by N-terminal extension with a 30-aa plant ER signal peptide (MATQRRANPSSLHLITVFSLLAAVVSAEVD) from Nicotiana plumbaginifolia and C-terminal extension with an ER retention signal (KDEL). The recombinant chimeric protein GA733-Fc was generated by fusing GA733 to the Fc fragment of human IgG1 (Val97-Gly328, GenBank accession no. “type”:”entrez-nucleotide”,”attrs”:”text”:”AY172957″,”term_id”:”27728676″,”term_text”:”AY172957″AY172957). The Lys279 (C-terminus of GA733) was followed by the Val97 (N-terminus of Fc fragment of human IgG1).

The GA733-Fc-encoding sequences were placed under the control of the enhanced cauliflower mosaic virus (CaMV) 35S promoter and tobacco etch viral 5��-leader sequence (TEV). The GA733, GA733-Fc, and GA733-FcK expression cassettes were subcloned into the HindIII sites of the binary plant transformation vector pBIN-Plus to yield pBI GA733K, pBI GA733-FcK, and PBI GA733-Fc, respectively (Figure 1(a)). Figure 1 Schematic diagram of plant expression vectors and recombinant proteins. (a) Gene expression cassettes of GA733K, GA733-FcK, and GA733-Fc. E/35S-P, the Cauliflower mosaic virus 35S promoter with duplicated enhancer region; TEV, untranslated leader sequence … 2.2. Plant Transformation Recombinant vectors were introduced into the Agrobacterium tumefaciens strain LBA4404 by electroporation.

Transgenic GSK-3 tobacco plants were generated by Agrobacterium-mediated transformation [11]. Transgenic tobacco lines were selected on Murashige and Skoog medium (Dachfu, Haarlem, Netherland) containing 100mg/L kanamycin. Transgenic plantlets were then transferred to soil in a growth chamber at a constant temperature of 23��C and 70% humidity and were maintained under a 14:10h light-dark cycle. Transgenic and nontransgenic N. tabacum plants were grown in a greenhouse under controlled conditions. 2.3.

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