The His-tag was removed and the concentration of purified fusion protein was measured with the Bradford assay. In fusion protein group, three of ten mice failed to develop tumor and all survived at the end of the research. The excess weight of tumors in fusion protein were obviously lighter than that in additional two organizations (t = 4.73, P = 0.044;t = 6.89, P = 0.040). These findings shown that EGFRvIII-HBcAg fusion protein triggered protective reactions against tumor expressing EGFRvIII. Intro Epidermal growth element receptor (EGFR) takes on an important part in tumor cell proliferation, differentiation and survival. Increasing evidences suggest that alterations within the EGFR gene may be as important as EGFR-overexpression to induce oncogenic effects [1-3]. The most common variation is an in-frame deletion of exons 2-7 in the mRNA, resulting in a truncated mutant (epidermal growth element receptor variant III, EGFRvIII). Even though EGFRvIII is lack of a portion of extracellular ligand-binding website and can not bind to its ligand, the tyrosine kinase in the intracellular portion can be constitutively triggered, therefore leading to receptor dimerization, autophosphorylation and activation of transmission transduction cascades[4]. Because EGFRvIII is present with a high frequency in several different types of tumor and has not been recognized in normal cells, it is an ideal target for tumor specific therapy[5,6]. Among methods directed to EGFRvIII, vaccine is definitely a encouraging strategy. Recombinant protein has been intensively studied like a vaccine on the basis of genetic executive technology. Compared with peptide vaccine, recombinant protein offers many advantages such as easy manipulation, mass production and low cost. The carrier of foreign epitope is important for building of recombinant protein. Hepatitis B core protein (HBcAg) is one of the most encouraging delivery vehicles for its high-density, immunogenic demonstration of foreign epitope and its production in various manifestation systems[7]. The e1 loop in the main determinant of the core antigen is considered as the most encouraging insertion site[8]. Pep-3, a 13-amino-acid peptide related to the amino Elaidic acid acid sequence of the EGFRvIII fusion junction (LEEKKGNYVVTDH), is an immunogenic peptide that was firstly reported Elaidic acid by Moscatello[9]. In this study, foreign epitope, encoding Pep-3, was put into the immunodominant e1 loop of the HBcAg to prepare the recombinant fusion protein. Next, the antigenicity and immunogenicity of the fusion protein were recognized in vitro. Elaidic acid The protective immune reactions against tumor was evaluated inside a murine model. Materials and methods Building of recombinant manifestation plasmids The genes encoding Pep-3, HBcAg amino acid resides from 1 to 71 and from 89 to 144 were amplified by PCR, and a set of primers were outlined in Table ?Table11. Table 1 Sequence of primers utilized for PCR amplification
GenePrimers sequenceRestriction sitePep-3Sense: 5′-CCTCGAGCTGGAGGAGAAGAAAGGTAATTATGTG-3′
Antisense: 5′-CGGATCCGTGATCTGTCACCACATAATTACC-3′Xhol
BamHIHBcAg1-71Sense:5′-GGAATTCATGATTACGCCAAGCTTGGCTGCAGAGTTCCATATG-3′
Antisense: 5′-CCCTCGAGTCCACCCCAGGTAGCTAGAGTC-3′EcoRI
XholHBcAg89-144Sense: 5′-CGGATCCGGTGGAGTCAACAACACTAATATGGGC-3′
Antisense: 5′-CCAAGCCTTCCGCGGAAGTGTTGATAGGAT-3BamHI
HindIII Open in a separate windowpane Recombinant prokaryotic manifestation plasmid Pep3- HBcAg/pET-28a(+) was constructed(Number ?constructed(Figure1),1), and then recognized by restriction enzymes and sequencing. Open in a separate window Number 1 Map of Pep3-HBcAg/pET-28a(+) prokaryotic manifestation plasmid. The three DNA fragments were ligated and subcloned into plasmid pGEMEX-1. Then fusion gene Pep3-HBcAg was digested with restriction enzymes EcoRI and SalI and ligated into the equal sites of the pET-28a(+) vector, yielding His-tagged Pep3-HBcAg/pET-28a(+). Manifestation and purification of the fusion protein in Escherichia coli Recombinant plasmid Pep3-HBcAg/pET-28a(+) was launched into Escherichia coli BL21 (DE3). Then isopropy–D-thiogalactoside (IPTG, Sigma) was added to induce fusion protein manifestation. The BL21 cells were harvested, supernatant and sediment were subjected for SDS-PAGE. As the fusion protein was confirmed to be present in inclusion body, a further lysis step was performed (8 M urea immediately). The supernatant was purified on a Ni2+-NTA SORBS2 affinity chromatography column (Novagen). The His-tag was eliminated and the concentration of purified fusion protein was measured with the Bradford assay. EGFRvIII-specific antibody (Zymed) was used to confirm the identity of the fusion protein. Immunization of mice and antibody detection Thirty 6-8-week-old female BALB/c mice were purchased from Medical Experimental Animal Center, Xi’an Jiaotong University or college. All studies were performed in accordance with the Institutional Animal Care and Use Committee (IACUC) of Xi’an Jiaotong University or college. Ten mice were subcutaneously injected with fusion protein (100 g/animal) emulsified in Freund’s total adjuvant (Sigma) on day time 0 and with the same amount of protein emulsified in Freund’s incomplete adjuvant on day time 7. The third and following boosters were carried out only with fusion protein.