Construction And Prokaryotic Expression Of Recombinant Gene G250Antigenic Peptide-HBcAg And The Immunogenicity Analysis Of The Fusion Protein

Research background and goalRenal cell carcinoma (RCC) is one of the most common malignant tumors of the urinary system, accounted for3%of all adults malignant tumor, the incidence of RCC has obvious rising trend and the death toll has increased annually in recent years. At present, chemothempy surgery is the major treatment for RCC and it is not sensitive to radiotherapy, chemotherapy and hormone therapy. However, RCC is one of the most immunoresponsive cancers in humans, immunotherapy exhibits a suitable basis for treatment. Tumor vaccine immune method to treat RCC had become the research focus of national scientists.G250is considered as a RCC-associated antigen and play an important role in RCC diagnosis, prognosis, and immunotherapy. G250is one of isomerases of the carbonic anhydrase family, which have the same genetic structure with Carbonic anhydrase IX (CAIX). G250was expressed in98%of RCC and ubiquitously expressed in the clear-cell subtype of RCC, whereas normal renal tissues do not express G250. Therefore, G250as a specific antigen for RCC and serves as a ideal target for RCC-specific tumor immunotherapy. With the deepening of the research on G250antigen, the G250peptide of amino acids249-268contains a cytotoxic T-lymphocyte epitope(CTL)and a helper T lymphocytes (Th) epitope, which able to induce both CD4+and CD8+T cell responses and generate anti-RCC activity. Therefore, G250peptide/249is a potential target for RCC-specific peptide vaccine immunotherapy. At present, on the basis of G250antigen peptide of RCC vaccine research have been completed the corresponding clinical report, however, G250peptide did not generate a ideal anti-RCC treatment efficiency due to the antigen peptide molecular weight is small and weak immunogenicity, vaccine vector play an important role in enhancethe antigen peptide immunogenicity, looking for the ideal vaccine vectors has becomed research hotspot on the basis of G250antigen peptide of RCC peptide vaccine research. Hepatitis B core antigen (HBcAg) has a natural ability of granular assembly and in experiment and clinical application research has already been confirmed that it can be used as the ideal vaccine vectors.Based on the above theoretical basis, in this study, the gene fragments of encoding mouse souse G250antigenic peptide/249wased inserted to remove the major immunodominant region (MIR) of the HBcAg and obtain the recombinant plasmid pET28a(+)/C-G250peptide-C, subsequently, we constructed G250peptid-HBcAg recombinant fusion protein and observed its immunogenicity.Research methods:1. Preparating G250peptide-HBcAg recombinant fusion protein:(1) The gene fragments of encoding G250antigenic peptide/249-268was amplified by PCR.(2) Construct recombinant plasmid pGEM-T/G250peptide.(3) the G250peptide was inserted to remove the major immunodominant region of the recombinant plasmid pGEMEX/HBcAg and obtain the recombinant plasmid pGEMEX/C-G250peptide-C.(4) For the replacement of the enzyme digestion sites, pGEMEX/C-G250peptide-C was used as a template, C-G250peptide-C gene fragment was amplified by PCR.(5) the C-G250peptide-C target gene was subcloned into the prokaryotic expression plasmid pET28a (+). IPTG was used to induce the fusion protein expression, the fusion protein was purified by Ni2+-NTA affinity chromatography, SDS-PAGE was used to identify the protein expression.2. Detecting immunogenicity of recombinant fusion protein:BALB/c mice were intraperitoneal injected with purified fusion protein, the mice serum were collected to measure the antibody titers to fusion protein, anti-G250peptide and anti-HBcAg wased measured by indirect ELISA.Research result1. The recombinant gene was confirmed to be correct by the restriction enzyme digestion and DNA sequencing. After prokaryotic expression and purified with Ni2+-NTA resin chromatography, the purity of final fusion protein reached a level higher than95%and its relative molecular mass was about22.35KDa.2. BALB/c mice were immunized intraperitoneally with fusion proteins, after four tmies of immunization, the titer of antibody can be detected in blood serum of all BALB/c mice, after six tmies of immunization, the titer of antibody in blood serum reached up to1:5.12×105, the titer of anti-G250peptide antibody reached up to1:6.4×104and the titer of anti-HBcAg peptide antibody was less than1:4×103.Conclusions1.The G250antigen peptide and HBcAg recombinant gene prokaryotic expression plasmid pET28a(+)C-G250peptide-C can be successfully constructed and was expressed in E. coli to produce fusion protein.2. The purified fusion protein has a strong immunogenicity and make mouse produce antibody with high titer and high specificity, HBcAg could effective enhance the immunogenicity of G250peptide/249. In addition, due to G250peptide/249replace the MIR area of HBcAg, the antigenicity of HBcAg have dropped significantly, the titer of anti-HBcAg peptide antibody was less than1:4×103.