Analysis And Anti-alignant Melanoma Effect Of Transmembrane Form Human Blood Group A Mimotope Vaccine

Malignant melanoma is a highly malignant and easy widespread metastasis tumor. It happened mainly in the white race, Australia has the highest incidence in the world. Immune therapy and biological therapy has become important clinical treatment methods of malignant melanoma because malignant melanoma cells has strong resistance to conventional radiotherapy and natural resistance to most chemotherapeutic drugs. How to treat more efficiently become a long and arduous undertaking that requires researchers and clinical workers to spare no effort to continue to work together.Anti-tumor effect of the body is very complex; non-specific mechanisms staggered with specific mechanisms, cellular immunity complement and coordinates with humoral immunity to implemente immune surveillance and anti-tumor effect. Humoral immunity plays anti-tumor effect mainly through CDC, ADCC effect, or conditioning phagocytosis when the body's immune system generates specific antibodies against the tumor antigen. Cellular immunity is another important mechanism for anti-tumor immunity mainly involved in T cells, NK cells, macrophages and dendritic cells. Anti-tumor immune mechanisms are extensive and successful applicated in clinical treatment. Immune escape of tumor cells may be involved in all aspects of the immune processes such as:tumor antigen expression, antigen recognition, processing, presenting, T cell proliferation, differentiation or immune response activation. Immune escape mechanism has great role in promoting the immune status, reversing tumor escape, designing new therapeutic strategies.In order to reversal tumor escape and eliminate tumors, a lot of tumor vaccines were designed to enhance the immunogenicity of tumor-specific antigens and induce anti-tumor immune response. The traditional vaccination method is based on the tumor cells, tumor extract or a composition of tumor cells to stimulate immune response. the most studied vaccines in malignant melanoma are following:①Gangliosides is glycosphingolipids exists mostly in the nerve cell membrane, and the ganglioside vaccine induce enhanced humoral immune response together with the adjuvant.②dendritic cell were cultured with various melanoma-associated antigen, peptide,or tumor lysate in vitro and then transfused patients.DC vaccine overcome the inefficient of antigen processing and presentation,and resolve the question of limited DC cells number in injection site of DNA vaccine.③DNA vaccine contains a variety of melanoma antigen gene using recombinant DNA plasmid or recombinant virus, the advantage of full-length DNA sequence of antigen can provide multiple epitopes with exempt from MHC restriction. Gene-modified tumor cell vaccine was used to strengthen anti-tumor effect by genetically transfecting endogenous gene such as cytokines, costimulatory molecules in tumor cells.④peptide vaccine has the advantage of high degree of tumor-specific, easy to preparation, and easy to monitor the immune response; the deficiency of peptide vaccine is that it only target a small number of epitopes expressed in a specific MHC haplotype in a limited patient population.⑤autologous tumor vaccine can provide a variety of individual-specific antigen, but requires a certain amount of living tumor samples to make vaccines, difficult to purified tumor cells. Allogeneic tumor vaccine contains more than one cell line obtained from different patients or multiple tumor cell lines to maximize the tumor antigen phenotype.Natural antibodies, such as anti-a-galactosidase (a-Gal) antibody, anti-ABO blood group antibodies and anti-Rha antibody, appear in serum without a series of antigen recognition, processing, presenting, T cell proliferation, differentiation and immune response activation processes. These antibodies can lead to strong hyperacute rejection of the immune response, transfusion reaction, or ABO incompatible organ transplantation for hyperacute rejection because of their powerful ability to complement activation, the stored anti-tumor antibodies would become a powerful weapon when combined with new anti-cancer vaccine. Researchers have applied naturally anti-a-Gal antibodies to tumor immunotherapy. Research shows that tumor cell in mice (α1,3-GT konckout) expression of a Gal antigens by transduction the al,3 galactosyltransferase gene (α1,3-GT) into tumor cells through adenoviral, tumor formation in mice do not express theα1,3-GT was significantly reduced compare With immunized miceAnother common natural antibody in vivo is anti-blood group antibody. If the ABO blood type is different from existing anti-blood group antibodies, there occur lethal antigen-antibody reaction, and activation of complement, mediated ABO incompatible organ transplantation for hyperacute rejection. In Our group previous studies, a clear anti-tumor effect such as clear post-inflammatory response within the tumor, and the emergence of tumor necrosis, tumor cell apoptosis after using human erythrocyte membrane immuniz animals. On thsis basis, it proposed that the antigen-antibody reaction occurs between the natural blood group and blood group antigens mimic peptide expressed on malignant melanoma cell surface, the complement-mediated cell killing effect destruct tumor cells.Fas expression in most benign and normal tissue is similar, but the Fas gene expression decreased or lost in malignant tumors to achieve immune evasion, so we can increase Fas expression in tumor cells to enhance anti-tumor effect, to achieve the treatment of cancer. Melanoma cell surface expression of Fas molecules, research shows that 16h after transfecting Fas gene into melanoma cells, Fas protein expression increased, there is apoptosis morphological changes of melanoma cells; in vivo experiments indicate that Fas expression in tumor cells significantly increased,the tumor weight was significantly lighter than the control group. Fas expression in the Jurkat human cells cell decreased after transduction of antisense can protect Jurkat cells exempt from Fas-mediated apoptosis. Although a single treatment can not completely control the tumor, Fas induced apoptosis has become a research hotspot in tumor cells.Macrophage inflammatory protein 3β(Mip3β) is a CC class of chemokines discovery by Rossi through bioinformatics methods in 1997. mature secreted Mip 3βform by cutting off the 21 amino acids signal peptide from The precursor protein gene of 98 amino acid residues. and its expression confined to the lymph nodes, thymus, appendix and other lymphoid tissues. Mip3βchemotactic with T cells, B cells, DC, NK cells and macrophages and other cells. Interactions between Mip3p and CCR7 play an important role in promoting DC cells and T cell contact and presenting antigens to stimulate an effective immune response. Activated mononuclear macrophages cause local inflammation response and inhibit tumor effect by secreting Mip-3α/β, MCP-1 and other chemokines, recruitment of more macrophages to and produce pro-inflammatory factor and a large variety of inflammatory mediators,Purpose and significanceNatural antibodies exsist abundance in serum without antigenic stimulation will become a powerful weapon against cancer when be fully utilized associated with anti-tumor vaccine.In this study, a human blood group A epitope mimic peptide screened by phage display can mimic human blood group A antigen to specific binding to anti-blood group A antibody.we observed CDC effect, ADCC ability and apoptosis of malignant melanoma cell line B16 simulated by transmembrane form human blood group A mimotope vaccine which fusing human blood group A mimotope with transmembrane domain, intracellular region of the Fas gene.we also observed immune response enhancement by Mip3β.This topic is an original and innovative research, and strives to provide new malignant melanoma vaccines, increase efficiency of biological treatment, and pave for new anti-tumor vaccine therapy in other tumors.Part 1 Mimotope peptide screening of blood group A antigenPurpose To screen mimotope peptide of blood group A antigen by phage display, and to explore a new way for blood antigen mimotope peptide vaccines.Methods The phage random peptide library was applied to 3 rounds "adsorption-elution-amplification" selection process by blood group A monoclonal antibodies.30 cloning were picked randomly, and analysed by enzyme-linked immunosorbent adsorption method (ELISA), and then sequenced to determine the blood group A antigen mimotope peptide.Results 12 positive clones were picked from 30 random clone after the phage enrichment and sequenced, the amino acid sequence Tyr-Val-Asp-Ser-PheLys-Ala-Arg-Ala--Val-Val-Arg was determined to be blood group A antigen mimotope peptide.Conclusion Blood group A antigen mimotope peptide screened by phage random twelve peptide library create condition for malignant melanoma prevention research.Part 2 Design and bioinformatics analysis of transmembrane form blood group A antigen mimotope peptide-Fas fusion geneObjective To design transmembrane form blood group A antigen mimotope peptide-Fas fusion gene and forecast the structure of its encoded protein, and to guide the experimental research in malignant melanoma B16 further.Methods mRNA of the signal peptide, exocellular section, transmembrane area and intracellular section of Fas genes identified from NCBI were used to design transmembrane form blood group A antigen mimotope peptide-Fas fusion gene. Fusion gene and the translation products sequence was compared using BLAST software, structure information and physicochemical properties of the fusion protein was analysed by Expert Protein Analysis System (ExPASY). Signal peptide, transmembrane region, the secondary structure and three-dimensional space conformation of the fusion protein were forcasted by the software SignalP, TMPRED, secpred_sopma and SWISS-MODEL.Results the fusion gene is constitute of 588 base pair, the fusion protein's relative theoretical molecular weight is 22.048 KDa and its isoelectric point is 9.36, the fusion protein has a protein signal peptide area, a transmembrane area and is composed of 47.18% alpha helix,12.82% outspread chain,36.92% the beta turn and 3.08% no rules curly. Conclusion the transform blood group A antigen mimotope peptide-Fas fusion gene has the same physicochemical properties as the Fas gene,such as signal peptide, secondary structure and three-dimensional space conformation, it has the properties to guide the fusion protein to expresse on tumore cell membrane surface.Part 3 construction of pIRES-P/Fas recombinat plasmid and its anti-malignant melanoma effect in vitroObjective To stable express chimeric transmembrane form vaccine of human blood group A mimotope on maligant melanoma cell line B16 and to investigate the in vitro cytotoxic effect of the vaccine.Methods the transform blood group A antigen mimotope peptide-Fas fusion gene was synthesised by chemical methods, cut and connected to the polyclonal sites A of the double expression plasmid pIRES, The recombinant plasmid was then transfected into malignant melanoma cells B16 through Lipofectamine 2000 after sequencing(pIRES-P/Fas group). The expression of mRNA and protein were detected by RT-PCR and Western blot. Complement dependent cytotoxicity assay (CDC) and Antihody-dependent cell-mediated cytotoxicity test (ADCC) on B16 cells was determined using cell counting Kit-8(CCK-8). The apoptosis effect was detected by flow cytometry. The malignant melanoma cells B16 transfected with empty pIRES plasmid was served as control group(pIRES group).Results the recombinated plasmid was proved successfully by enzyme cutting and sequencing. Specific bands were both detected at the expected place by RT-PCR, The recombinant mimotope/Fas fusion protein exhibited specific binding activity with anti-A antibody shown by Western blotting analysis. The CDC difference was statistically significant between different recombinated plasmid groups (F=7895, P<0.001). Multiple comparison results suggest that appropriate serum dilution degrees was 1:32. The ADCC difference was statistically significant between different recombinated plasmid groups (F=677.817, P<0.001), Multiple comparison results suggest that appropriate antibody concentration was 20μg/ml. The apoptosis difference was statistically significant between different recombinated plasmid groups(F=358.075,P<0.001). Multiple comparison results suggest that appropriate antibody concentration was 5μg/ml.Conclusion the transmembrane form vaccine of human blood group A mimotope could be expressed in B16 cell line stablely and mediate ADCC, CDC and apoptosis of B16 cells in vitro. This vaccine may be a promising candidate for potential malignant melanoma therapy.Part 4 construction of pIRES-P/Fas-Mip 3βrecombinat plasmid and its anti-malignant melanoma cells effect in vitroObjective To stable express chimeric transmembrane form vaccine pIRES-P/Fas-Mip 3βrecombinat plasmid in maligant melanoma cell line B16 and to investigate the in vitro cytotoxic effect of the vaccine.Methods The Mip 3βgene was cloned from human tonsillitis cell, cut and connected to the polyclonal sites B of the double expression plasmid pIRES, The recombinant plasmid was then transfected into malignant melanoma cells B16 through Lipofectamine 2000 after sequencing (pIRES-P/Fas-Mip 3βgroup and pIRES-Mip 3βgroup). The expression of mRNA and protein were detected by RT-PCR and Western blot. Complement dependent cytotoxicity assay (CDC) and Antihody-dependent cell-mediated cytotoxicity test (ADCC) on B16 cells was determined using Cell Counting Kit-8(CCK-8). The apoptosis effect was detected by flow cytometry. Experimental group:pIRES-P/Fas- Mip 30 group, pIRES-P/Fas group, pIRES-Mip 3βgroup, pIRES group.Results Specific bands were both detected at the expected place by RT-PCR, The CDC difference was statistically significant among different recombinated plasmid groups(F=1079,P<0.001). The ADCC difference was statistically significant among different recombinated plasmid groups (F=97.597,P<0.001). The apoptosis difference was statistically significant among different recombinated plasmid groups (F=146.943,P<0.001). Multiple comparison results suggest that the difference of CDC, ADCC and apoptosis were not statistically significant between pIRES-P/Fas group and pIRES-P/Fas- Mip 3βgroup. Conclusion chemotactic factor Mip3βfailed to enhance the CDC, ADCC and apoptosis effect mediated by recombinate plasmid pIRES-P/Fas, consider Mip3βfailed to effectively chemotaxis the T cells, monocytes, dendritic cells and neutrophil in vitro environment.