title: Construction of random tumor transcriptome expression library for creating and selecting novel tumor antigens.
abstract: Novel tumor antigens are necessary for the development of efficient tumor vaccines for overcoming the immunotolerance and immunosuppression induced by tumors. Here, we developed a novel strategy to create tumor antigens by construction of random tumor transcriptome expression library (RTTEL). The complementary DNA (cDNA) from S180 sarcoma was used as template for arbitrarily amplifying gene fragments with random primers by PCR, then ligated to the C-terminal of HSP65 in a plasmid pET28a-HSP for constructing RTTEL in Escherichia coli. A novel antigen of A5 was selected from RTTEL with the strongest immunotherapeutic effects on S180 sarcoma. Adoptive immunotherapy with anti-A5 sera also inhibited tumor growth, further confirming the key antitumor roles of A5-specific antibodies in mice. A5 contains a sequence similar to protein-L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1). The antisera of A5 were verified to cross-react with PCMT1 by Western blotting assay and vice versa. Both anti-A5 sera and anti-PCMT1 sera could induce antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity toward S180 cells by in vitro assay. Further assay with fluorescent staining showed that PCMT1 is detectable on the surface of S180 cells. Summary, the strategy to construct RTTEL is potential for creating and screening novel tumor antigens to develop efficient tumor vaccines. By RTTEL, we successfully created a protein antigen of A5 with significant immunotherapeutic effects on S180 sarcoma by induction of antibodies targeting for PCMT1.
authors.affiliation: College of Life Science and Technology, Huazhong Agricultural University, No. 1 Shizishan Street, Hongshan District, Wuhan, 430070, HuBei Province, China.
authors.forename: Huizhun
authors.initials: H
authors.lastname: Zhao
authors.affiliation: College of Life Science and Technology, Huazhong Agricultural University, No. 1 Shizishan Street, Hongshan District, Wuhan, 430070, HuBei Province, China.
authors.forename: Xiuyun
authors.initials: X
authors.lastname: Zhao
authors.affiliation: College of Life Science, Hubei University, 430062, Wuhan, China.
authors.forename: Peng
authors.initials: P
authors.lastname: Du
authors.affiliation: College of Life Science and Technology, Huazhong Agricultural University, No. 1 Shizishan Street, Hongshan District, Wuhan, 430070, HuBei Province, China. qigaofu@mail.hzau.edu.cn.
authors.forename: Gaofu
authors.initials: G
authors.lastname: Qi
mesh_terms: D000818:Animals; D000920:Antibody-Dependent Cell Cytotoxicity / Q000187:drug effects / Q000276:immunology; D000951:Antigens, Neoplasm / Q000235:genetics* / Q000276:immunology; D015153:Blotting, Western; D045744:Cell Line, Tumor; D003165:Complement System Proteins / Q000276:immunology; D003602:Cytotoxicity, Immunologic / Q000187:drug effects / Q000276:immunology; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics* / Q000276:immunology; D015723:Gene Library; D007106:Immune Sera / Q000276:immunology / Q000494:pharmacology; D007114:Immunization / Q000379:methods; D008297:Male; D008807:Mice, Inbred BALB C; D018613:Microscopy, Confocal; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase / Q000235:genetics / Q000276:immunology; D012510:Sarcoma 180 / Q000235:genetics* / Q000276:immunology / Q000628:therapy; D059467:Transcriptome / Q000235:genetics* / Q000276:immunology
chemical_list: D000951:Antigens, Neoplasm; D007106:Immune Sera; D003165:Complement System Proteins; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase
keywords: Humoral immunity.; Protein-L-isoaspartate (D-aspartate) O-methyltransferase; Random tumor transcriptome expression library; S180 sarcoma; Tumor antigen