Construction Of Tumor Genes Randomly Expression Library For Creating Novel Anti-Sarcoma Tumor Antigens

Active immunotherapy is aimed to elicit specific host immune responses against selected tumor antigens, which are mainly used for development of cancer vaccines. However, results of clinical studies on therapeutic cancer vaccines were not lucky. This failure was due to several reasons, including immunotolerance induced by tumor antigens, immunosuppression induced by tumors and other reasons. In order to overcome these obstacles, novel tumor antigens are necessary. Novel tumor antigens are necessary for developing efficient anti-tumor vaccines to overcome immunotolerance and immunosuppression induced by tumors. Here, we developed a novel strategy for creating anti-tumor protein antigens by constructing a tumor transcriptome expression library.(1) Construction of the sarcoma S180 tumor genes random Ly expression library: The c DNA from S180 sarcoma tumor was used as template for amplifying gene fragments with random primers by PCR, then inserted into the C- terminal of HSP65 in a expression plasmid p ET- 28a- HSP for constructing a tumor transcriptome expression library in Escherichia coli BL21(DE3).(2) Screening transformants with anti-tumor activity from sarcoma S180 tumor genes random Ly expression library: By SDS-PAGE, 100 positive transformants were selected with the expressed fusion proteins more than 65 k Da from the total 537 transformants. We obtained the transformant A5 with the best anti-tumor effect by 3 screening by immuned with culture and its inhibitory rate was(79.15 + 1.74)%.(3) Verification of immunotherapy effects of recombinant proteins on sarcoma tumor: Strain A5 expressed a molecular weight of 81 k Da fusion protein HSP-A5. Sequencing the sequence of A5, removing HSP65 gene sequence and construct a recombinant plasmid p ET- 28a- A5 which expressed a 16 k Da protein expression of A5. Test the immune anti-tumor effect of purified fusion protein HSP-A5 and A5 and results show that the inhibition rates of proteins HSP-A5 and A5 were(62.40 + 1.91)% and(58.74 + 0.91)%, respectively, and there was significant difference between the PBS control group(p<0.01) or HSP65 control group(p<0.01).(4) The study of antitumor mechanism I: The humoral immune response: detect the Ig G antibody Ig G titer in serum of mice immunized with recombinant protein HSP-A5 and A5 by ELISA. The results showed that the titer of Ig G antibody against HSP-A5 was 25600× in HSP-A5 group, the titer of Ig G antibody against A5 was 3200× in HSP-A5 group, and the mice immunized with A5 the serum Ig G antibody titer of anti A5 was 1600×. Antibodies to A5 protein HSP-A5 and A5 can stimulate mice to produce high titer antibodies A5, there by inhibiting the growth of sarcoma S180. In addition,the adoptive immunotherapy test with anti-HSP-A5 serum showed that the experimental group of inhibitory rate was(34.67±1.23)%, the positive control Taxol(36.73±1.18)%, and there were no significant difference. These data confirmed the key anti- tumor role of A5-specific antibodies. The cellular immune response: in order to verify whether the proteins HSP-A5 and A5 can stimulate the immune response against mouse sarcoma S180 cells, we detected the intracellular cytokines TNF-α and IFN-γ of T lymphocyte by FACS,and the proliferation effect of A5 specific lymphocyte, and the cytolytic activity of cytotoxic T lymphocyte. Theses investigations showed A5 could not elicit strong cellular immune response towards tumor.(5) Detection of cross immune reaction of protein A5 and PCMT1: The A5 protein contained a sequence similar to protein L-isoaspartate(D-aspartate) O-methyltransferase(PCMT1), thus the anti-A5 antibodies might bind to PCMT1 to neutralize its activity, resulting in an increased tumor susceptibility to apoptosis. In order to verify that, firstly expressed protein PCMT1(32 k Da), then Western Blotting, ADCC, CDC, and staining sarcoma S180 cells by immunofluorescence. The results showed that A5 and PCMT1 protein can stimulate the body to produce antibodies against A5 and Ig G similarly, and the killing activity of ADCC and CDC had no significant difference. The target antigen in combination with A5 and PCMT1 specific antibodies are all anchored on the surface of S180 sarcoma cells. The above results, to verify the existence of closely relationship to the anti-tumor mechanism of protein A5 and PCMT1.In this study, screening a novel tumor antigen by the constructio of tumor genes random Ly expression library, and study the anti-tumor mechanism, then we obtained the antigen protein A5 with good anti-tumor effect achieved mainly by stimulating humoral immune response. Collectively, we successfully developed a novel strategy for creating and selecting new tumor antigens by constructing a tumor transcriptome expression library. This method may be able to find a new valuable tumor antigen for the development of tumor vaccine.