Expressing Beta-galactosidase Galb16 Tumor Model And Its Preliminary Application In The Study Of Anti-tumor Immunity

In the research described in this dissertation, a mouse melanoma model expressing P -galactosidase was established. Then an animal experiment was designed and finished to test the model in the study of tumor immunotherapy. The whole research work contained several parts: construction of recombinant vector p3gal containing P -galactosidase code gene, transfection with p3gal into mouse melanoma B16 cells to obtain galB16 cells expressing P -galactosidase, establishment of galB16 melanoma model and animal experiment on the model.The P -galactosidase code gene was obtained from pSV- P -Galactosidase control vector(promega) by digestion with Hind and Bam restriction endonuclease. The recombinant vector p3gal was constructed by inserting the P -galactosidase code gene into the MCS of plasmid pcDNA3 between Hindlll and BamHI sites. After digestion identification with restriction endonuclease and sequence identification, the p3gal vector was extracted and purified with Qiagen plasmid purification kit. Transient transfection with purified p3gal into mouse melanoma B16 cells and following X-Gal in situ staining suggested that the recombinant vector expressed P -galactosidase in eukaryotic cells.The p3gal vector then stably transfected B16 cells. Through selection with 500 g/ml G418 and in situ X-Gal staining, the melanoma cell line galB16, stably expressing galactosidase was obtained. The galB16 cells showed blue through optical microscope after X-Gal statining.The melanoma model was successfully established after inoculation in mouse with galB16 cells. PCR identification showed that galB16 tumor genomic DNA containing fragment of 3 -galactosidase code gene. In situ X-Gal staining indicated that the tumor cells expressed P -galactosidase in vivo. The above results suggested that the melanoma model, galB16, expressing P -galactosidase had been established successfully.Based on the model, an animal experiment was designed for mouse tumor immunotherapy. Twenty mice were randomly assigned to four parallel groups. Theyreceived i.m. injection with saline, DNA vaccine p3gal(100g/mouse), adjuvant CpGl826(20g/mouse), or p3gal+CpG1826 respectively. It showed that the DNA vaccine containing P -galactosidase gene could protect mice against the galB 16 tumor challenge. In addition, when combining with the adjuvant CpG 1826, the effect was increased prominently. 3 -galactosidase could be regarded as the endogenic antigen of the established galB16 melanoma model. Apparently, the model could be used to study cancer immunotherapy, particularly study immunotherapy strategies, immunotherapy adjuvants and so on.