Construction Of Recombinant Plasmid Her2-HSP70and Its Anti-tumor Study

BackgroundBreast cancer is one of the most common malignancies in women, with an increasing morbidityyear by year. Breast cancer at advanced stages is unsensitive to conventional treatments. Immunotherapyaims to stimulate and enhance immune function, selectively recognise Her2positive tumor cells and killthem specially. By this way, tumor cells are controlled and cleaned. Numerous data show thatimmunotherapy coupled with conventional treatments could benefit the outcome of tumor treatment. Her2is a member of growth factor receptor family, and is over expressed in25-30%of invasive breast cancer.Her2associates with the metastasis and poor prognosis of tumors. Meanwhile, Her2is low expressed innormal tissues, which makes it the target of tumor immnobiothrapy.Numerous literatures showed that HSP70was a potent molecular adjuvant, could be the vectorsof the anti-tumor-peptide. It played an important role in the process of recognizing, presenting antigens andalso took part in activating CD8+T cells. In that HSP70shares a quite alike multi-peptide binding grooveswith MHC, It facilitates MHC to recognizing and presenting tumor-specific peptide antigens. HSP can actas a vector of tumor-specific peptide-antigens just like MHC, which coupled with her2taking part inrecognizing and presenting antigens. In this study, adjuvant HSP70and Her2gene were connected toconstruct pcDNA3.1/Her2-HSP70recombinant plasmids, and in vivo, the capacity in inducing immuneresponse and curing mouse breast cancer were observed. We hope to establish gene vaccines for breastcancer in this study and provide experimental and theoretical evidence for the treatment to breast cancer.ObjectiveConstruct pcDNA3.1/Her2-HSP70recombinant plasmids, and observed the capacity of them toinduce immune response and the function to tumor immunotherapy.MethodsThe full length CDS of human breast cancer cell Her2/neu (neu) and heat shock protein70(HSP70) were amplified by reverse transcript poly enzyme chain reaction (RT-PCR). PcDNA3.1/neu,PcDNA3.1/hsp70, and PcDNA3.1/neu-hsp70recombinant plasmids were constructed and used to transfect mouse breast cancer cell line (4T-1) to get cell lines stably expressing relevant proteins which weredetected by western blot (WB).4T-1/Her2+cells were injected subcutaneously into mice to establishmouse breast cancer models which were divided into5groups randomly,one group received injection ofgene vaccine PcDNA3.1/neu-hsp70, and other groups challenged with PcDNA3.1/neu,PcDNA3.1/hsp70,pcDNA3.1or phosphate buffer as control. The reagents were administrated every7days,for3times, and were assisted by adjuvant bacillus calmette-gurin(BCG) and granulocyte-macrophagecolony stimulating factor (GM-CSF).During this process, the growth of tumors was observed. The micebearing tumors were killed and tumors were isolated and weighted. Tumor tissues were sliced the sectionswere stained with hematoxylin eosin(HE).The changes of surface markers (such as CD3,CD4,CD8)of Tcells in mouse splenocytes and the anti-HER2antibody in mouse sera were detected by flowcytometry(FCM).The quantity of IL-4and IFN-γwas measured by enzyme-linked immunosorbentassay(ELISA).ResultsPcDNA3.1/neu, PcDNA3.1/hsp70, PcDNA3.1/neu-hsp70recombinant plasmids were established,and the quality of them were verified by electrophoresis after double-enzyme digesting. The recombinantplasmids were confirmed the same as designed after the base-pair of which were sequenced. A stablytransfected4T-1/her2+cell line was obtained. Molecular weight of proteins translated by the mRNAswhich were transcribed by the recombinant plasmids was69kDa. HER2was found using WB in the4T-1/her2+cell line, while it was not found in the4T-1cell line transfected with PcDNA3.1and the wildtype. In vivo, when PcDNA3.1/neu-hsp70recombinant plasmids combining with the adjuvant BCG andGM-CSF were used to vaccinate BalB/c mice, the immunoresponse of T cell was enhanced, the expressionof INF-γ was increased,and the tumors of the mouse models were rejected. Tissue slices of tumorsdisplay a phenomenon that the volume of cells was decreased, the growth of cells was suppressed and themesenchymal stroma was richer. After treated with her2-hsp70gene vaccine and adjuvants, the sera of micewere analyzed using the FCM. We found that the proportion of CD8+T cells was increased, the ratio ofCD8+T cells and CD4+T cells was decreased, which indicates HSP70combining adjuvants playedimportant roles in presenting tumor-antigens and activating CD8+T cells.Conclusion The cooperation of her2-hsp70gene vaccine and adjuvants generated strong anti-tumor effects.Xenogeneic antigen HER2broke the anti-tumor immune tolerance. HSP70augmented antigen presenting.BCG and GM-CSF stimulated the production of the cytokine INF-γ efficiently. ThePcDNA3.1/neu-hsp70recombinant plasmid gene vaccine was of excellent use in mouse breast cancertherapy.