| Tumor vaccine is an important approach for tumor biological therapy. By taking advantage of the initiative immunity to the specific antigen of tumor, it can activate and reinforce immune response to the tumor. It was reported that epidermis Growth Factor Receptor2gene which encodes a transmembrane protein of185KD,namely HER2/neu, is overexpressed in about25%of invasive breast cancers and is associated with poor disease free survival and resistance to certain chemotherapeutic agents. HER2/neu protein possesses endogenous tyros ine kinase activity, which makes it the perfect target molecular for the tumor therapy. On the other hand, TF antigen, one of the glycophorin antigens, which expressed in87.5%breast cancer, is another target molecular for tumor therapy. Currently, coryne bacterium parvum enriched with TF antigen is one of biological drugs for breast cancer treatment.HER2/neu is a glycoprotein, which make it diffcult to protein vaccine, therefore, DNA vaccine is a good choice. However, low immunogenicity of the DNA vaccine limits it application for tumor therapy. Since antigen presenting cells (APC)play a center role in immunological response of DNA vaccine, targetly delivery of DNA vaccine into the APC which increases the expression level of antigen encodes by DNA vaccine, and improves the presentation efficiency of endo-expressed antigen are the ultimate ways to reinforce the immune activity of the DNA vaccine.Hence, we propose a new vaccine devoloping strategy for preparing HER2/neu DNA vaccine by using bacterial ghost (BG) as a delivery carrier. At first, BG can be recognized and phagocytosed by APC, make it as an efficient DNA delivery carrier by loading plasmid DNA in it. Secondly, the HER2/neu DNA will be linked with MHC-Ⅱ invariant chain(mIi), which makes the endo-expressed HER2/neu antigens directly bind to MHC-Ⅱ molecules and efficiently presented on the surface of APC, so as to improve the presentation efficiency. This strategy is belived to enhance the HER2/neu mediated immune response by impove both antigen exprssion and presentation efficiency in APC. On the other hand, abundant TF antigens were detected on the suface of E coli. We hypothesizes that, this new strategy can also enhance the killing effect on breast cancer by target both HER2/neu and TF antigen on the surface of tumor. The aim of this study is to prove our proposal. Here are the preliminary results.1. Preparing the E. coli BG, plasmid DNA loading and transfection of APC with BG.E. coli was lysized by E gene by increasing the temperature to42℃, the lysed bacteria were collected and rinsed by PBS buffer. TF antigen on the surface of E coli BG was detected with FITC-PNA and monitored by the FACS, and results showed all the BGs were TF anitgens positive. Plasmid DNA was loaded in BG, and the loading efficicency was greatly improved after optimization of BG and DNA ratios. Under scanning electron microscope and transmission electron microscope, BG was found to be empty bacterial possessed integrity morphology and membrane structure of E coli. After being loaded with plasmid DNA, DNA signal could be observed in BG. The DNA loading efficiency was monitored by the FACS and almost100%BG showed plasmid DNA signal. APC cells were transfected fluorescent labeled BG, and fluorescent signal was determined by FACS. The results showed that almost100%cells contained BG. The results indicated that BG be good DNA carrier which could be efficiently taken by APC.2. Construction of mli-linked HER2/neu plasmid.The total RNA of human breast cancer cell line SKBR-3was isolated and HER2/neu cDNA was obtained by RT-PCR. We sequently subcloned the mouse mli sequence and the segment encoding the first receptor region of HER2/neu into the pVAXI plasmid and recombinant plasmid was refered to as pVAXI-mIi-HER2/neu. Besides, full length HER2/neu cDNA was also subcloned into the pCDNA3.1-EGFP-HYGRO plasmid and the obtained plasmid was termed as pCDNA3.1-EGFP-HYGRO-HER2/neu which was transfected into the4T1cell line later. 3. Mouse breast cancer model stably expressed luciferase gene and HER2/neu geneTumor grafts of Murine breast tumor cells are good models for human breast cancer. However, because of the difference between murine neu gene and human HER2/neu gene, these cells are not suitable for evaluation of HER2/neu mediated immune response. The4T1cell line was sequently transfected with luciferase reporter gene and pCDNA3.1-EGFP-HYGRO-HER2/neu. After G418and hygromycin selection, the tranfectant which stably expressed the HER2/neu and luciferase gene was obtained and termed as4T1-Luc-HER2/neu. Tumor graft experiments showed that the transfected cells had similar in vivo tumorgenesis and metastasis to wild-type4T1cells. The cells will be suitable for study HER2/neu mediated immune response. Furthermore, the proliferation and metastasis of the tumor graft can be observed with bioluminescence imaging technology.4. Animal experimentThe BALB/c mice were intramuscular injected with free plasmid or plasmid loaded in BG for4times, by a week interval. One week after last vaccine injection, the4T1-Luc-HER2/neu cells were inoculated into BALB/c mice(1×106per mouse).The volumes of tumor grafts were determined, and the proliferation and metastasis of the tumor grafts were also observed with bioluminescence imaging technology. The results showed that mIi-linked HER2/neu plasmid was better in inhibiting tumor growth then HER2/neu plasmid and BG could enhance the immunity of pVAX I-mli-HER2/neu plasmid. To our surprise, BG itself exhibited a very high efficiency in inhibiting tumor growth.In summary, mIi-linked HER2/neu plasmid was constructed, murine breast cancer model that expressed HER2/neu and luciferase gene was established and several techniques for delivery of DNA vaccine targeting TF and HER2/neu antigen were develop in this study. Furthermore, preliminary but promise results which supporting our hypothesis were got. All of results make the foundation for development of novel DNA vaccine for breast cencer。... |
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