| Objective:The death and recurrence rates of HCC is rather high as its high mutation rate,immunity escaption,weak antigenicity and abundant blood supplement. None of available therapies has significantly improved the dismal prognosis of patients with unresectable HCC. DNA vaccine application in prophylaxis and therapies has made a big progress and some of them is being on trial. To aim to AFP, TAA frequently expressed at high level in HCC, we constructed chimeric DNA vaccine of mAFP and chaperon of Mt.HSP70 for researching its targeting effectiveness. C57BL/6J mice were used for object as the level and change of AFP in serum just as human being. Moreover, mAFP eukaryotic expressed plamid with EGFP was constructed and transformed into Salmonella. For investigating distribution and mAFP expression of recombinant attenuated vaccine in vivo, oral immunization was performed in mice. These were groundworks for exploring immunity arosing by fused DNA vaccine and oral live vaccine. Methods:1．hAFP and mAFPexpression in HCC cells: RT-PCR was used to identify whether mAFP was expressed in EL4,Hepa1-6 and liver of immature C57BL/6J. Implanted tumor was confirmed by HE staining and immunohistochemical staining. Immunohistochemical staining was performed to detect hAFP in HCC tissue； 2．Construction and expression of chimeric plasmids of mAFP and Mt.HSP70: mAFP gene was gained by RT-PCR from Hepa1-6 cell line. pcDNA3.1-AFP,pcDNA3.1-HSP70,pcDNA3.1-AFP-HSP70 were constructed by molecular clone techniques. Plasmids were transfected into COS-7 cells with Lipofectamine 2000 after being confirmed by restriction enzyme digestion and DNA sequencing. Protein expressed in cells was detected by RT-PCR and immunocytochemical staining. Furthermore, immunohistochemical staining was performed to assay protein expressed in muscles after i.m. nude DNA； 3. Immune response induced by chimeric plasmids of mAFP and Mt.HSP70: Facilitated DNA immunization was performed by injecting plasmids into quadriceps muscles of mice at 3 different sites in a final volume of 100μL 0.25% Bupivacaine and 0.1% Methylphydroxy- benzoate. 100μg nude DNA was injected into the same sites 3 days after pretreatment. Booster immunization was performed as the first 1 month later. Venous blood was collected from orbital vein for assaying hepatic function. Splenocytes derived from immunized mice were stimulated by EL4/EL4(mAFP)inactivated by ultrasound. IL-4,IFN-γin supernatant were assayed by ELISA. AFP specific CTL activity was assayed by LDH method. Anti-tumor effect was evaluated by tumor size after injecting recombinant DNA into mice bearing Hepa1-6 tumor； 4. Distribution and expression of DNA vaccine carried by attenuated Salmonella typhimurium in mice: mAFP eukaryotic plasmids contained EGFP were constructed by molecular clone techniques. These plasmids were transfected into COS-7 cells with Lipofectamine 2000 after confirming by restriction enzyme digestion. They were introduced into attenuated Salmonella typhimurium-SL5928 (flagellin knocked out) by electroporation. Stability of vaccine was inspected by comparing restriction enzyme digestion charts of plasmids purified from Salmonella after being cultivated 50 generations continuously in medium containing/or no antibiotics. Growth,spirit,diarrhea,death were acted as markers to verdict its safety in mice after taking bacteria with serial CFU counts. 4,7,14,21 days after oral,liver,spleen,ileocecal colon were sectioned and observed by fluorescence microscope. Whether mAFP was expressed in tissues was judged by observing fluorescence. Results:hAFP and mAFPexpression in HCC cells: Specific bands could be amplified from total RNA of Hepa1-6 cells and liver of C57BL/6J mice by RT-PCR. No bands from EL4 cells. HE staining of tumor implanted with Hepa1-6 accorded with characteristics of tumor tissue and immunohistochemical staining for AFP was positive. It was also positive in HCC tissue.Construction and expression of chime...
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