Anti-Tumor Research Of Recombinant Salmonella Carrying Granulysin And Murine Interleukin-12 Genes

Cancer is one of the leading causes of death in the world. Almost 7.6 million people die of cancer each year worldwide and more than 70% of those deaths occurred in low and middle income countries. In China, almost 1.5 million people die of cancer every year and cancer has become the major cause of death. With the development of molecular biology and biotechnology, cancer gene therapy following surgery, chemotherapy and radiation therapy, has become a promising therapeutic method in recent years.The granulysin is a cytolytic molecule expressed by human CTL and NK cells with activity against a variety of tumors and microbes. IL-12 as a heterodimeric Th1 cytokine that promote the proliferation of T cells, NK cells, and tumor-infiltrating lymphocytes, has been shown antitumoral effects relying upon immunomodulatory properties. In this study, we constructed a eucaryotic co-expression plasmid carrying human granulysin and murine IL-12 genes, constructed recombinant salmonella carrying the co-expression plasmid, and observed anti-tumor effect of the recombinant salmonella in murine melanoma model by intratumoral and oral administration.PartⅠConstruction and expression of the eucaryotic co-expression plasmid carrying human 9 kDa GLS gene and mIL-12 geneObjective: To Construct the eucaryotic co-expression plasmid pBudCE4.1-S9K/mIL-12 carrying human 9 kDa granulysin gene and murine IL-12 gene, and detect expression products in eucaryotic cell and cell culture supernatant.Methods: The primer pairs including granulysin leader peptide DNA sequence were designed to amplify 9 kDa granulysin from the plasmid pZM03 carrying granulysin gene by polymerase chain reaction (PCR). PCR product was directed cloning into eucaryotic co-expression plasmid pBudCE4.1 to construct plasmid pBudCE4.1-S9K.The pBudCE4.1-S9K plasmid identified by DNA sequencing was transfected into murine macrophage (RAW264.7), and its expression products in cell were dectected by reverse transcriptase PCR (RT-PCR) and immunocytochemistry. Murine IL-12 gene from plasmid pZMO2 was subcloned into pBudCE4.1-S9K to construct eucaryotic co-expression plasmid pBudCE4.1-S9K/mIL-12. In transfected cells and culture supernatant, the expression of GLS and mIL-12 was detected by RT-PCR, enzyme-linked immunosorbent assay (ELISA) and Dot-ELISA.Result:9 kDa granulysin gene fragment including leader peptide gene was obtained from pZM03 plasmid by PCR. The plasmid pBudCE4.1-S9K was identified by restrict endonuclease digestion and DNA sequencing respectively. About 267bp 9kDa granulusin gene fragment was amplified from transfected cells by RT-PCR. The brown granules were detected in transfected cells by immunocytochemistry. The specific bands for GLS and mIL-12 were detected by RT-PCR in RAW264.7 cells transfected with pBudCE4.1-S9K/mIL-12. Secretary products of granulysin and mIL-12 were detected in culture supernatant by ELISA.Conclusion : Eukaryotic co-expression plasmid pBudCE4.1-S9K/mIL-12 carrying human GLS and mIL-12 genes was constructed successfully . The recombinant plasmid may express in secretory form in murine macrophage.PartⅡAnti-tumor effect of pBudCE4.1-S9K/mIL-12 in vitroObjective : To observe the anti-tumor effect of pBudCE4.1-S9K/mIL-12 on B16 cells in vitro.Methods:The expression of GLS was detected by RT-PCR in B16 cells transfected with pBudCE4.1-S9K/mIL-12. Restrain proliferation effect on B16 cells was detected by MTT assay. The apoptosis of B16 cells was detected by Flow Cytometry (FCM), Hoechst 33258 staining and AO/EB staining.Result: The specific band for GLS was detected by RT-PCR in B16 cells transfected with pBudCE4.1-S9K/mIL-12. The granulysin can restrain proliferation of tumor cell, and the rate of proliferation is about 34.27%. The apoptic rate of tumor cells transfected with pBudCE4.1-S9K/mIL-12 is 21.02%, which is 15.57% higher than that of control cells by FCM. In hoechst 33258 staining, the apoptic rate is 31.40±4.01%. Nucleus were concentrated and were stained bright blue in apoptic cells. In AO/EB staining, the tumor cells transfected with pBudCE4.1-S9K/mIL-12 were stained orange and showed morphological changes such as membrane outstanding at 48-72h. The mean apoptic rate is 32.80±4.83%.Conclusion: The anti-tumor effect was observed in B16 cells transfected with pBudCE4.1-S9K/mIL-12 in vitro.PartⅢConstruction of the recombinant salmonella carrying human 9 kDa granulysin and mIL-12 genesObjective: To construct recombinant salmonella carrying pBudCE4.1-S9K/mIL-12, and to identify the ability of submitting plasmids to eukaryotic cell. Methods: The pBudCE4.1-S9K/mIL-12 was transfected to attenuated salmonella strain SL7207 competence by electrotransformation. The recombinant salmonella was screened in low-salt LB medium (Zeocin 40μg/ml) and the plasmid was extracted for PCR identification. The recombinant salmonella was serial subcultured in low-salt LB medium to observe the stablity for plasmid-bearing. Other recombinant salmonella were constructed including pBudCE4.1 , pBudCE4.1-S9K and pBudCE4.1/mIL-12 plasmid for control test. The recombinant salmonella carrying pEGFP-C1 or pEGFP-C1/S9K which can express enhance green fluorescent protein or enhance green fluorescent protein-9kDa granulysin fusion protein, was investigated for the ability of submitting plasmids to eukaryotic cell. Murine macrophage cells (RAW264.7) were infected by the pEGFP-C1 and pEGFP-C1/S9K recombinant salmonella, and green fluorescence protein was detected. The expression of GLS in macrophage cells and tumor cells infected with pBudCE4.1-S9K recombinant salmonella was detected by RT-PCR.Result: The plasmid pBudCE4.1-S9K/mIL-12 may be obtained from recombinant salmonella and the specific bands for GLS and mIL-12 were detected by PCR.The recombinant salmonella can be subcultured at least 15 generations in low-salt LB plat without lossing the plasmids. Serial recombinant salmonella were constructed including pBudCE4.1 ,pBudCE4.1-S9K and pBudCE4.1/mIL-12 plasmid. Green fluorescence was observed in macrophages infected by recombinant salmonella carrying pEGFP-C1 or pEGFP-C1/S9K. About 267bp GLS specific gene fragment was detected by RT-PCR in macrophages and tumor cells infected with recombinant salmonella carrying pBudCE4.1-S9K.Conclusion: Recombinant salmonella carrying pBudCE4.1-S9K/mIL-12 was successful constructed. The recombinant salmonella can su-bmitte plasmids to the eukaryotic cell and infect the tumor cells.PartⅣAnti-tumor effect of recombinant salmonella carrying pBudCE4.1-S9K/mIL-12 by local intratumoral administrationObjective: To establish murine melanoma model and observe anti-tumor effect of recombinant salmonella carrying pBudCE4.1-S9K/mIL-12 in murine melanoma model by local intratumoral administration.Methods: C57BL/6J inbred line mouse as model animal was inoculated with 106 B16 cells subcutaneously in right forelimb oxter, and the model was confirmed by pathological detection. The optimal dose and time for innoculation were invistgated by treating model mice in different concentrations (103, 105, 107 and 109 bacteria each mouse) and times (1, 4, 7 and 10 day). The mice were treated with recombinant salmonella by intratumoral administration in optimal concentration and time. The tumor-bearing mice were random divided into six groups and were treated with phosphate buffered solution (PBS), salmonella, pBudCE4.1 recombinant salmonella, pBudCE4.1-S9K recombinant salmonella, pBudCE4.1/mIL-12 recombinant salmonella, and pBudCE4.1-S9K/mIL-12 recombinant salmonella respectively. At the next day after receiving B16 cell innoculation, each mouse was treated with 107 bacteria and repeated every week for 4 times. Tumor-bearing mice were observed every two days, and tumor size was measured precisely in sliding caliper. Survival time was monitored. The level of IFN-γand IL-12 in serum was evaluated by ELISA. Pathological changes of tumor were observed in HE staining.Result: The murine melanoma was established in C57BL/6J mice, and melanin was observed by pathological detection. The optimal dose of recombinant salmonella therapy was 1×107 bacteria each mouse, and the optimal time for therapy was 1 day after tumor cell innoculation. Receiving the treatment of pBudCE4.1-S9K/mIL-12 recombinant salmonella, tumor-bearing mice showed smaller tumor size than that in PBS group and salmonella group, and the level of IFN-γand IL-12 in serum was higher. The necrosis, cytolysis and lymphocyte infiltration were observed in tumor tissue.Conclusion: The murine melanoma model was successful established. The anti-tumor effect of recombinant salmonella carrying pBudCE4.1-S9K/mIL-12 in murine melanoma model was observed by local intratumoral administration.PartⅤAnti-tumor effect of recombinant salmonella carrying pBudCE4.1-S9K/mIL-12 by oral administrationObjective: To observe anti-tumor effect of recombinant salmonella carrying pBudCE4.1-S9K/mIL-12 in murine melanoma model by oral administration.Methods: The tumor-bearing mice were treated with recombinant salmonella carrying pEGFP-C1 by oral administration. The expression of enhance green fluorscence protein was observed in tumor tissue. The tumor-bearing mice were random divided into six groups and were treated with phosphate buffered solution (PBS), salmonella, pBudCE4.1 recombinant salmonella, pBudCE4.1-S9K recombinant salmonella, pBudCE4.1/mIL-12 recombinant salmonella, and pBudCE4.1-S9K/mIL-12 recombinant salmonella respectively. At the next day after receiving B16 cell innoculation, each mouse was treated with 108 bacteria and repeated every week for 4 times. Control group was treated with 0.1ml PBS. Tumor -bearing mice were observed every two days, and tumor size was measured precisely in sliding caliper. Survival time was monitored. The level of IFN-γand IL-12 in serum was evaluated by ELISA. Pathological changes of tumor were observed in HE staining. Result: The expression of enhance green fluorscence protein was observed in tumor tissue. Receiving the treatment of pBudCE4.1-S9K/mIL-12 recombinant salmonella, tumor-bearing mice showed smaller tumor size than that in PBS group and salmonella group, and the level of IFN-γand IL-12 in serum was higher. The necrosis, cytolysis and lymphocyte infiltration were observed in tumor tissue.Conclusion: The anti-tumor effect of recombinant salmonella carrying pBudCE4.1-S9K/mIL-12 in murine melanoma model was observed by oral administration.