Screening And Identification Of Anti-tumor Immune Adjuvants Based On RMBP-NAP

Research Background:Melanoma is a common malignancy in skin cancer.The incidence of melanoma has risen sharply in recent years in the world.In China,there are about 20,000 new cases a year.However,in recent years,the immunotherapy of melanoma has made great progress,and tumor immunotherapy has become a new important treatment.Non-specific immunostimulants affect the adaptive immune response by participating in innate immunity,the innate immune response plays an important role in the initiation,regulation and adaptation of the adaptive immune response.When extraneous contents which were called pathogen-associated molecular pattern(PAMP)such as microorganisms,polysaccharides,proteins,nucleic acids,and small molecules enter the body,it is the pattern recognition receptor(PRR)immune cells that recognize,stimulate,and enhance the body's immune function,encouraging the immune system to kill cancer cells,destroy tumors,and play an anti-tumor role.Different pathogen-associated molecular pattern stimulate pattern recognition receptors on the surface of innate immune cells,producing different cytokines of Th1 or Th2.These different cytokines further regulate the differentiation of specific immune cells and thus affect the type of adaptive immune response.Toll-like receptor(TLR)agonists which can trigger rapid innate immune response and then induce adaptive immune response,have been used as the anti-cancer agents in immunotherapy of cancer.Dendritic cells(DC)plays a key role in TLR mediated immune response.Bone marrow derived dendritic cells(BMDC)are the most functional antigen presenting cells(APC)in the body,which have different biological activities in different periods.Immature DC has strong migration and endocytosis ability.Mature DC has dendritic form,with the antigen uptake level decreased,but the antigen processing and presentation level increased.It is the central link to initiate,regulate and maintain the immune response,and plays an important role in the body's anti-tumor immunity.When stimulated by exogenous TLR agonists,TLR signals on the DC surface are activated to induce DC maturation.Different TLRs interact with corresponding transporters to activate downstream transcription factors through My D88-dependent and non-dependent signal transduction,and further up-regulate the expression of cytokines such as Il-12 and IFN-? through NF-?B or MAPK signaltransduction pathways.Promote the differentiation of Th0 into Th1,and dominate the immune response of Th1,so as to help clear the tumor.Preliminary studies have shown that as a microbe derived TLR2 agonist,rMBP-NAP interacts with TLR2 to activate a signaling pathway that mediates the innate immune response indicating a significant anti-tumor effect.Based on the fusion protein rMBP-NAP,this study used bioinformatics and genetic engineering methods to cut off the spiral segments of the secondary structure of HP-NAP from the C-terminal one by one and construct new fragments S1,S2,S3 and S4,and then connect these new fragments with rMBP-NAP to create new fusion proteins rMBP-NAP-S1,rMBP-NAP-S2,rMBP-NAP-S3 and rMBP-NAP-S4.Fusion protein rMBP-NAP-S1,showed the optimal effect on activating BMDCs,and the therapeutic effect of rMBP-NAP-S1 on melanoma B16 in situ tumor model in mice was evaluated.It will lay a theoretical and experimental foundation for the development of novel rMBP-NAP related anti-tumor immunomodulator.Objective:Based on the early stage of the project of rMBP-NAP antitumor effect of study,we further explore of the protein structure,a series of novel fusion proteins rMBP-NAP-S based on the structure of rMBP-NAP were constructed and expressed.The optimal candidate protein was decided through BMDC activation assay,and its anti-tumor effect was detected by in situ tumor model of mouse melanin B16,so as to further study the anti-tumor mechanism of rMBP-NAP and identify the effective functional fragment of its effect.Research method:1.rMBP-NAP gene was amplified by PCR,and the fusion genes NAP-S1,NAP-S2,NAP-S3 and NAP-S4 were obtained by overlapping extension.The recombinant plasmids pMal-c2X-NAP-S1,pMal-c2X-NAP-S2,pMal-c2X-NAP-S3 and pMal-c2X-NAP-S4 were successfully constructed by genetic engineering.IPTG induced expression and affinity chromatography purified the fusion proteins rMBP-NAP-S1,rMBP-NAP-S2,rMBP-NAP-S3 and rMBP-NAP-S4.SDS-PAGE protein glue was used to identify the protein induction form,expression form and purification form.2.The purity of BMDC was determined by flow cytometry using mouse granulocyte-macrophage colony stimulating factor(rm GM-CSF)combined with interleukinin(rm IL-4).3.The relative expression levels of IL-12p40,IFN-?,NF-?B in BMDC stimulated by the fusion proteins rMBP-NAP-S1,rMBP-NAP-S2,rMBP-NAP-S3 and rMBP-NAP-S4 for 2 h and 16 h were detected by fluorescence quantitative PCR(qRT-PCR)respectively.The relative expression levels of cytokines(IL-12p40,IFN-?,NF-?B)and cell surface markers(CD80,CD86,MHC?)in BMDC stimulated by the fusion proteins rMBP-NAP-S1 at different concentrations were detected by qRT-PCR.4.Flow cytometry were used to detect the expression of BMDC markers(CD80,CD86,MHC?)in BMDC stimulated by rMBP-NAP-S1.The relative expression levels of TLR(TLR2,TLR4)in BMDC stimulated by rMBP-NAP-S1 were detected by qRT-PCR.Enzyme-linked immunosorbent assay(ELISA)was used to detect the expression of cytokines(IL-6,IL-12,IFN-?,TNF-?)in the supernatant after the stimulation of BMDC by the fusion protein rMBP-NAP-S1.Western blot was used to detect the expression of related proteins(TLR2,TLR4,My D88,NF-?B)in the signaling pathway of BMDC stimulated by the fusion protein rMBP-NAP-S1.5.The in situ tumor model of mouse melanin B16 was established to evaluate the anti-tumor immune effect of the fusion protein rMBP-NAP-S1 on the model,and the changes in tumor volume and weight during the whole experiment were counted to draw the tumor growth curve.Research result:1.The prokaryotic expression vectors pMal-c2X-NAP-S1,pMal-c2X-NAP-S2,pMal-c2X-NAP-S3 and pMal-c2X-NAP-S4 were successfully constructed.Fusion protein rMBP-NAP-S1,rMBP-NAP-S2,rMBP-NAP-S3 and rMBP-NAP-S4 were obtained by IPTG induction expression and affinity chromatography purification and separation.2.The purity of BMDC was more than 80% by flow cytometry,the cell purity was high and in good condition,which could be used for subsequent detection.3.qRT-PCR results showed that the relative expression levels of IL-12p40,IFN-?,NF-?B,CD80,CD86 and MHC? were increased after the fusion protein rMBP-NAP-S1(200 ?g/m L)stimulated BMDC for 16 h.The fusion proteinrMBP-NAP-S1 was selected as the best candidate protein.4.The expression levels of cell surface markers(CD80,CD86 and MHC?),were significantly increased after the BMDC was stimulated by the fusion protein rMBP-NAP-S1(200 ?g/m L)for 16 h by flow cytometry.qRT-PCR results showed that m RNA expression levels of TLR2 and TLR4 were increased.ELISA results showed that TNF-?,IFN-?,IL-6 and IL-12 protein levels were increased.Western blot analysis showed that rMBP-NAP-S1 was transduced through My D88 signaling pathway,and the protein levels of TLR4 did not change significantly,while the expression levels of TLR2,My D88 and NF-?B increased significantly.5.The results of the treatment of mouse melanoma B16 in situ tumor model showed that the fusion protein rMBP-NAP-S1 had a certain inhibitory effect on the growth of melanoma.Analysis conclusion:In this study,recombinant plasmids pMal-c2X-NAP-S1,pMal-c2X-NAP-S2,pMal-c2X-NAP-S3 and pMal-c2X-NAP-S4 were successfully constructed,and fusion proteins rMBP-NAP-S1,rMBP-NAP-S2,rMBP-NAP-S3 and rMBP-NAP-S4 were purified by induction of expression.The fusion protein rMBP-NAP-S1 was selected as the best candidate protein for tumor immunotherapy when the stimulation time was16 h at the concentration of 200 ?g/m L.The stimulating effect of rMBP-NAP-S1 on BMDC is mainly to regulate the expression of cytokines through the TLR2-My D88-NF-?B signaling pathway,so as to play a role in immune regulation.In situ melanoma model of mouse melanin B16 was used to verify that the fusion protein rMBP-NAP-S1 could inhibit melanoma growth.