The Experimental Study Of Specific Cytotoxic T Lymphocytes Response Induced By Ub-HBc Ag-CTP Fusion Protein

Hepatitis B virus(HBV) infection is still a severe health problem, there are approximately 350 million chronic hepatitis B virus carriers worldwide. There is no way to clear HBV thoroughly at present, so that the virus persists in liver cells, which will lead to chronic infection, and be secondary to liver cirrhosis and hepatocellular carcinoma, even death. The HBV specific cytotoxic T lymphocyte(CTL) is thought to be the key role in eliminating the virus.Ubiquitin-proteasome system(UPS) is a kind of selective protein degradation system which widely exists in eukaryotic cells and dependent on ATP. It is an important protein quality control system in eukaryotic cells, and plays an important role in maintaining the normal physiological function of cells and even the entire organism. Ubiquitinated protein is recognized by proteasome complex and degradation to a number of small peptides, which could combine with major histocompatibility complex(MHC)-I and was identified by specific CD8+ T lymphocytes to induce specific CTL immune responses. Considerable studies have shown that, after ubiquitinated, the protein can be degradation and presented significantly inside cells, thereby enhance the immune response.As a result of the existence of membrane barrier, proteins and peptides is difficult to enter the cells under normal circumstance. Some proteins were independent of receptor and penetrated through the cellular membrance. The peptides with transduction domain, which penetrate through the cellular membrance, is termed as cellpenetrating peptide(CPP). Nowadays, the TAT protein from HIV-1 is the mostunderstood and extensively used among cell penetrating peptides. Theproteintransduction domain(PTD) is a core region of HIV-Tat protein, whichcould penetrate through the cellular membrance. Cytoplasmic transduction peptide(CTP) is derived from PTD, is a novel, deliberately designed transduction protein that is used to ensure an efficient cytoplasmic delivery of biomolecules. The purpose of this study was to study the effect of specific cytotoxic T lymphocyte induced by Ub-HBc Ag-CTP fusion protein through it promoting the maturation of DC to induce specific CTLs in vitro and immunizing BALB/c mice in vivo, based on constructing Ub-HBc Ag-CTP fusion gene plasmid and expressing and purifying the protein.This experiment was composed of three parts:(1) Expression and purification of Ub-HBc Ag-CTP fusion protein and the effect of it on inducing the maturation of murine bone marrow-derived dendritic cells in vitro;(2) Effect of Ub-HBc Ag-CTP fusion protein on promoting T lymphocytes proliferation and inducing specific CTL response in vitro;(3) Specific CTL response induced by Ub-HBc Ag-CTP fusion protein in BALB/c mice. Part 1 Expression and purification of Ub-HBc Ag-CTP fusion proteinand the effect of it on inducing the maturation of murine bonemarrow-derived dendritic cells in vitroObjective: To construct a prokaryotic expression vector of Ub-HBc Ag-CTP fusion protein, and identify the expression and purification of the recombinant protein. Meanwhile, to observe the effect of Ub-HBc Ag-CTP inducing murine bone marrow-derived dendritic cell maturation in vitro.Methods: A pair of primers were designed based on fusion gene Ub-HBc Ag in pc DNA3.1(-)-Ub-HBc Ag with the forward primer having CTP gene. The fusion gene Ub-HBc Ag was amplified by PCR, then the product was cloned into p MAL-c2 X, a prokaryotic expression vector. The correct vector was transformed into E.coli BL21(DE) to express the fusion protein after identified by colony PCR and sequence test. The fusion protein was purified and identified by western blotting. And the control proteins HBc Ag-CTP and Ub-HBc Ag were also contructed and expressed. Bone marrow-derived dendritic cells isolated from BALB/c mice were cocultured with recombinant IL-4 and GM-CSF for 5 days. Fusion proteins Ub-HBc Ag-CTP, HBc Ag-CTP, Ub-HBc Ag or HBc Ag were added to induce DC maturation. The distribution and localization of intracellular immunofluorescence were observed by confocal microscopy with fluorescence intensity assaying quantitatively. DCs phenotypes was analyzed via flow cytometry(FCM). IL-12p70 levelin the supernatant was tested via enzyme linked immunosorbent assay(ELISA).Results: 820 bp, 590 bp and 780 bp bands were obtained by PCR, and genes were cloned into the p MAL-c2 X plasmid which were transfected into DE3 and expressed successfully. The colony PCR were obtained and identified to be fusion proteins Ub-HBc Ag-CTP, HBc Ag-CTP and Ub-HBc Ag. DCs were cultured and identified successfully. Immunofluorescence confirmed that the protein of Ub-HBc Ag-CTP could penetrate the membrane into the cytoplasm of DCs, but not into the nucleus. DCs surface molecules, such as CD80, CD83, CD86 and MHC-I was upregulated by Ub-HBc Ag-CTP. IL-12p70 level induced by Ub-HBc Ag-CTP was significantly higher than those control groups.Conclusion: Fusion protein Ub-HBc Ag-CTP and the control proteins were successfully synthesized. Ub-HBc Ag-CTP had the ability of penetrating and locating in the cytoplasm of DCs. Fusion protein Ub-HBc Ag-CTP could upregulate the expression of costimulatory molecules on cell surface of DCs, and enhance the ability of DCs to stimulate IL-12p70 production.Part 2 Effect of Ub-HBc Ag-CTP fusion protein on promoting Tlymphocytes proliferation and inducing specificCTL response in vitroObjective: To observe the effect of Ub-HBc Ag-CTP-sensitized DCs on promoting T lymphocyte proliferation and inducing specific cytotoxic T lymphocyte in vitro.Methods: DCs were cultured and induced maturation by different fusion proteins, and cocultured with T cells. The levels of IFN-γ, IL-2, IL-4 and IL-10 in the supernatants were detected by ELISA. The intracellular cytokine of stimulated T cells was analyzed via flow cytometry. The proliferation of T lymphocytes was performed by using cell counting kit-8(CCK-8). The specific CTL activity was detected via lactate dehydrogenase(LDH) release assay.Results: The levels of IFN-γ and IL-2 induced by Ub-HBc Ag-CTP were higher than other groups. The amount of CTLs induced by Ub-HBc Ag-CTP fusion protein was more than others by the analysis of intracellular cytokine of proliferative T cells. The proliferation of T lymphocytes induced by Ub-HBc Ag-CTP was much higher than that in other groups. Specific CTL killing activity was higher than the control groups.Conclusion: DCs sensitized by Ub-HBc Ag-CTP fusion protein can stimulate Th1 cytokine secretion and increase cytotoxic T lymphocytes generation effectively. Also, the specific CTL activity was enhanced.Part 3 Specific CTL response induced by Ub-HBc Ag-CTP fusionprotein in BALB/c miceObjective: To observe the effect of specific cytotoxic T lymphocyte induced by Ub-HBc Ag-CTP fusion protein in BALB/c mice and mechanisms probably involved in, and to investigate the relationship between CTL induced by Ub-HBc Ag-CTP fusion protein and JAK/STAT signaling pathway.Methods: BALB/c mice were divided into 5 groups. Mice were immunized intramuscularly in the left tibialis with 50μg Ub-HBc Ag-CTP, HBc Ag-CTP, Ub-HBc Ag, HBc Ag, respectively and 100μl PBS thrice at an interval of 1 week. Afterward, the mice were sacrificed. Seplenocytes were collected at 7 days after the third immunization was administered. ELISA was performed to detect the levels of cytokines secreted by T lymphocytes. The intracellular cytokine of proliferative T cells was detecteded by FCM and specific T lymphocytes were detected by enzyme-linked immunospot assay(ELISPOT). The proliferation of T lymphocytes was performed by using CCK-8 and the specific CTL activity was measured by a LDH release assay. The expression of each molecule in JAK/STAT signaling pathway was detected by western blotting.Results: Ub-HBc Ag-CTP fusion protein not only can stimulate Th1 cytokine secretion, but also induce higher specific CTL activity detected by flow cytometry and ELISPOT. The proliferation of T lymphocytes was much higher in Ub-HBc Ag-CTP group than in other groups. The expression of Jak2, Tyk2, STAT1 and STAT4 was significantly upregulated in BALB/c mice immunized with Ub-HBc Ag-CTP compared with other group. However, the expression of Jak1, Jak3 and STAT6 had no statistical difference in mice immunized with all of the fusion proteins or PBS.Conclusion: Ub-HBc Ag-CTP fusion protein could not only enhance the percentages of CTLs, but also induce robust specific CTL activity in vivo, which was probably associated with activation of the JAK/STAT signaling pathway.