The Study of Recombinant Plasmid CpG-HBcAg Immune Effects on BALB/c Mouse and Human Dendritic Cells
|School||Huazhong University of Science and Technology|
|Keywords||CpG motifs Hepatitis B core antigen Dendritic cells Th1/Th2 cytokines TLR9|
Hepatitis B is a common infectious diseases, according to statistics worldwide 350 million chronic carriers of hepatitis B virus. Develop into chronic hepatitis B patients with cirrhosis of the risk is relatively high, which is hepatocellular carcinoma and non-neoplastic complications of cirrhosis caused the main reason for the higher mortality rate. Currently, the treatment of chronic hepatitis B is the only effective way of continuing the system IFN-a treatment. However, only 1/3 of patients with chronic hepatitis B can achieve sustained response. Nucleoside analogues such as lamivudine make serum HBV DNA in rapid decline, but also make the liver in patients with histological improvement. But after cessation of treatment often leads to disease relapse quickly, but long-term treatment often causes selective variation of the virus occurred. For these situations we need to find a new method of treatment. Although the pathogenesis of chronic hepatitis B has not yet fully understood, but there is still a consensus that liver damage is immune-mediated. Specific immunotherapy approach is to replace as much as possible the use of interferon or antiviral drugs to enhance the efficacy of the treatment of chronic hepatitis B patients role. Methyl cytosine guanine dinucleotide sequence of certain side (CpG motif) was first found in the bacterial DNA, its innate and acquired immune systems have different stimulation. Some of which help to strengthen the role of antigen-specific Th1-type responses adjuvant activity. For example, CpG DNA to promote 95% B cell proliferation and immunoglobulin secretion (Ig) and cytokines, the B cells to be protected from apoptosis, all of which contribute to the formation of a strong humoral response. CpG DNA also directly activates monocytes, macrophages and dendritic cells to secrete Th1 type cytokines, which in turn induce T cells and NK cells to secrete more cytokines. Overall, CpG induced Th1-type cytokines, notably interleukin -12 (IL-12) and the IFN-g, little secretion of Th2 cytokines, such Th1-type cytokines may enhance the humoral and T cell mediated immune response. In animal studies, CpGODN with various antigens has been shown to be effective Th1 combination vaccine adjuvant. For example, mice immunized by intramuscular injection of antigen and CpGODN will have a strong cytotoxic T lymphocyte (CTL) and the main IgG2a antibodies, i.e., Th1-type response. Because Th1-type immune response is considered to be cleared hepatitis B virus infection necessary response, CpGODN with recombinant HBcAg is likely to be an effective therapeutic vaccine for the treatment of chronic hepatitis B patients. Therefore, in our study, we first constructed successfully HBcAg and CpG-containing recombinant eukaryotic expression plasmid, intramuscular injection of recombinant plasmid pZeoSV2 () / CpG-HBcAg (ISS) immunized BALB / c mice, study its effect on BALB / c mice mice immunization. Meanwhile, the recombinant plasmid pEGFP-N1/CpG-HBcAg (ISS) transfected human dendritic cells, the researchers transfected DC surface molecule expression changes and plasmids for DC immune effects. In addition, DC after transfection supernatant induced hepatocellular carcinoma cell line HepG2, study apoptosis in HepG2 cells and its mechanism. Finally, the study TLR9 in various chronic viral titer hepatitis B, hepatitis C patients with peripheral mononuclear cells expression. Purpose 1. Build pZeoSV2 () / CpG-HBcAg (ISS) and pEGFP-N1/CpG-HBcAg (ISS) eukaryotic expression vector for the development of hepatitis B therapeutic vaccine basis. 2. Explore the recombinant plasmid pZeoSV2 () / CpG-HBcAg (ISS) in BALB / c mice were immunized role. 3. Explore the recombinant plasmid pEGFP-N1/CpG-HBcAg (ISS) was transfected into human peripheral blood monocyte-derived dendritic cells (DC), the expression of its surface molecules and immune function. 4. Explore the recombinant plasmid pEGFP-N1/CpG-HBcAg (ISS) was transfected into human peripheral blood monocyte-derived dendritic cells, the cell culture supernatant induced apoptosis in HepG2 cells and its mechanism. 5. Research TLR9 receptor at different viral load chronic hepatitis B, hepatitis C patients with peripheral blood mononuclear cells (PMBC) expression. Method 1. According to the hepatitis B virus core antigen gene sequences were designed and synthesized three pairs of primers were introduced in the primers for the human, mouse-sensitive and non-CpG CpG fragment fragment by PCR from the serum of patients with chronic HBV DNA was amplified hepatitis B core antigen gene fragment was amplified product with eukaryotic expression plasmid pZeoSV2 () and pEGFP-N1 to construct recombinant pZeoSV2 () / CpG-HBcAg (ISS) and pEGFP-N1/CpG-HBcAg (ISS), were digested , PCR and sequencing. 2. Eukaryotic expression plasmid pZeoSV2 () / CpG-HBcAg (ISSb, c), to be immunized BALB / c mice, ELISA assay immunized mouse serum HBcAb, IFN-γ, IL-2, IL-12, IL-4 and IL-10 levels. 3. Eukaryotic expression plasmid pEGFP-N1/CpG-HBcAg (ISSa, c), transfected human peripheral blood DC. Flow cytometry was used to detect the transfection of DC surface expression of CD80 and CD86. By ELISA after transfection DC culture supernatant IFN-γ, IL-12, IL-4 and IL-10 levels. 4. Eukaryotic expression plasmid pEGFP-N1/CpG-HBcAg (ISSa, c), transfected human peripheral blood DC, with a culture supernatant of HepG2 induced apoptosis. Supernatant was detected induced apoptosis of HepG2 changes. 5. Detection of chronic hepatitis B, hepatitis C viral load in patients, and to analyze its relationship with TLR9 expression. TLR9 protein expression levels by flow cytometry. The study group comprised 90 patients (60 patients with chronic hepatitis B, 30 patients with chronic hepatitis C) and 20 normal healthy controls. Results 1. Hepatitis B core antigen gene in vitro amplification product size of about 530bp. The constructed pZeoSV2 () / CpG-HBcAg (ISS) and pEGFP-N1/CpG-HBcAg (ISS) recombinants by double enzyme digestion and PCR identification, consistent with the expected size of the fragments; sequencing results with full HBcAg in GenBank consistent with long sequences, suggesting pZeoSV2 () / CpG-HBcAg (ISS) and pEGFP-N1/CpG-HBcAg (ISS) eukaryotic expression vector was constructed correctly. 2. pZeoSV2 () / CpG-HBcAg (ISSb, c) the recombinant plasmid could induce BALB / c mice to produce specific antibodies HBcAb, pZeoSV2 () / CpG-HBcAg (ISSb) group than pZeoSV2 () / CpG-HBcAg (ISSc) group produced anti-HBc titers were significantly higher (P <0.01), and their immune BALB / c mice could make Th1-type cytokines IFN-γ, IL-2 and IL-12 expression in enhanced inhibition of Th2 cells factor IL-4 and IL-10 production. 3. pEGFP-N1/CpG-HBcAg (ISSa) DC transfected with the expression of CD80 and CD86 were significantly higher (P <0.01). After transfection supernatant Th1-type cytokines IFN-γ and IL-12 expression enhanced (P <0.01), Th2-type cytokines IL-4 and IL-10 expression decreased (P <0.05). 4. pEGFP-N1/CpG-HBcAg (ISSa) group culture supernatant of HepG2 cells induce apoptosis, with the incubation time, gradually increase the rate of apoptosis [0h: (2.8 ± 0.8)%, 6h: (7.6 ± 1.0)%, 12h: (9.2 ± 1.1)%, 18h: (12.6 ± 1.2)%, 24h: (17.7 ± 0.7)%, P <0.05]. 5. The results showed that compared with normal control group of hepatitis B, hepatitis C virus chronic infection leads to decreased levels of TLR9 protein expression (P <0.05). TLR9 protein levels in serum of chronic hepatitis B and chronic hepatitis C viral load in serum viral load was negatively correlated (r = -0.632, r = -0.909, P <0.01). Conclusions 1. Successfully constructed eukaryotic expression vector pZeoSV2 () / CpG-HBcAg (ISS) and pEGFP-N1/CpG-HBcAg (ISS), the function of CpG research and treatment of hepatitis B vaccine provides the material basis. 2. pZeoSV2 () / CpG-HBcAg (ISSb, c) HBcAb recombinant mice generated a significant role in promoting containing CpG-HBcAg (ISSb) recombinant plasmid can significantly enhance the expression of Th1-type cytokines, inhibition of Th2-type cells factor expression. 3. Recombinant plasmid pEGFP-N1/CpG-HBcAg (ISSa) transfected DC can increase the expression of costimulatory molecules, and can induce Th1-type cytokines (IFN-γ and IL-12) production, inhibition of Th2 cytokines ( IL-4 and IL-10) production. 4. Recombinant plasmid pEGFP-N1/CpG-HBcAg (ISSa) transfected supernatants can significantly promote apoptosis of HepG2 cells. 5. Chronic hepatitis B, hepatitis C patients PMBC of TLR9 protein levels decreased, and with serum viral load was negatively correlated with detection of viral replication role.