抗乙型肝炎病毒免疫机制的相关研究
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摘要
目的：检测不同基因型的HBV PreS1多肽的免疫反应，评估其作为一种新的入胞抑制剂用于治疗急慢性HBV感染对机体免疫系统可能造成的影响。方法：1、用不同的免疫方案免疫Balb／C小鼠。2、ELISA检测多肽诱导机体产生特异性的Anti-PreS1抗体及其交叉反应。3、ELISA检测特异性的Anti-PreS1抗体是否能结合纯化的病毒颗粒，进一步用免疫沉淀，Real time PCR等检测Anti-PreS1是否具有识别并清除病毒颗粒的作用。4、收集不同免疫状态的慢性HBV患者血清，ELISA检测是否存在可识别多肽的特异性抗体，PCR测序检测血清基因型背景。
结果：1、多肽可诱导特异性抗体产生。2、不同基因型的多肽免疫后得到的抗体具有交叉反应。3、特异性的Anti-PreS1抗体能够识别并结合病毒颗粒，具有一定的中和作用。
结论：体内实验证实HBV PreS1多肽可诱导机体产生特异性的抗体，抗体具有识别及结合病毒的能力，推测其可能有部分中和作用，参与病毒清除。由于有特异性抗体产生，故不适合作为临床治疗性药物长期使用。
Objective: To detect the immune reaction of HBV PreS1 peptides with different genotype and evaluate the potential influence for host immunity of the peptides as a new entry inhibitors for the treatment of acute and chronic hepatitis B virus infection.
Methods: 1. Immunization of Balb/C mice with different methods. 2. Measurement of the HBV PreS1 peptides induced specific antibody response and the crossing reaction with ELISA. 3. Detection of the Anti-PreS1 antibody binding with the purified hepatitis B virus particles with ELISA. Detection of the antibody recognizing purified virus particles and its role in virus clearance with immunoprecipitation, Real Time PCR and so on. 4. Detection of specific Anti-preS1 peptides antibody in the serum of chronic HBV infectious patients in different immunization status with ELISA. 5. Analysis of virus genotype of different patient background with PCR.
Results: 1. HBV preS1 peptides could induce specific antibody in Balb/C mice. 2. The Anti-preS1 for different genotype could have crossing reaction. 3. The Anti-PreS1 could recognize purified hepatitis B virus particles and bind with them.
Conclusion: HBV PreS1 peptides could induce the specific Anti-PreS1 that could recognize hepatitis B virus particles and bind with them, which indicated that the specific antibody have ability of clearing virus to a certain extent. As the peptides could induce production of specific antibody, it maybe not fit for clinic long-term treatment.
结果：1、多肽可诱导特异性抗体产生。2、不同基因型的多肽免疫后得到的抗体具有交叉反应。3、特异性的Anti-PreS1抗体能够识别并结合病毒颗粒，具有一定的中和作用。
结论：体内实验证实HBV PreS1多肽可诱导机体产生特异性的抗体，抗体具有识别及结合病毒的能力，推测其可能有部分中和作用，参与病毒清除。由于有特异性抗体产生，故不适合作为临床治疗性药物长期使用。
Objective: To detect the immune reaction of HBV PreS1 peptides with different genotype and evaluate the potential influence for host immunity of the peptides as a new entry inhibitors for the treatment of acute and chronic hepatitis B virus infection.
Methods: 1. Immunization of Balb/C mice with different methods. 2. Measurement of the HBV PreS1 peptides induced specific antibody response and the crossing reaction with ELISA. 3. Detection of the Anti-PreS1 antibody binding with the purified hepatitis B virus particles with ELISA. Detection of the antibody recognizing purified virus particles and its role in virus clearance with immunoprecipitation, Real Time PCR and so on. 4. Detection of specific Anti-preS1 peptides antibody in the serum of chronic HBV infectious patients in different immunization status with ELISA. 5. Analysis of virus genotype of different patient background with PCR.
Results: 1. HBV preS1 peptides could induce specific antibody in Balb/C mice. 2. The Anti-preS1 for different genotype could have crossing reaction. 3. The Anti-PreS1 could recognize purified hepatitis B virus particles and bind with them.
Conclusion: HBV PreS1 peptides could induce the specific Anti-PreS1 that could recognize hepatitis B virus particles and bind with them, which indicated that the specific antibody have ability of clearing virus to a certain extent. As the peptides could induce production of specific antibody, it maybe not fit for clinic long-term treatment.
引文
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20．Gripon P， Diot C， Theze N， Fourel I， Loreal O， Brechot C， Guguen-Guillouzo C．Hepatitis B virus infection of adult human hepatocytes cultured in the presence of dimethyl sulfoxide．J Virol．1988；62： 4136-43．
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22. Petersen J, Burda MR, Dandri M, Rogler CE.Transplantation of human hepatocytes in immunodeficient UPA mice: a model for the study of hepatitis B virus. Methods Mol Med. 2004;96:253-60.
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24. Engelke M, Mills K, Seitz S, Simon P, Gripon P, Schnolzer M, Urban S. Characterization of a hepatitis B and hepatitis delta virus receptor binding site. Hepatology. 2006;43:750-60.
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6、 Petit MA， Strick N， Dubanchet S， Capel F， Neurath AR．Inhibitory activity of monoclonal antibody F35．25 on the interaction between hepatocytes (HepG2 cells) and preS1-specific ligands．Mol Immunol．1991；28：517-21．
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8、 Le Seyec J， Chouteau P， Cannie I， Guguen-Guillouzo C， Gripon P．Role of the pre-S2 domain of the large envelope protein in hepatitis B virus assembly and infectivity．J Virol 1998；72 ：5573-5578．
9、 Gripon P， Cannie I， Urban S．Efficient inhibition of hepatitis B virus infection by acylated peptides derived from the large viral surface protein．J Virol．2005； 79：1613-22．
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20．Gripon P， Diot C， Theze N， Fourel I， Loreal O， Brechot C， Guguen-Guillouzo C．Hepatitis B virus infection of adult human hepatocytes cultured in the presence of dimethyl sulfoxide．J Virol．1988；62： 4136-43．
21．Neurath AR， Seto B， Strick N．Antibodies to synthetic peptides from the preS 1 region of the hepatitis B virus (HBV) envelope (env) protein are virus-neutralizing and protective．Vaccine．1989：7：234-6
22. Petersen J, Burda MR, Dandri M, Rogler CE.Transplantation of human hepatocytes in immunodeficient UPA mice: a model for the study of hepatitis B virus. Methods Mol Med. 2004;96:253-60.
23. Petersen J, Dandri M, Mier W, Lutgehetmann M, Volz T, von Weizsacker F, Haberkorn U, Fischer L, Pollok JM, Erbes B, Seitz S, Urban S. Prevention of hepatitis B virus infection in vivo by entry inhibitors derived from the large envelope protein. Nat Biotechnol. 2008:335-41.
24. Engelke M, Mills K, Seitz S, Simon P, Gripon P, Schnolzer M, Urban S. Characterization of a hepatitis B and hepatitis delta virus receptor binding site. Hepatology. 2006;43:750-60.
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