|
|
|
| Packaging and identification of ubiquitinated HBcAg fusion gene and Tapasin gene expression recombinant lentivirus vectors |
| DAI Shenglan QIU Caiyu YAO Jun XU Yaping |
| Department of Gastroenterology, Affiliated People′s Hospital of Jiangsu University, Jiangsu Province, Zhenjiang 212002, China |
|
|
|
Abstract Objective To construct and package a recombinant lentivirus stably expressing the ubiquitinated HBcAg fusion gene and the Tapasin gene and to lay the foundation for the experimental study of immunotherapy for chronic hepatitis B. Methods The Ub-HBcAg gene was amplified with designing primers by polymerase chain reaction (PCR). The purified Ub-HBcAg fragment was cloned into the lentivirus vector pLenti-CMV-HA-3Flag-P2A-EGFP which was ligated by restriction endonuclease. The product pLenti-CMV-Ub-HBcAg-HA-3Flag-P2A-EGFP was transformed into E. coli DH5α cells. The positive clones were chosen for PCR identification and direct sequencing analysis. Then, T2A-Tapasin sequence was chemically synthesized and cloned into pLenti-CMV-Ub-HBcAg-HA-3Flag-P2A-EGFP. The product recombinant pLenti-CMV-Ub-HBcAg-HA-T2A-Tapasin-3Flag-P2A-EGFP plasmid was confirmed by direct gene sequencing. Lentiviral particles of LV-Ub-HBcAg/Tapasin were produced by triple transfection of 293T cells with pLenti-CMV-Ub-HBcAg-HA-T2A-Tapasin-3Flag-P2A-EGFP using packaging plasmids pLP1, pLP2 and envelope plasmid pLP/VSVG. The recombinant lentiviral particles were transfected into human embryonic kidney 293T cells. The titer of lentivirus was detected by real-time fluorescent quantitative PCR and the expression of the target gene was determined by Western blot. Results Recombinant lentivirus vectors expressing ubiquitinated HBcAg fusion gene and Tapasin gene were successfully constructed and packaged. The titer was 3.25×108 TU/mL detected by real-time fluorescent quantitative PCR. Conclusion The stable expressing of ubiquitinated HBcAg fusion gene and Tapasin gene recombinant lentivirus vectors are successfully established, providing experimental basis for immunotherapy of chronic hepatitis B.
|
|
|
|
|
|
[1] Trepo C，Chan HL，Lok A. Hepatitis B virus infection [J]. Lancet，2014，384（9959）：2053-2063.
[2] Sijts EJ，Kloetzel PM. The role of the proteasome in the generation of MHC class Ⅰ ligands and immune responses [J]. Cell Mol Life Sci，2011，68（9）：1491-1502.
[3] Tang TJ，De Man RA，Kusters JG，et al. Intrahepatic CD8 T-lymphocytes and HBV core expression in relation to response to antiviral therapy for chronic hepatitis B patient [J]. J Med Virol，2004，72（2）：215-222.
[4] 沈楠，余永胜，奚敏，等.强制泛素化乙肝病毒核心抗原融合基因表达质粒的构建[J].中国现代医学杂志，2010， 20（5）：699-701.
[5] Ning LH，Hao J，Liao ZL，et al. A survey on the current trends in the management of hepatitis B in China [J]. Eur J Gastroenterol Hepatol，2012，24（8）：884-889.
[6] Rehermann B. Pathogenesis of chronic viral hepatitis：differential roles of T cells and NK cells [J]. Nat Med，2013， 19（7）：859-868.
[7] Bertoletti A，Ferrari C. Innate and adaptive immune responses in chronic hepatitis B virus infections：towards restoration of immune control of viral infection [J]. Gut，2012，61（12）：1754-1764.
[8] Asabe S，Wieland SF，Chattopadhyay PK，et al. The size of the viral inoculum contributes to the outcome of hepatitis B virus infection [J]. J Virol，2009，83（19）：9652-9662.
[9] Wang Y，Chen K，Wu Z，et al. Immunizations with hepatitis B viral antigens and a TLR7/8 agonist adjuvant induce antigen-specific immune responses in HBV-transgenic mice [J]. Int J Infect Dis，2014，29：31-36.
[10] Akbar SM，Furukawa S，Horiike N，et al. Safety and immunogenicity of hepatitis B surface antigen-pulsed dendritic cells in patients with chronic hepatitis B [J]. J Viral Hepat，2011，18（6）：408-414.
[11] Miller DS，Halpern M，Kotlarski I，et al. Vaccination of ducks with a whole-cell vaccine expressing duck hepatitis B virus core antigen elicits antiviral immune responses that enable rapid resolution of de novo infection [J]. Virology，2006，348（2）：297-308.
[12] Yin Y，Wu C，Song J，et al. DNA immunization with fusion of CTLA-4 to hepatitis B virus（HBV）core protein enhanced Th2 type responses and cleared HBV with an accelerated kinetic [J]. PLoS One，2011，6（7）：e22524.
[13] Fu F，Li X，Lang Y，et al. Co-expression of ubiquitin gene and capsid protein gene enhances the potency of DNA immunization of PCV2 in mice [J]. Virol J，2011，8：264-272.
[14] Hinz A，Jedamzick J，Herbring V，et al. Assembly and function of the major histocompatibility complex（MHC）Ⅰ peptide-loading complex are conserved across higher vertebrates [J]. J Biol Chem，2014，289（48）：33109-33117.
[15] Hulpke S，Tomioka M，Kremmer E，et al. Direct evidence that the N-terminal extensions of the TAP complex act as autonomous interaction scaffolds for the assembly of the MHC Ⅰ peptide-loading complex [J]. Cell Mol Life Sci，2012，69（19）：3317-3327.
[16] Hermann C，Van Hateren A，Trautwein N，et al. TAPBPR alters MHC class Ⅰ peptide presentation by functioning as a peptide exchange catalyst [J]. Elife，2015，4：e09617.
[17] Tang Y，Wang J，Zhang Y，et al. Correlation between low tapasin expression and impaired CD8+ Tcell function in patients with chronic hepatitis B [J]. Mol Med Rep，2016， 14（4）：3315-3322.
[18] Chen X，Tang Y，Zhang Y，et al. Tapasin modification on the intracellular epitope HBcAg18-27 enhances HBV-specific CTL immune response and inhibits hepatitis B virus replication in vivo [J]. Lab Invest，2014，94（5）：478-490.
[19] Dai S，Zhuo M，Song L，et al. Dendritic cell-based vaccination with lentiviral vectors encoding ubiquitinated hepatitis B core antigen enhances hepatitis B virus-specific immune responses in vivo [J]. Acta Biochim Biophys Sin（Shanghai），2015，47（11）：870-879.
[20] Dai S，Zhuo M，Song L，et al. Lentiviral vector encoding ubiquitinated hepatitis B core antigen induces potent cellular immune responses and therapeutic immunity in HBV transgenic mice [J]. Immunobiology，2016，221（7）：813-821. |
|
|
|
