HBV 乙型肝炎造模-rAAV

HBV rAAV载体的特点

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技术细节

• 操作要求

  rAAV作为基因治疗载体的安全性在1994年被FDA认可，其生物安全性为一级（BSL-1），与质粒DNA的安全级别相同，建议使用BSL-2实验室和ClassⅡ生物安全柜。

• 储存要求

  • 将病毒储存在 -80°C冰箱， 并在操作过程中将其置于冰上。
  • 建议对使用量提前进行预估，派真生物出货时按照预定的分装要求进行分装，避免收到货后再次冻融分装影响滴度准确性。如需再次分装使用，推荐使用内壁硅化程度良好的PCR管，或者低蛋白结合率的病毒专用保存管。
  • 如需稀释AAV-HBV,请将病毒置于冰浴融解后，使用Tris缓冲液稀释。

• AAV-HBV小鼠造模实验设计

  重组腺相关病毒（rAAV）已经被广泛应用于动物体内实验和细胞研究中。但对于体内试验，注射部位如何选择，注射多少病毒量，注射后多久检测等问题，因为研究领域、研究的动物不同，而很难有简单通用的注射方法，实验中还需要具体探索最佳条件：

  强烈建议在正式实验之前，对AAV-HBV进行3~4个注射量的梯度测试，每只小鼠依次按最低要求递增。比如：1E+11 GC；2E+11 GC；3E+11 GC 做3个梯度稀释；

• 客户案例

  新型抗HBV治疗方法的研究

  客户发表文章：Meng C, Sun S, Liang Y, et al. Engineered anti-PDL1 with IFNαtargets both immunoinhibitory and activating signals in the liver to break HBV immune tolerance. Gut 2023; 72: 1544-1554.

  病毒类型：rAAV8-HBV ayw，D基因型

  注射方式：C57BL/6小鼠，尾静脉注射

  注射剂量：1 E 11 GC

  检测时间：35天

  

• 相关文献

  1. Gao L, Yang J, Feng J, Liu Z, Dong Y, Luo J, Yu L, Wang J, Fan H, Ma W and Liu T (2022)PreS/2-21-Guided siRNA Nanoparticles Target to Inhibit Hepatitis B Virus Infection and Replication. Front. Immunol. 13:856463. doi: 10.3389/fimmu.2022.856463.
  2. Jing Miao, Xiqin Yang, Xuwei Shang, Zhe Gao, Qian Li, Yun Hong, Jiaying Wu, Tingting Meng, Hong Yuan, Fuqiang Hu. Hepatocyte-targeting and microenvironmentally responsive glycolipid-like polymer micelles for gene therapy of hepatitis B. Molecular Therapy - Nucleic Acids,Volume 24, 2021, Pages 127-139.
  3. Xu W, Niu Y, Ai X, Xia C, Geng P, Zhu H, Zhou W, Huang H, Shi X. Liver-Targeted Nanoparticles Facilitate the Bioavailability and Anti-HBV Efficacy of Baicalin In Vitro and In Vivo. Biomedicines. 2022; 10(4):900.
  4.Wang X, Zhang Y, Ben Y, Qiu C, Wu J, Zhang W, Wan Y. Anti-HBc IgG Responses Occurring at the Early Phase of Infection Correlate Negatively with HBV Replication in a Mouse Model. Viruses. 2022; 14(9):2011.
  5. Yue Wang, Yaxian Mei, Zhenghong Ao, Yuanzhi Chen, Yichao Jiang, Xiaoqing Chen, Ruoyao Qi, Baorong Fu, Jixian Tang, Mujin Fang, Min You, Tianying Zhang, Quan Yuan, Wenxin Luo, Ningshao Xia. A broad-spectrum nanobody targeting the C-terminus of the hepatitis B surface antigen for chronic hepatitis B infection therapy. Antiviral Research, Volume 199, 2022, 105265.
  6. Xiu-Qing Pang, Xing Li, Wei-Hang Zhu, Run-Kai Huang, Zhi-shuo Mo, Ze-Xuan Huang, Yuan Zhang, Dong-Ying Xie, Zhi-Liang Gao. LAG3+ erythroid progenitor cells inhibit HBsAg seroclearance during finite pegylated interferon treatment through LAG3 and TGF-β. Antiviral Research, Volume 213, 2023, 105592.
  7. Meng C, Sun S, Liang Y, et al. Engineered anti-PDL1 with IFNα targets both immunoinhibitory and activating signals in the liver to break HBV immune tolerance. Gut 2023; 72: 1544-1554.
  8. Y. Jiang, X. Chen, X. Ye, C. Wen, T. Xu, C. Yu, W. Ning, G. Wang, X. Xiang, X. Liu, Y. Wang, Y. Chen, X. Liu, C. Shi, C. Liu, Q. Yuan, Y. Chen, T. Zhang, W. Luo, N. Xia. A Dual-domain Engineered Antibody for Efficient HBV Suppression and Immune Responses Restoration. Adv. Sci. 2024, 11, 2305316.
  9. Wang, Y., Guo, L., Shi, J. et al. Interferon stimulated immune profile changes in a  humanized mouse model of HBV infection. Nat Commun 14, 7393 (2023).