乙型肝炎病毒B、C、D基因型AAV-HBV小鼠模型构建与病毒学特征比较  

Construction of hepatitis B virus B,C,D AAV-HBV mouse models and comparison of virological features

作　　者：郑晓文 冯成千 李华鹏 刘土花 古国铭 康桦华 徐志宏 胡凤玉[1] 李锋 ZHENG Xiao⁃wen;FENG Cheng⁃qian;LI Hua⁃peng;LIU Tu⁃hua;GU Guo⁃ming;KANG Hua⁃hua;XU Zhi⁃hong;HU Feng⁃yu;LI Feng(Guangzhou Eignth People’s Hospital,Guangzhou Medical University,Guangzhou,Guangdong 510060;Guangzhou Paizhen Biological Co.,Ltd,Guangzhou,Guangdong 510663;Guangzhou Xinyi Biotechnology Co.,Ltd,Guangzhou,Guangdong 510640;Institute of Animal Health,Guangdong Acadamy of Agrioultural Sciences,Guangzhou,Guangdong 510640,China)

机构地区：[1]广州医科大学附属广州市第八人民医院,广东广州510060 [2]广州派真生物技术有限公司,广东广州510663 [3]广州欣意生物技术有限公司,广东广州510640 [4]广东省农业科学院动物卫生研究所,广东广州510640

出　　处：《热带医学杂志》2020年第3期297-302,308,共7页Journal of Tropical Medicine

基　　金：国家自然科学基金(81670536,81200947);十三五国家科技重大专项(2017ZX10202203⁃004⁃002,2018ZX10301404⁃003⁃002)。

摘　　要：目的建立乙型肝炎病毒(HBV)B、C基因型中国流行株腺相关病毒介导的乙肝病毒(AAV-HBV)小鼠模型,并研究其病毒学变化特征。方法以B、C基因型HBV流行株序列为基础,并参照常用D基因型病毒序列,分别构建出携带1.3×基因组的腺相关病毒载体质粒:p AAV-HBV1.3×(B)、pAAV-HBV1.3×(C)和pAAV-HBV1.3×(D)。通过三质粒共转染方法在293细胞中包装成三种成熟的腺相关病毒载体rAAV-HBV 1.3(B)、rAAV-HBV 1.3(C)与rAAV-HBV1.3(D)。C57BL/J小鼠通过尾静脉注射相同剂量的AAV-HBV病毒载体,在注射后不同时间点,用ELISA方法检测血清中HBsAg和HBeAg表达水平,用荧光定量PCR(Q-PCR)方法检测小鼠血清中HBV DNA拷贝数。结果新构建B、C基因型两株病毒有更高HBsAg表达水平,B基因型小鼠血清中HBsAg检测值(均值)波动在957.67~2 590.04 IU/mL,C基因型小鼠血清HBsAg波动在725.21~3 195.16 IU/mL,D基因型小鼠血清HBsAg波动在153.4~531.8 IU/mL。B基因型小鼠血清中,HBsAg与HBV DNA比值最高,D基因型最低。新构建B、C基因型两株病毒表现出与常用D基因型病毒不同的血清动力学特征。B、C基因型小鼠的HBsAg和HBV DNA可在1周内快速达到较高水平,并维持稳定,在第3~4周表现出波动下降,随后表达水平回升。D基因型小鼠HBsAg在10 d内达到峰值,然后不断降低,HBV DNA在第3~4周也有明显波动。B、C、D三种基因型HBeAg在第1周较低,在两周达到峰值,并保持稳定。结论基于我国流行B、C基因型序列,成功建立了两株AAV-HBV小鼠模型,为研究B、C基因型病毒提供了新工具。Objective To generate mouse models carrying hepatitis B virus(HBV)genotype B and C virus which are dominant in China using adeno-associated virus(AAV),and characterize their viral biomarker profiles in mouse models.Methods pAAV-HBV1.3×(B),p AAV-HBV1.3×(C)and pAAV-HBV1.3×(D),containing 1.3-fold length of HBV genome were constructed based on the sequences from HBV genotype B,C islolated from clinical samples,and on genotype D sequence,respectively. Reconbiant AAV-HBV(rAAV-HBV)was packaged in 293 T cell culture through three plamid cotransfection. Then,rAAV-HBV1.3(B),rAAV-HBV1.3(C)and rAAV-HBV1.3(D)were tail-veinly injected into C57 BL/J separately. Blood samples were collected at different time points. Serum HBsAg and HBeAg were measured by enzymelinked immunosorbent assay(ELISA)and HBV DNA was measured by quantitative PCR(Q-PCR)method. Results The newly generated HBV B and C genotype mouses expressed high levels of HBsAg ranging from 957.67 to 2 590.04 IU/mL in B genotype,from 725.21 to 3 195.16 IU/mL for C genotype,and from 153.4 to 531.8 IU/mL for D genotype. B genotype mice had the highest ratio of HBsAg to HBV DNA while D genotype mice had the lowest. Newly generated B and C genotype mice expressed different serum viral biomarker profiles,compared to the commonly used D genotype. The HBsAg and HBV DNA of B and C genotype mice peaked within 1 stweek quickly and maintained at high levels thenafter,but with a flunction between week 2 and week 4. The HBsAg and HBV DNA in D genotype mice peaked late within 10 days,and the HBsAg kept consistent but gradually decreased while the DNA level showed a flunction between week 2 and week 4. The HBeAg levels of B,C,D genotype mice were low within the 1 stweek,then peaked in week 2 and kept stable thenafter.Conclusion We have successfully generated AAV-HBV mouse models based on the sequences of HBV B and C genotypes isolated in China,our study provided new tools for studying B and C genotype HBV.

关 键 词：乙型肝炎病毒 AAV-HBV B、C基因型 

分 类 号：R512.62[医药卫生—内科学]