YMDD突变株HBV转基因小鼠的制备及检测

摘要

Objective To establish the hepatitis B virus (HBV) transgenie mice with YMDD mutation, and provide an animal model for research of HBV prevention and therapeutic approach. Methods 1. 3 copies HBV genome containing YMDD mutation associated with lamivudine resistance was injected into the zygote of FVB/N female mice by microinjection. Integration and passage of exogenous gene in transgenie mice was confirmed by PCR, The expression of HBsAg in liver and kidney tissues in transgenie mice was identified by ELISA and immunohistochemistry. Results A total of 3401 zygotes were injected and 269 Fo pups were bora PCR analysis indicated that 33 out of 269 pups were positive, and the integrating rate of exogenous gene was 12.3% in Fo. Fluorescent quantitative PCR showed that HBV DNA was weakly positive in serum samples in 9 transgenie mice, less than 103 copies/ml The expression of HBsAg in transgenie mice was observed in liver and kidney tissues by immunohistochemistry, and it was higher in kidney than in liver. The target gene was detected by PCR in 27.6% of 47 F1 offsprings. The expression of HBsAg could be observed in liver and kidney tissues in F1, which was similar to that in F0. Conclusion 1. 3 copies HBV transgenie mice model containing YMDD mutation associated with lamivudine resistance is successfully produced by microinjection, and HBsAg expression can be transmitted through germline cells.%目的 建立YMDD耐药突变株HBV转基因小鼠,为乙肝防治研究提供转基因动物模型.方法 采用受精卵显微注射法,将带有YMDD突变的对拉米夫定有耐药性的1.3拷贝HBV基因注入FVB/N单细胞受精卵的原核内,制备YMDD耐药突变株HBV转基因小鼠.采用PCR检测外源基因的整合和传代情况,采用ELISA和免疫组化等方法 检测HBsAg在肝、肾中的复制和表达情况.结果 注射受精卵3401枚,产269只F0代仔鼠,PCR阳性33只,外源基因的整合率12.3%.9只转基因鼠血清HBVDNA弱阳性,拷贝数低于103拷贝/ml;免疫组化结果 显示肝组织和肾组织均有HBsAg表达,且肾组织中的表达强于肝组织.经传代产下47只F1代转基因鼠,目的基因FCR阳性率为27.6%,且肝组织和肾组织中HBsAg均有表达,分布特性与F0代一致.结论成功制备出体内有复制表达而且可以传代YMDD耐药突变株HBV的转基因小鼠.