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目的制备C型HBV转基因小鼠,为乙肝的防治研究工作提供较好的动物模型。方法采用受精卵显微注射法,制备HBV转基因小鼠,用PCR,ELISA,荧光定量PCR和免疫组化的方法检测HBV基因在转基因小鼠体内的整合、复制和表达情况。结果注射受精卵2282枚,筛选注射后成活的受精卵共2024枚,注射成活率88.7%。注射后受精卵共移植假孕雌鼠72只,其中有59只怀孕,假孕雌鼠妊娠率82%,共产下F0代小鼠185只,PCR检测共有19只小鼠阳性,PCR阳性率为10.3%,荧光定量PCR血清HBV DNA显示6只小鼠有复制,其拷贝数为102～103copies/L之间;经传代产下F1代小鼠96只,PCR检测HBV DNA阳性33只,PCR阳性率为34.4%,荧光定量PCR血清HBV DNA显示10只小鼠有复制;随机分别取3只F0代和F1代小鼠的肝脏和肾脏HBsAg免疫组化均为阳性。结论制备的C型HBV转基因小鼠在体内有复制和表达,并且可以遗传给下一代。
Objective To prepare C type HBV transgenic mice to provide a good animal model for the prevention and treatment of hepatitis B. Methods HBV transgenic mice were prepared by microinjection of zygotes. The integration, replication and expression of HBV gene in transgenic mice were detected by PCR, ELISA, fluorescence quantitative PCR and immunohistochemistry. Results A total of 2282 fertilized eggs were injected. A total of 2024 fertilized eggs were selected after inoculation, and the survival rate of injections was 88.7%. After inoculation, 72 pregnant women with fertilized eggs were co-transplanted, of which 59 were pregnant and 82 were pregnant. The pregnancy rate of fake pregnant women was 82%. A total of 185 F0 mice were co-produced. A total of 19 mice were positive by PCR. The positive rate of PCR was 10.3%. Fluorescence quantitative PCR serum HBV DNA showed that there were 6 mice replicating with 102 ~ 103 copies / L copy number. 96 F1 mice were passaged, PCR positive HBV DNA was detected by PCR, The rate was 34.4%. Fluorescent quantitative PCR serum HBV DNA showed that 10 mice were duplicated. The liver and kidney of three F0 generation and F1 generation mice were randomly selected to be immunohistochemically positive for HBsAg. Conclusion The prepared C type HBV transgenic mice are replicated and expressed in vivo and can be passed on to the next generation.