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目的:建立C型HBV转基因小鼠,为乙肝防治研究提供实验动物模型.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和免疫组化的方法研究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2840枚,筛选注射后成活的受精卵共2256枚,注射成活率79.4%.注射后受精卵共移植假孕雌鼠85只,其中有42只怀孕,假孕雌鼠妊娠率49%,共产下F0代小鼠168只,PCR检测共有14只小鼠阳性,其中ELISA检测血清HBsAg阳性5只,HBeAg均阴性.荧光定量PCR血清HBV DNA显示,2只小鼠的拷贝数分别为3.12×105copies/L,1.98×105copies/L,经传代产下F1代小鼠61只,PCR检测HBV DNA阳性5只,ELISA检测HBsAg阳性4只,其中肝脏HBsAg免疫组化阳性2只,HBcAg均阴性,且肝组织表达HB-sAg为胞浆型.结论:构建的C型HBV基因不但可以在转基因小鼠体内进行复制表达,而且可以遗传给下一代小鼠.
OBJECTIVE: To establish a C transgenic HBV transgenic mouse model for the study of hepatitis B prevention and treatment.Methods: HBV transgenic mice were prepared by microinjection of zygotes and analyzed by PCR, ELISA, fluorescence quantitative PCR and immunohistochemistry HBV gene integration, replication and expression in transgenic mice.Results: 2840 eggs were injected, and 2256 survived fertilized eggs were screened and the survival rate of injection was 79.4% .Pregnant female rats There were 85 pregnant women and pregnant women with pregnancy rate of 49% and 168 pregnant women of F0 generation were co-produced. A total of 14 mice were detected by PCR, of which 5 were positive for HBsAg by ELISA and negative for HBeAg. Quantitative PCR serum HBV DNA showed that the copy number of the two mice was 3.12 × 105 copies / L and 1.98 × 105 copies / L, respectively, 61 F1 mice were passaged, 5 were positive for HBV DNA by PCR, and HBsAg was detected by ELISA 2 were HBsAg positive in the liver, HBcAg was negative, and the HBsAg expression in the liver was cytoplasm.Conclusion: The constructed HBV C gene not only can be replicated and expressed in the transgenic mice, but also Can be passed on to the next generation of mice.